Ferrous ion-EDTA-stimulated phospholipid peroxidation. A reaction changing from alkoxyl-radical- to hydroxyl-radical-dependent initiation.
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ABSTRACT:
SUBMITTER: Gutteridge JM
PROVIDER: S-EPMC1144503 | biostudies-other | 1984 Dec
REPOSITORIES: biostudies-other
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Project description:Hydroxyl radicals (OH.) in free solution react with scavengers at rates predictable from their known second-order rate constants. However, when OH. radicals are produced in biological systems by metal-ion-dependent Fenton-type reactions scavengers do not always appear to conform to these established rate constants. The detector molecules deoxyribose and benzoate were used to study damage by OH. involving a hydrogen-abstraction reaction and an aromatic hydroxylation. In the presence of EDTA the rate constant for the reaction of scavengers with OH. was generally higher than in the absence of EDTA. This radiomimetic effect of EDTA can be explained by the removal of iron from the detector molecule, where it brings about a site-specific reaction, by EDTA allowing more OH. radicals to escape into free solution to react with added scavengers. The deoxyribose assay, although chemically complex, in the presence of EDTA appears to give a simple and cheap method of obtaining rate constants for OH. reactions that compare well with those obtained by using pulse radiolysis.
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Project description:1. Rat hepatocytes suspended in 0.25 M-sucrose were electropermeabilized. This completely disrupted their plasma-membrane permeability barrier. 2. The endoplasmic reticulum in electroporated hepatocytes appeared morphologically preserved and maintained its permeability barrier as evidenced by electron-microscopic examination and latency measurements on luminal reticular enzymes. 3. Upon aerobic incubation with an NADPH-generating system and iron/ADP, porated hepatocytes peroxidized their membrane lipids at rates similar to those of matched microsomal preparations. 4. When hepatocytes were incubated with iron/EDTA and azide, radical formation detectable with dimethyl sulphoxide (DMSO) was only 10-20% that shown by microsomes. Omitting azide abolished hepatocyte reactivity with DMSO completely. Effects of hydroxyl-radical (.OH) scavengers and of added catalase suggest that the radical detected by DMSO is .OH. 5. Cytosolic inhibitor(s) from hepatocytes seemed to be a major factor limiting .OH formation. These were macromolecular, but showed a degree of heat-stability. Dialysis largely abolished inhibition, but this could be restored again by adding GSH. 6. Since .OH formation in hepatocytes seems to be much more stringently prevented than lipid peroxidation, free-radical damage originating from intracellular redox systems seems more likely to take the form of lipid peroxidation.
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Project description:Lipid peroxidation within human arterial lesions is thought to play an important role in the development of atherosclerosis. Peroxidation can be accelerated by the presence of 'catalytic' iron or copper ions. Gruel samples from advanced atherosclerotic lesions in the abdominal aortae of human cadavers were tested for pro-oxidant properties. All samples contained bleomycin-detectable iron and phenanthroline-detectable copper. Almost all gruel samples stimulated peroxidation of rat liver microsomes, and this was usually inhibited by the iron-ion chelator desferrioxamine. Some samples stimulated formation of hydroxyl radicals from H2O2 in the presence of ascorbate, a reaction again inhibited by desferrioxamine. We conclude that the interior of human advanced atherosclerotic lesions is a highly pro-oxidant environment, and that the use of copper or iron ions to promote peroxidation of low-density lipoproteins in vitro may be a valid model for events in the arterial wall.
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Project description:The rate of formation of dichloride anions (Cl2•-) in dilute aqueous solutions of HCl (2-100 mmol·kg-1) was measured by the technique of pulse radiolysis over the temperature range of 288-373 K. The obtained Arrhenius dependence shows a concentration averaged activation energy of 7.3 ± 1.8 kJ·mol-1, being half of that expected from the mechanism assuming the •OHCl- intermediate and supporting the ionic equilibrium-based mechanism, i.e., the formation of Cl2•- in the reaction of •OH with a hydronium-chloride (Cl-·H3O+) contact ion pair. Assuming diffusion-controlled encounter of the hydronium and chloride ions and including the effect of the ionic atmosphere, we showed that the reciprocal of τ, the lifetime of (Cl-·H3O+), follows an Arrhenius dependence with an activation energy of 23 ± 4 kJ·mol-1, independent of the acid concentration. This result indicates that the contact pair is stabilized by hydrogen bonding interaction of the solvent molecules. We also found that at a fixed temperature, τ is noticeably increased in less-concentrated solutions (mHCl < 0.01 m). Since this concentration effect is particularly pronounced at near ambient temperatures, the increasing pair lifetime may result from the solvent cage effect enhanced by the presence of large supramolecular structures (patches) formed by continuously connected four-bonded water molecules.
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Project description:Eukaryotic translation initiation factor eIF5B is a ribosome-dependent GTPase that mediates displacement of initiation factors from the 40S ribosomal subunit in 48S initiation complexes and joining of 40S and 60S subunits. Here, we determined eIF5B's position on 80S ribosomes by directed hydroxyl radical cleavage. In the resulting model, eIF5B is located in the intersubunit cleft of the 80S ribosome: domain 1 is positioned near the GTPase activating center of the 60S subunit, domain 2 interacts with the 40S subunit (helices 3, 5 and the base of helix 15 of 18S rRNA and ribosomal protein (rp) rpS23), domain 3 is sandwiched between subunits and directly contacts several ribosomal elements including Helix 95 of 28S rRNA and helix 44 of 18S rRNA, domain 4 is near the peptidyl-transferase center and its helical subdomain contacts rpL10E. The cleavage data also indicate that binding of eIF5B might induce conformational changes in both subunits, with ribosomal segments wrapping around the factor. Some of these changes could also occur upon binding of other translational GTPases, and may contribute to factor recognition.
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Project description:Hydroxyl radical protein footprinting (HRPF) by fast photochemical oxidation of proteins (FPOP) is a powerful benchtop tool used to probe protein structure, interactions, and conformational changes in solution. However, the reproducibility of all HRPF techniques is limited by the ability to deliver a defined concentration of hydroxyl radicals to the protein. This ability is impacted by both the amount of radical generated and the presence of radical scavengers in solution. In order to compare HRPF data from sample to sample, a hydroxyl radical dosimeter is needed that can measure the effective concentration of radical that is delivered to the protein, after accounting for both differences in hydroxyl radical generation and nonanalyte radical consumption. Here, we test three radical dosimeters (Alexa Fluor 488, terepthalic acid, and adenine) for their ability to quantitatively measure the effective radical dose under the high radical concentration conditions of FPOP. Adenine has a quantitative relationship between UV spectrophotometric response, effective hydroxyl radical dose delivered, and peptide and protein oxidation levels over the range of radical concentrations typically encountered in FPOP. The simplicity of an adenine-based dosimeter allows for convenient and flexible incorporation into FPOP applications, and the ability to accurately measure the delivered radical dose will enable reproducible and reliable FPOP across a variety of platforms and applications.
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Project description:Lipid peroxidation occurs in the context of many physiological processes but is greatly increased in various pathological situations. A consequence of phospholipid peroxidation is the generation of oxidation-specific epitopes, such as phosphocholine of oxidized phospholipids and malondialdehyde, which form neo-self determinants on dying cells and oxidized low-density lipoproteins. In this review we discuss evidence demonstrating that pattern recognition receptors of the innate immune system recognize oxidation-specific epitopes as endogenous damage-associated molecular patterns, allowing the host to identify dangerous biological waste. Oxidation-specific epitopes are important targets of both cellular and soluble pattern recognition receptors, including toll-like and scavenger receptors, C-reactive protein, complement factor H, and innate natural IgM antibodies. This recognition allows the innate immune system to mediate important physiological house keeping functions, for example by promoting the removal of dying cells and oxidized molecules. Once this system is malfunctional or overwhelmed the development of diseases, such as atherosclerosis and age-related macular degeneration is favored. Understanding the molecular components and mechanisms involved in this process, will help the identification of individuals with increased risk of developing chronic inflammation, and indicate novel points for therapeutic intervention. This article is part of a Special Issue entitled: Oxidized phospholipids-their properties and interactions with proteins.
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Project description:Ferroptosis is widely involved in degenerative diseases in various tissues including kidney, liver and brain, and is a targetable vulnerability in multiple primary and therapy-resistant cancers. Accumulation of phospholipid hydroperoxides in cellular membranes is the hallmark and rate-limiting step of ferroptosis; however, the enzymes contributing to lipid peroxidation remain poorly characterized. Using genome-wide, CRISPR-Cas9-mediated suppressor screens, we identify cytochrome P450 oxidoreductase (POR) as necessary for ferroptotic cell death in cancer cells exhibiting inherent and induced susceptibility to ferroptosis. By genetic depletion of POR in cancer cells, we reveal that POR contributes to ferroptosis across a wide range of lineages and cell states, and in response to distinct mechanisms of ferroptosis induction. Using systematic lipidomic profiling, we further map POR's activity to the lipid peroxidation step in ferroptosis. Hence, our work suggests that POR is a key mediator of ferroptosis and potential druggable target for developing antiferroptosis therapeutics.
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Project description:The universally conserved eukaryotic initiation factor (eIF), eIF1A, plays multiple roles throughout initiation: it stimulates eIF2/GTP/Met-tRNA(i)(Met) attachment to 40S ribosomal subunits, scanning, start codon selection and subunit joining. Its bacterial ortholog IF1 consists of an oligonucleotide/oligosaccharide-binding (OB) domain, whereas eIF1A additionally contains a helical subdomain, N-terminal tail (NTT) and C-terminal tail (CTT). The NTT and CTT both enhance ribosomal recruitment of eIF2/GTP/Met-tRNA(i)(Met), but have opposite effects on the stringency of start codon selection: the CTT increases, whereas the NTT decreases it. Here, we determined the position of eIF1A on the 40S subunit by directed hydroxyl radical cleavage. eIF1A's OB domain binds in the A site, similar to IF1, whereas the helical subdomain contacts the head, forming a bridge over the mRNA channel. The NTT and CTT both thread under Met-tRNA(i)(Met) reaching into the P-site. The NTT threads closer to the mRNA channel. In the proposed model, the NTT does not clash with either mRNA or Met-tRNA(i)(Met), consistent with its suggested role in promoting the 'closed' conformation of ribosomal complexes upon start codon recognition. In contrast, eIF1A-CTT appears to interfere with the P-site tRNA-head interaction in the 'closed' complex and is likely ejected from the P-site upon start codon recognition.
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Project description:Methods of structural mass spectrometry have become more popular to study protein structure and dynamics. Among them, fast photochemical oxidation of proteins (FPOP) has several advantages such as irreversibility of modifications and more facile determination of the site of modification with single residue resolution. In the present study, FPOP analysis was applied to study the hemoglobin (Hb) – haptoglobin (Hp) complex allowing identification of respective regions altered upon the complex formation. The footprinting using a timsTOF Pro mass spectrometer allowed to obtain structural information for 84 and 76 residues in Hp and Hb, respectively, including statistically significant differences in the modification extent below 0.3 %. The most affected residues upon the complex formation were Met76 and Tyr140 in Hbα, and Tyr280 and Trp284 in Hpβ. The data allowed determination of amino acids directly involved in Hb – Hp interactions and those located outside of the interaction interface yet affected by the complex formation. Also, previously modeled interaction between Hb βTrp37 and Hp βPhe292 was not confirmed by our data.
2021-12-30 | PXD021621 | Pride