Inactivation of Glutathione Peroxidase by Peroxynitrite

Glutathione peroxidase (GSH-Px) is inactivated on exposure to peroxynitrite under physiologically relevant conditions. Stopped-flow kinetic studies show that the reaction between peroxynitrite and GSH-Px is first-order in each of the reactants, with an apparent second-order rate constant of 4.5 ± 0.2 × 10⁴M⁻¹s⁻¹per monomer unit of enzyme. In good agreement with this value, GSH-Px inactivation experiments afford an apparent second-order rate constant of 1.8 ± 0.1 × 10⁴M⁻¹s⁻¹per monomer unit of enzyme. The hydroxyl radical scavengers mannitol, DMSO, and benzoate (at 100 mM) afford only 8–12% protection of the enzyme, while addition of 25 mM bicarbonate results in 55% protection. The minimal protection by hydroxyl radical scavengers indicates, as expected, that hydroxyl radicals are not involved in the inactivation. Protection by bicarbonate occurs because peroxynitrite is rapidly trapped by CO₂to form the adduct nitrosoperoxycarbonate (ONOOCO⁻₂), and/or other reactive species that preferentially decompose to nitrate rather than react with GSH-Px. The close agreement between the rate constants obtained from enzyme inactivation and from stopped-flow kinetics experiments suggests that the mechanism of the reaction between peroxynitrite and GSH-Px involves the oxidation of the ionized selenol of the selenocysteine residue in the enzyme's active site (E-Se⁻) by peroxynitrite. This reaction does not simply involve formation of the selenenic acid, E-SeOH, because E-SeOH is an intermediate in the catalytic cycle of the enzyme, and thus its formation cannot explain the inactivation we observe. Thus, the ionized selenol in the active site is transformed into a form of selenium that cannot easily be reduced back to the selenol.     

Structural Basis for Recognition of the Pore-Forming Toxin Intermedilysin by Human Complement Receptor CD59

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The Autophagic Pathway: A Cell Survival Strategy Against the Bacterial Pore-Forming Toxin Vibrio Cholerae Cytolysin

Vibrio cholerae is the causative agent of cholera in humans. In addition to the critical virulence factors cholera toxin and toxin co-regulated pilus, V. cholerae secretes V. cholerae cytolysin (VCC), a pore-forming exotoxin able to induce cell lysis and extensive vacuolation. We have shown that this vacuolation is related to the activation of autophagy in response to VCC action. Furthermore, we found that the autophagic pathway was required to protect cells upon VCC intoxication. Based on additional data presented here, we propose a model aimed to explain the mechanism of cell protection. We postulate that VCC-induced autophagic vacuoles, which display features of multivesicular bodies and enclose the toxin, are implicated in cell defense through VCC degradation involving fusion with lysosomes.

Addendum to:

Protective Role of Autophagy Against Vibrio cholerae Cytolysin, a Pore-Forming Toxin from V. cholerae

M.G. Gutierrez, H.A. Saka, I. Chinen, F.C.M. Zoppino, T. Yoshimori, J.L. Bocco and M.I. Colombo

Proc Natl Acad Sci USA 2007; 104:1829-34     

Cys31, Cys47, and Cys195 in BinA Are Essential for Toxicity of a Binary Toxin from Bacillus sphaericus

The mosquito larvicidal binary toxin produced by Bacillus sphaericus is composed of 2 proteins called BinA and BinB. While BinB acts as specificity determinant, BinA is expected to bind to BinB, translocates into cytosol, and exerts its activity via an unknown mechanism. To study the role of cysteine in BinA, 3 cysteine residues were substituted by alanine and serine. Substitution at Cys195 significantly reduced the toxin activity, whereas substitution at Cys31 and Cys47 abolished its toxicity. Intrinsic fluorescent analysis suggested that all mutant proteins should have similar tertiary structure to that of the wild type. Analysis of the mutant protein on sodium dodecyl sulfate–polyacrylamide gel electrophoresis with and without a reducing agent indicated that all 3 cysteine residues were not involved in disulfide bond formation within the BinA molecule. This is the first report to demonstrate that cysteine residues at 3 positions in BinA are required for full toxicity of the binary toxin. They may play a critical role during oligomerization or interaction between BinA and BinB to form the active complex.     

Peroxynitrite scavenging by different antioxidants. Part I: convenient assay.

A convenient "tube" assay to quantify relative antioxidant activities in aqueous solutions has been developed. Peroxynitrite was employed as a biologically relevant source of radicals with Pyrogallol Red as a detecting molecule. A variety of compounds have been examined, namely polyphenols, uric acid, glutathione, and ascorbic acid. Competition kinetics were observed for the majority of examined compounds, except thymol and ascorbic acid. Pyrogallol Red was fully protected by ascorbic acid against the bleaching by peroxynitrite until its total consumption. The deviation from competition kinetics in the case of thymol was due to the formation of radicals from thymol and their subsequent reaction with Pyrogallol Red. Quercetin was the most efficient scavenger of free radicals. The measurements of relative antioxidant activities using Pyrogallol Red and other detecting molecules, such as gallocyanine and carminic acid, were in fair agreement. The assay was successfully used for a screening of antioxidant activity of plant extracts of unknown composition.     

Macrophages Aggravate Hypoxia-Induced Cardiac Microvascular Endothelial Cell Injury via Peroxynitrite: Protection by Tongxinluo

Abstract

Activated macrophages contribute to endothelial dysfunction; however, it is unclear how peroxynitrite contributes to macrophage-mediated human cardiac microvascular endothelial cell (HCMEC) injury in hypoxia. In macrophage-HCMEC co-cultures subjected to hypoxia, there was an increase in hypoxia-inducible factor (HIF)-1α, HIF-2α, inducible nitric oxide synthase (iNOS), endothelin-converting enzyme (ECE)-1 and cyclooxygenase-2 (COX-2), and concomitant decrease in prostacyclin synthase (PGIS). This was mimicked by a peroxynitrite donor and attenuated by its decomposition catalyst. Tongxinluo (TXL) could decrease HIF-2α, iNOS, ECE-1 and COX-2 and increase PGIS in a dose-dependent manner, with increase of vascular endothelial growth factor. The protein alterations verified the remarkably affected mRNAs, indicating that the effects of TXL were similar to but better than that of peroxynitrite decomposition catalyst. Furthermore, TXL inhibited macrophage-mediated nitrotyrosine accumulation and attenuated HCMEC injury. The results suggest that peroxynitrite contributes to macrophage-mediated HCMEC injury in hypoxia, and TXL attenuates HCMEC injury mainly by inhibiting peroxynitrite.     

Effect of Human Serum Albumin Upon the Permeabilizing Activity of Sticholysin II, a Pore Forming Toxin from Stichodactyla heliantus

Sticholysin II (St II) is a haemolytic toxin isolated from the sea anemone Stichodactyla helianthus. The high haemolytic activity of this toxin is strongly dependent on the red cell status and the macromolecule conformation. In the present communication we evaluate the effect of human serum albumin on St II haemolytic activity and its capacity to form pores in the bilayer of synthetic liposomes. St II retains its pore forming capacity in the presence of large concentrations (up to 500 μM) of human serum albumin. This effect is observed both in its capacity to produce red blood cells haemolysis and to generate functional pores in liposomes. In particular, the capacity of the toxin to lyse red blood cells increases in the presence of human serum albumin (HSA). Regarding the rate of the pore forming process, it is moderately decreased in liposomes and in red blood cells, in spite of an almost total coverage of the interface by albumin. All the data obtained in red cells and model membranes show that St II remains lytically active even in the presence of high HSA concentrations. This stubbornness can explain why the toxin is able to exert its haemolytic activity on membranes immersed in complex plasma matrixes such as those present in living organisms.     

Kinetics of the Thermal Inactivation of Type E Clostridium botulinum Toxin

Rate of inactivation curves for the "free" toxin, prototoxin, or activated toxin in crude filtrates of Clostridium botulinum type E were nonlinear, consisting of a fast inactivating rate followed by a slow inactivating rate. Thermodynamic parameters were calculated over a temperature range of 125 to 145 F (51.7 to 62.8 C) for the two different inactivation rates. Energy of activation was low at the lower temperature and high at the higher temperature. The thermal requirement for inactivating similar concentrations of the "free" toxin, prototoxin, or activated toxin was considered to be the same.     

Effects of Mutations Within Surface-Exposed Loops in the Pore-Forming Domain of the Cry9Ca Insecticidal Toxin of Bacillus thuringiensis

The pore-forming domain of Bacillus thuringiensis insecticidal Cry toxins is formed of seven amphipathic α-helices. Because pore formation is thought to involve conformational changes within this domain, the possible role of its interhelical loops in this crucial step was investigated with Cry9Ca double mutants, which all share the previously characterized R164A mutation, using a combination of homology modeling, bioassays and electrophysiological measurements. The mutations either introduced, neutralized or reversed an electrical charge carried by a single residue of one of the domain I loops. The ability of the 28 Cry9Ca double mutants to depolarize the apical membrane of freshly isolated Manduca sexta larval midguts was tested in the presence of either midgut juice or a cocktail of protease inhibitors because these conditions had been shown earlier to greatly enhance pore formation by Cry9Ca and its R164A single-site mutant. Most mutants retained toxicity toward neonate larvae and a pore-forming ability in the electrophysiological assay, which were comparable to those of their parental toxin. In contrast, mutants F130D, L186D and V189D were very poorly toxic and practically inactive in vitro. On the other hand, mutant E129A depolarized the midgut membrane efficiently despite a considerably reduced toxicity, and mutant Q192E displayed a reduced depolarizing ability while conserving a near wild-type toxicity. These results suggest that the conditions found in the insect midgut, including high ionic strength, contribute to minimizing the influence of surface charges on the ability of Cry9Ca and probably other B. thuringiensis toxins to form pores within their target membrane.     

Protection of Vaccinia from Heat Inactivation by Nucleotide Triphosphates

The presence of adenosine triphosphate, guanosine triphosphate, cytosine triphosphate, or uridine triphosphate reduced the rate of inactivation of vaccinia when heated at 50 C. The virus-associated nucleoside triphosphate phosphohydrolases (adenosine triphosphatase, guanosine triphosphatase, cytosine triphosphatase, and uridine triphosphatase) and ribonucleic acid polymerase were also protected from heat inactivation by these compounds. These obervations are best explained by postulating that ribonucleoside triphosphates bind to enzymes in the virus particle, and that these enzyme-substrate complexes are more resistant to thermal denaturation than are the enzymes without their substrates. The kinetics of heat inactivation of the vaccinia ATP phosphohydrolase activity is biphasic, suggesting that there are two proteins in the vaccinia particle that have this enzyme activity but they have different kinetics of heat inactivation. Any of the vaccinia-associated nucleotide phosphohydrolase activities are protected from heat inactivation by the presence of any one of the respective nucleoside triphosphates. This observation suggests that there is a single enzymatic site in vaccinia that is able to react with any ribonucleoside triphosphate.     

Cloning, Functional Characterization, and Mode of Action of a Novel Insecticidal Pore-Forming Toxin, Sphaericolysin, Produced by Bacillus sphaericus

An insecticidal protein produced by Bacillus sphaericus A3-2 was purified to elucidate its structure and mode of action. The active principle purified from the culture broth of A3-2 was a protein with a molecular mass of 53 kDa that rapidly intoxicated German cockroaches (Blattela germanica) at a dose of about 100 ng when injected. The insecticidal protein sphaericolysin possessed the undecapeptide motif of cholesterol-dependent cytolysins and had a unique N-terminal sequence. The recombinant protein expressed in Escherichia coli was equally as potent as the native protein. Sphaericolysin-induced hemolysis resulted from the protein's pore-forming action. This activity as well as the insecticidal activity was markedly reduced by a Y159A mutation. Also, coapplication of sphaericolysin with cholesterol abolished the insecticidal action, suggesting that cholesterol binding plays an important role in insecticidal activity. Sphaericolysin-lysed neurons dissociated from the thoracic ganglia of the German cockroaches. In addition, sphaericolysin's activity in ganglia was suppressed by the Y159A mutation. The sphaericolysin-induced damage to the cockroach ganglia was greater than the damage to the ganglia of common cutworms (Spodoptera litura), which accounts, at least in part, for the higher sensitivity to sphaericolysin displayed by the cockroaches than that displayed by cutworms.     

1H, 13C, and 15N NMR assignments of the actinoporin Sticholysin I

Sticholysin I is an actinoporin, a pore forming toxin, of 176 aminoacids produced by the sea anemone Stichodactyla heliantus. Isotopically labelled ¹³C/¹⁵N recombinant protein was produced in E. coli. Here we report the complete NMR ¹⁵N, ¹³C and ¹H chemical shifts assignments of Stn I at pH 4.0 and 25°C (BMRB No. 15927).     

Inactivation of Bacillus cereus beta-lactamase I by 6 beta-bromopenicillanic acid: kinetics

The kinetics of the inactivation of Bacillus cereus beta-lactamase I by 6 beta-bromopenicillanic acid are described. Loss of beta-lactamase activity is accompanied by a decrease in protein fluorescence, by the appearance of a protein-bound chromophore at 326 nm, and by loss of tritium from 6 alpha-[3H]-6 beta-bromopenicillanic acid. It is shown that all of the above changes probably have the same rate-determining step. The inactivation reaction is competitively inhibited by cephalosporin C, a competitive inhibitor of this enzyme, and by covalently bound clavulanic acid, suggesting that 6 beta-bromopenicillanic acid reacts directly with the beta-lactamase active site. It is proposed that this inhibitor reacts initially as a normal substrate and that the rate-determining step of the inactivation is acylation of the enzyme. A rapid irreversible inactivation reaction rather than normal hydrolysis of the acyl-enzyme then follows acylation; 6 beta-bromopenicillanic acid is thus a suicide substrate.     

Protection of Measles Virus by Sulfate Ions Against Thermal Inactivation.

Rapp, Fred (Baylor University College of Medicine, Houston, Tex.), Janet S. Butel, and Craig Wallis. Protection of measles virus by sulfate ions against thermal inactivation. J. Bacteriol. 90:132-135. 1965.-The infectivity of measles virus in water is rapidly destroyed at temperatures of 37 C and above. More than 50% of the infectivity is lost after 1 hr at 25 C, and almost 90% loss of infectivity occurs within 24 hr at 4 C. Magnesium chloride enhances the inactivation of the virus at all temperatures tested. Addition of either magnesium or sodium sulfate protects the virus against thermal inactivation. The stabilizing effect is demonstrable at temperatures ranging from 4 to 56 C, but is especially pronounced through 45 C. Prolonged storage (up to 6 weeks) of the virulent virus at 4 C in 1 m magnesium sulfate permits retention of substantial infectivity, whereas storage at 4 C in either water or 1 m magnesium chloride results in a loss of infectivity approximating 99% after 2 weeks. Magnesium chloride also enhances inactivation of the attenuated vaccine strain of measles virus. The attenuated virus, however, is strongly protected by magnesium sulfate against thermal inactivation, and retention of infectivity for long periods of time at 4 C seems feasible when the virus is kept in 1 m magnesium sulfate.     

Regulation of the Pore-Forming Activity of Cecropin A by Local Anesthetics

The influence of local anesthetics on the regulation of the channel-forming activity of the antimicrobial peptide cecropin A has been investigated. The mean current flowing through the single cecropin channels i_sc was determined, and steady-state transmembrane current induced by cecropin AI_∞ was measured. It has been shown that the introduction of 1 mM of bupivacaine, benzocaine or 0.3 mM of tetracaine into the membrane bathing solution results in a decrease in i_sc and I_∞. At the same time, the addition of 1 mM lidocaine or procaine to the membrane-bathing solutions does not lead to a significant change in i_sc and I_∞. Comparison of the absolute values and the sign of the change in the boundary potential of negatively charged membranes and relative changes of i_sc and I_∞ after addition of local anesthetics shows that neither parameter correlates with the membrane boundary potential. The results of studying the effect of tested local anesthetics on the phase transition of membrane lipids allow us to conclude that the observed changes of i_sc and I_∞ are due to modulation of the elastic properties of the membrane.     

WWP-1 Is a Novel Modulator of the DAF-2 Insulin-Like Signaling Network Involved in Pore-Forming Toxin Cellular Defenses in Caenorhabditis elegans

Pore-forming toxins (PFTs) are the single largest class of bacterial virulence factors. The DAF-2 insulin/insulin-like growth factor-1 signaling pathway, which regulates lifespan and stress resistance in Caenorhabditis elegans, is known to mutate to resistance to pathogenic bacteria. However, its role in responses against bacterial toxins and PFTs is as yet unexplored. Here we reveal that reduction of the DAF-2 insulin-like pathway confers the resistance of Caenorhabditis elegans to cytolitic crystal (Cry) PFTs produced by Bacillus thuringiensis. In contrast to the canonical DAF-2 insulin-like signaling pathway previously defined for aging and pathogenesis, the PFT response pathway diverges at 3-phosphoinositide-dependent kinase 1 (PDK-1) and appears to feed into a novel insulin-like pathway signal arm defined by the WW domain Protein 1 (WWP-1). In addition, we also find that WWP-1 not only plays an important role in the intrinsic cellular defense (INCED) against PFTs but also is involved in innate immunity against pathogenic bacteria Pseudomonas aeruginosa and in lifespan regulation. Taken together, our data suggest that WWP-1 and DAF-16 function in parallel within the fundamental DAF-2 insulin/IGF-1 signaling network to regulate fundamental cellular responses in C. elegans.     

Agonist-Selective Protection of the Opioid Receptor-Coupled G Proteins from Inactivation by 5''-p-fluorosulphonylbenzoyl Guanosine

The guanine nucleotide analogue, 5'-p-fluorosulphonylbenzoyl guanosine (FSBG), can react covalently with GTP-binding proteins (G proteins). In rat brain membranes, FSBG causes a time-dependent loss of beta,gamma-imido[8-3H]guanosine 5'-triphosphate binding sites. Using 1 mM FSBG, the guanyl nucleotide modulation of opioid agonist binding is abolished, whereas the guanyl nucleotide sensitivity of neurotensin binding is retained. The action of FSBG can be prevented by the presence of opioid agonists, but not the antagonist naloxone. Iodoacetamide treatment of membranes in the presence of agonist, but not antagonist, can attenuate the action of FSBG in blocking guanyl nucleotide modulation of opioid agonist binding. These results suggest that FSBG covalently modifies essential thiol groups, whose exposure to the reagent is modified by agonist occupancy of the receptor, on a species of G protein linked to opioid receptors, but not on a species of G protein linked to neurotensin receptors. Thus, FSBG may have selectivity for the forms of Gi or Go, proteins associated with opioid receptors.