Redox Biochemistry and Chemistry最新文献

Mitochondria are main sources of biological hydroperoxides, including hydrogen peroxide, peroxynitrite and various organic hydroperoxides. Most of these species are involved in the regulation of cellular functions when formed at low, physiological levels. Additionally, they can cause oxidative damage when formed at higher rates, eventually leading to mitochondrial disfunction and cytotoxicity. Different peroxidases sense the levels and catalyze the reduction of mitochondrial hydroperoxides. Among them, peroxiredoxin 3 and peroxiredoxin 5 decompose most hydrogen peroxide, peroxynitrite and free fatty acid hydroperoxides formed in the mitochondrial matrix. Kinetic considerations indicate that the role of selenol-dependent glutathione peroxidases in the reduction of these soluble hydroperoxides in mitochondria would be secondary. Glutathione peroxidase 4, which has a unique phospholipid hydroperoxide peroxidase activity, is only expressed in the mitochondria of selected tissues. Peroxiredoxin 3 catalyzes the reduction of hydroperoxides, but is also hyperoxidized and inactivated by them, in particular by free fatty acid hydroperoxides which react at high rate constants. Indeed, computer-assisted simulations support that free fatty acid hydroperoxides significantly contribute to Prdx3 hyperoxidation under biologically-relevant conditions. In addition, kinetic data indicate that hydroperoxides may partially diffuse to the cytosol. Several open questions regarding the oxidizing substrate specificities of mitochondrial peroxiredoxins and their modulation by CO₂ are presented. Thus, peroxiredoxins 3 and 5 are the main sensors of mitochondrial hydroperoxides, provide protection from their excess and also determine the ability of these reactive species to diffuse through mitochondria; these combined actions of the mitochondrial peroxiredoxins impact redox regulation and outcomes of physiological or pathological processes.

Cellular respiration is highly regulated, changes dynamically in response to the microenvironment of individual cells and during differentiation and differs between cell and tissue types. Too little cell respiration can cause an accumulation of reductants, leading to reductive stress, while inefficient respiration, that causes a build-up of reactive oxygen species (ROS), can result in oxidative stress. Most of the discussion of this central redox dichotomy has centred around oxidative stress because the damaging effects of cellular oxidants on DNA, lipids and proteins are well-established, and have been shown to contribute to health issues including, mitochondrial and cardiovascular diseases, tumorigenesis, and to the effects of ageing. Much less attention has been paid to cellular reductive stress. Nevertheless, excessive levels of key cellular reductants including NADH, NADPH and glutathione, as well as an imbalance in protein thiols, and insufficient levels of ROS to maintain cell signalling pathways, can be harmful to cells and result in poor health outcomes. Recently, cellular mechanisms that sense and regulate cellular reductive stress associated with low ROS levels have been identified. In addition, plasma membrane electron transport has been shown to be a key player in cellular redox homeostasis involving NAD(P)H/NAD(P)⁺ ratios. It is now well-established that the plasma membrane contains coenzyme Q-mediated electron transport pathways capable of oxidizing intracellular NAD(P)H and reducing extracellular electron acceptors such as molecular oxygen. A better understanding of the origins, cellular and subcellular compartmentalization and regulation of cellular reductants could lead to the development of new anticancer strategies.

Mycoredoxins (Mrxs) are a group of small dithiol oxidoreductases that share a conserved CXXC active site sequence motif resembling glutaredoxins. They are commonly found in saprophytic microorganisms, including Actinobacteria such as Mycobacterium tuberculosis. Among the known mycoredoxins, Corynebacterium glutamicum (Cg) Mrx1, featuring a conserved CVQC active site, functions as a mycothiol-dependent monothiol oxidoreductase. On the other hand, Mrx2, also known as NrdH-redoxin and containing the same CVQC motif, exhibits dithiol oxidoreductase properties and receives electrons from thioredoxin reductase (TrxR). Recently, it has been reported that CgMrx3, featuring a CGSC motif, acts as a thioredoxin, although its structural and biophysical characteristics remain unexplored.

In this study, we successfully determined the X-ray structure of CgMrx3 at a resolution of 1.7 Å, revealing a swapped dimer arrangement. We compared the structure of CgMrx3 with those of CgMrx1 and CgMrx2 and with the AlphaFold2 predicted structure of Mrx3 from Mycobaterium tuberculosis (MtMrx3). We correlated the number of hydrogen bonds accepted by the nucleophilic cysteines with the relatively low pKa's determined for MtMrx3 and CgMrx3. Finally, we showed that CgMrx3 has DsbA-like oxidase activity. Taken together, our results provide valuable insights into the structural and functional characteristics of Mrx3, thereby enhancing our understanding of mycoredoxin-dependent redox biology.

Fenamic acids are a group of non-steroidal anti-inflammatory drugs (NSAIDs) that are among the most common drugs prescribed globally. However, they have been associated with many adverse effects, such as agranulocytosis, neutropenia, hepatotoxicity, and nephrotoxicity. The interactions between peroxidase enzymes and fenamic acid-like NSAIDs cause the formation of reactive species, potentially involved in side effects. The aim of this study was to investigate the neutrophil myeloperoxidase (MPO)-mediated bioactivation of fenamic acids based on N-phenylanthranilic acid (NPA) and its four drug analogues: flufenamic acid (FFA), mefenamic acid (MFA), meclofenamic acid (MCFA), and tolfenamic acid (TFA). We hypothesized that the enzymatic oxidation of fenamic acids by MPO/hydrogen peroxide (H₂O₂) would produce reactive metabolites, cause oxidative damage and induce cytotoxicity. We utilized UV–Vis spectrophotometry, liquid chromatography-mass spectrometry (LC-MS), and electron paramagnetic spin resonance (EPR) spectroscopy using purified MPO from human neutrophils. In addition, in vitro studies were performed with MPO-containing human promyelocytic leukemia (HL-60) cells for cytotoxicity and immuno-spin trapping to detect protein-free radicals. UV–Vis spectrophotometry revealed that MPO oxidized the fenamic acids. LC-MS analyses revealed the formation of dimers, hydroxylated, and quinoneimine species, and glutathione (GSH) conjugates. EPR spin trapping with DMPO using GSH revealed that fenamic acids produced glutathionyl radicals in a concentration-dependent manner. We also detected the formation of protein-free radicals in HL-60 cells, which correlated with cytotoxicity. Despite the minor structural differences between the fenamic acids, there were variations in their oxidation potential. These findings revealed a correlation between pro-oxidant metabolite reactivity and cytotoxicity caused by fenamic acid NSAIDs.

In mammals, heme peroxidases are well known to generate oxidized (pseudo)halide products such as hypochlorous acid, hypobromous acid, oxidized iodine species, and hypothiocyanite. In addition, inter(pseudo)halogens are also oxidized (pseudo)halide compounds where two or more different (pseudo)halides are combined within a molecule without participation of other atoms. However, the information of this group of chemicals as potential products of peroxidases is limited and very fragmentary. In this review, we summarize current knowledge about chemical properties of inter(pseudo)halogens, their role as products of peroxidase-mediated conversions, and possible applications of these compounds in antimicrobial defense. The major focus is directed on bromyl chloride, cyanogen halides, and some products derived from interaction of oxidized iodine with thiocyanate.

Myeloperoxidase and eosinophil peroxidase exert their antimicrobial functions through the oxidative actions of their hypohalous acid products. Plasmalogen phospholipids are particularly susceptible to oxidation of their vinyl ether functional group by hypohalous acids. This produces a family of halogenated lipid products with pro-inflammatory roles and potential biomarker utility. The initial product of plasmalogen oxidation by HOCl is 2-chlorofatty aldehyde, which has been shown to play a key role at the blood-endothelium interface. In vitro and in vivo studies indicate increased endothelial barrier permeability, neutrophil chemotaxis, neutrophil and platelet adherence to endothelium, and promotion of erythrocyte lysis as some of its effects. These effects may be due to protein modification by 2-chlorofatty aldehyde. 2-Chlorofatty aldehyde is metabolized by host dehydrogenases to 2-chlorofatty acid. While it is less chemically reactive, 2-chlorofatty acid has partial overlap of pro-inflammatory effects with 2-chlorofatty aldehyde and unique actions such as induction of neutrophil extracellular trap formation. The stability of 2-chlorofatty acid in plasma also makes it well-suited as a biomarker of HOCl generation, and its plasma levels may be predictive of disease outcomes. 2-Bromofatty aldehydes and acids are produced analogously from HOBr reaction with plasmalogens. Their functions have yet to be well-elucidated, though similarities with chlorolipids have been observed, and increased reactivity with proteins is expected through enhanced electrophilicity of the alpha carbon. Altogether, these halogenated lipids represent underexplored mediators of diseases involving excess hypohalous acid production.

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.