Redox Biochemistry and Chemistry最新文献

Electron paramagnetic resonance (EPR) spectroscopy is unique in providing robust information about free radicals, transition metal ions and metalloenzymes, which are crucial players in redox processes. EPR had a major role in advancing the redox biology field during the 20th century, but the interest in this methodology considerably decreased in recent years. Here, we discuss potential reasons for this decline as well as potential reasons for maintaining the mind open to the many possibilities brought by EPR and associated methodologies to the redox field. We present the fundamentals of EPR using pictorial images and minimal physicochemical language. We also present EPR derived methodologies developed to detect radical metabolites, that is, direct EPR of solutions (static and continuous-flow), direct EPR of frozen solutions, spin-trapping and spin-scavenging, showing examples and discussing the advantages and drawbacks of each one. Finally, we discuss the EPR spectra of metalloproteins and metal ion complexes of biological interest, which are more complex than those of radical metabolites in solution. In addition to introduce EPR methodologies to those new to the redox field, our goal is to show that these methodologies can contribute to advance the field.

In the nucleus, histones are essential in the packaging of DNA and the regulation of gene expression. These histones can also be released to the extracellular space by mechanisms such as necrosis and neutrophil extracellular trap (NET) formation. Histones are cytotoxic and cause sterile inflammation, and as a result, have been implicated in tissue damage in several pathologies, including atherosclerosis. Myeloperoxidase (MPO) is also present on NETs, which is catalytically active and able to produce hypochlorous acid (HOCl). This could modify histones and alter their extracellular reactivity. In this study, we compared the reactivity of histones with and without modification by HOCl with primary human coronary artery smooth muscle cells (HCASMCs). Histones induced a loss in viability and cell death primarily by apoptosis, which was attenuated on modification of the histones by HOCl. Exposure of HCASMCs to histones also resulted in the increased expression of the pro-inflammatory genes monocyte chemoattractant protein-1 (MCP-1), interleukin 6 (IL-6), and vascular cell adhesion molecule-1 (VCAM-1) and a decrease in intracellular thiols. In addition, there were changes in the expression of the stress related gene heme oxygenase-1 (HO-1). Modification of the histones with HOCl had no significant influence on changes in gene expression or thiol loss, in contrast to the cytotoxicity studies. Together, these studies provide new insight into the pathways by which histones could promote vascular dysfunction, which could be relevant to inflammatory diseases, such as atherosclerosis and sepsis, which are associated with elevated NET release and high circulating histones, respectively.

Retinitis pigmentosa (RP) is a disease characterised by photoreceptor cell death. It can be initiated by mutations in a number of different genes, primarily affecting rods, which will die first, resulting in loss of night vision. The secondary death of cones then leads to loss of visual acuity and blindness. We set out to investigate whether increased mitochondrial reactive oxygen species (ROS) formation, plays a role in this sequential photoreceptor degeneration. To do this we measured mitochondrial H₂O₂ production within mouse eyes in vivo using the mass spectrometric probe MitoB. We found higher levels of mitochondrial ROS that preceded photoreceptor loss in four mouse models of RP: Pde6b^rd1/rd1; Prhp2^rds/rds; RPGR^−/−; Cln6^nclf. In contrast, there was no increase in mitochondrial ROS in loss of function models of vision loss (GNAT^−/−, OGC), or where vision loss was not due to photoreceptor death (Cln3). Upregulation of Nrf2 transcriptional activity with dimethylfumarate (DMF) lowered mitochondrial ROS in RPGR^−/− mice. These findings have important implications for the mechanism and treatment of RP.

When neutrophils phagocytose bacteria, they release myeloperoxidase (MPO) into phagosomes to catalyse the conversion of superoxide to the potent antimicrobial oxidant hypochlorous acid (HOCl). Here we show that within neutrophils, MPO is inactivated by HOCl. In this study, we aimed to identify the effects of HOCl on the structure and function of MPO, and determine the enzyme's susceptibility to oxidative inactivation during phagocytosis. When hydrogen peroxide was added to a neutrophil granule extract containing chloride, MPO activity was rapidly lost in a HOCl-dependent reaction. With high concentrations of hydrogen peroxide, western blotting demonstrated that MPO was both fragmented and converted to high molecular weight aggregates. Using the purified enzyme, we showed that HOCl generated by MPO inactivated the enzyme by destroying its prosthetic heme groups and releasing iron. MPO protein was additionally modified by forming high molecular weight aggregates. Before inactivation occurred, MPO chlorinated itself to convert most of its amine groups to dichloramines. When human neutrophils phagocytosed Staphylococcus aureus, they released MPO that was largely inactivated in a process that required production of superoxide. Enzyme inactivation occurred inside neutrophils because it was not blocked when extracellular HOCl was scavenged with methionine. The inactivated enzyme contained a chlorinated tyrosine residue, establishing that it had reacted with HOCl. Our results demonstrate that MPO will substantially inactivate itself during phagocytosis, which may limit oxidant production inside phagosomes. Other neutrophil proteins are also likely to be inactivated. The chloramines formed on neutrophil proteins may contribute to the bactericidal milieu of the phagosome.

It has been established that hydrogen peroxide (H₂O₂) acts as a signalling messenger by triggering the reversible oxidation of redox-regulated proteins via a redox-relay provided by peroxiredoxins (Prdxs). Exceptionally high reactivity of Prdxs with H₂O₂ exceeding other thiols by orders of magnitude places Prdxs as sensors of H₂O₂ and distributers of oxidizing equivalents to specific thiol targets which can't be oxidized by H₂O₂ directly. By this mechanism the oxidative stress response can be achieved.

Despite its involvement in oxidative stress responses, H₂O₂ is continuously generated as a normal metabolite necessary for regular cell functioning. The challenge lies in understanding how the Prdx-dependent redox relay can differentiate between basal levels of H₂O₂ and excessive amounts that lead to oxidative stress.

Peroxymonocarbonate, an oxidant formed when H₂O₂ reacts with CO₂/HCO₃⁻, emerges as a potent cellular oxidant. The peroxymonocarbonate formation could be catalysed and then consumed at localised sites by certain thiol proteins. This mechanism could prevent H₂O₂ from reacting with Prdx, thereby averting the redox-relayed activation of regulatory thiol proteins and subsequent oxidative stress response below a certain level of H₂O₂.

Melatonin (N-acetyl-5-methoxytryptamine, MLT), an evolutionarily conserved indoleamine, is known to act as an antioxidant. However, the evidence indicating the role of MLT as a powerful chain-breaking antioxidant by scavenging peroxyl radical remains controversial. The radical-scavenging rate of MLT determined in this study in methanol using galvinoxyl radical (GO^•) was much lower than that of an α-tocopherol model compound. The acceleration of the GO^•-scavenging reaction by MLT was observed in the presence of magnesium ion (Mg²⁺), a bio-related redox-inactive metal ion, suggesting that this reaction may proceed via a rate-determining electron transfer followed by proton transfer. The coordination of Mg²⁺ to the carbonyl oxygen in the one-electron reduced species of GO^• (GO^–) may stabilize the product, resulting in the acceleration of the electron-transfer process. We also demonstrated that prophylactically administrated MLT efficiently inhibited the lipid peroxide-derived protein modification, which can be detected by a sensitive marker, N^ε-(hexanoyl)lysine adduct, in the plasma of X-irradiated mice. The relatively weak GO^•-scavenging activity of MLT suggests that the ameliorative effect of MLT against in vivo lipid peroxidation does not result from the direct scavenging of lipid peroxyl radicals by MLT. Therefore, the observed superior protective efficiency of MLT against in vivo lipid peroxidation may partly support the earlier studies, which reported the synergistic antioxidative effect of the metabolites of MLT.

Polyunsaturated fatty acids (PUFA) are particularly susceptible to free radical-induced lipid peroxidation (LPO). Specific deuteration at bis-allylic positions of PUFA (D-PUFA) has been recently proposed as a way to inhibit the LPO. Here, a high mass resolution untargeted lipidomic analysis protocol was applied to examine the changes in the lipidome of keratinocytes supplemented with bis-allylic deuterated linoleic acid (D₂-LA). Incorporation of D₂-LA occurs preferentially in membrane phospholipids such as phosphatidylcholine and phosphatidylethanolamine, followed by triglycerides. However, the relative contribution of D₂-LA among membrane lipids is highest in cardiolipin (60%) followed by its precursor phosphatidylglycerol (50%). Cardiolipins are enriched in PUFA and exclusively located in mitochondrial membranes, thus representing major targets for lipid peroxidation. These findings indicate that D₂-LA supplementation is linked to the preservation of mitochondrial function under oxidative stress. Finally, our study highlights the suitability of high mass resolution lipidomic analysis to investigate lipid metabolism at the level of individual molecular species in stable isotope tracing experiments.

The absorption of ionizing radiation initiates redox reactions, producing chemical species resulting from single electron loss or electron gain. Radiation chemists have developed methods to study individual redox species selectively and to monitor their reactions in real time. This has provided an enormous resource of kinetic, thermodynamic and spectroscopic information concerning the characteristics and reactions of free radicals and their redox reactions, mainly in aqueous solution. While the techniques are specialized and exploiting them is certainly more difficult than initiating redox changes by simple mixing of two chemicals or adding a reagent to a biological target, it is useful to gain an understanding of the basic mechanisms and approaches involved in exploiting radiation chemistry in the wider context of redox reactions in biochemistry, chemistry, and biology. This should enable readers both to appreciate the reliance which can be placed on the kinetic and other information resulting from such studies, as well as identify potential new applications of the technique which might be exploited in their research, by seeking partners who have access to the necessary specialized equipment or just basic irradiation facilities. This review outlines how radiation can be used to initiate selective redox reactions, mainly in water, and helps point readers to resources which should be useful in considering such reactions in a wider context.

Cysteine residues are the most favored targets for oxidation by hypochlorous acid and other reactive halogen species. The end-products of cysteine oxidation are usually considered to be reversibly formed disulfides and the more highly oxidized sulfinic and sulfonic acids. However, reactive halogen species are capable of generating additional products in which cysteine is cross-linked to other amino acids. Here we have treated a range of peptides with hypochlorous acid (HOCl) and hypobromous acid (HOBr), and used mass spectrometry to demonstrate sulfenamide, sulfinamide and sulfonamide formation with lysine residues, as well as –S(O)- and –S(O₂)- linkages with tyrosine, tryptophan and arginine residues. The -(SO₂)- products were more prevalent with HOCl than HOBr, reflecting its higher oxidizing ability. There was also considerable variation between peptides in efficiency of cross-linking compared with other modifications. The –S(O)- and –S(O₂)- forms were much more resistant than the disulfide to reduction by dithiothreitol. Using calprotectin as a representative cysteine-containing protein, we show that a range of products containing each of these cross-links is formed when the protein is treated with HOCl. Two of the identified cysteine-lysine calprotectin cross-links were also detected in bronchoalveolar lavage fluid from children with cystic fibrosis. Our results imply that cross-linked species would be formed when cysteine-containing proteins are exposed to reactive halogen species, with the nature of the specific products depending on structural features around the cysteine residue. Cross-linking could have a modulatory effect on protein function or be detrimental in causing oligomerization and aggregation.