Proteins are major targets for radicals and two-electron oxidants in biological systems due to their abundance and high rate constants for reaction. With highly reactive radicals damage occurs at ...multiple side-chain and backbone sites. Less reactive species show greater selectivity with regard to the residues targeted and their spatial location. Modification can result in increased side-chain hydrophilicity, side-chain and backbone fragmentation, aggregation via covalent cross-linking or hydrophobic interactions, protein unfolding and altered conformation, altered interactions with biological partners and modified turnover. In the presence of O2, high yields of peroxyl radicals and peroxides (protein peroxidation) are formed; the latter account for up to 70% of the initial oxidant flux. Protein peroxides can oxidize both proteins and other targets. One-electron reduction results in additional radicals and chain reactions with alcohols and carbonyls as major products; the latter are commonly used markers of protein damage. Direct oxidation of cysteine (and less commonly) methionine residues is a major reaction; this is typically faster than with H2O2, and results in altered protein activity and function. Unlike H2O2, which is rapidly removed by protective enzymes, protein peroxides are only slowly removed, and catabolism is a major fate. Although turnover of modified proteins by proteasomal and lysosomal enzymes, and other proteases (e.g. mitochondrial Lon), can be efficient, protein hydroperoxides inhibit these pathways and this may contribute to the accumulation of modified proteins in cells. Available evidence supports an association between protein oxidation and multiple human pathologies, but whether this link is causal remains to be established.
Exposure of biological molecules to oxidants is inevitable and therefore commonplace. Oxidative stress in cells arises from both external agents and endogenous processes that generate reactive ...species, either purposely (e.g. during pathogen killing or enzymatic reactions) or accidentally (e.g. exposure to radiation, pollutants, drugs, or chemicals). As proteins are highly abundant and react rapidly with many oxidants, they are highly susceptible to, and major targets of, oxidative damage. This can result in changes to protein structure, function, and turnover and to loss or (occasional) gain of activity. Accumulation of oxidatively-modified proteins, due to either increased generation or decreased removal, has been associated with both aging and multiple diseases. Different oxidants generate a broad, and sometimes characteristic, spectrum of post-translational modifications. The kinetics (rates) of damage formation also vary dramatically. There is a pressing need for reliable and robust methods that can detect, identify, and quantify the products formed on amino acids, peptides, and proteins, especially in complex systems. This review summarizes several advances in our understanding of this complex chemistry and highlights methods that are available to detect oxidative modifications—at the amino acid, peptide, or protein level—and their nature, quantity, and position within a peptide sequence. Although considerable progress has been made in the development and application of new techniques, it is clear that further development is required to fully assess the relative importance of protein oxidation and to determine whether an oxidation is a cause, or merely a consequence, of injurious processes.
•Electron paramagnetic resonance (EPR) can detect radicals in biological systems.•Spin traps are typically used to examine low steady-state levels of radicals.•This article reviews spin trapping ...methods – both advantages and pitfalls.•Complementary radical detection methods are discussed.
Electron paramagnetic resonance (EPR) spectroscopy (also known as electron spin resonance, ESR, or electron magnetic resonance, EMR, spectroscopy) is often described as the “gold standard” for the detection and characterisation of radicals in chemical, biological and medical systems. The article reviews aspects of EPR spectroscopy and discusses how this methodology and related techniques can be used to obtain useful information from biological systems. Consideration is given to the direct detection of radicals, the use of spin traps and the detection of nitric oxide, and the advantages and pitfalls of various approaches. When used with care, this technique can provide a huge amount of valuable data on the presence of radicals, their identity and information on their concentration, structure, mobility and interactions. It is however a technique that has limitations, and the novice user needs to understand the various pitfalls and shortcomings of the method to avoid making significant errors.
Heme peroxidases are a major source of reactive oxidants at sites of inflammation in biological systems. The formation of some of these oxidants (e.g. hypochlorous acid, HOCl) is important in the ...innate immune response of activated neutrophils and leukocytes to invading pathogens (e.g. bacteria, yeasts, fungi parasites), and responsible for the anti-microbial activity present in excreted fluids (e.g. hypothiocyanous acid, HOSCN, generated by lactoperoxidase). Other oxidants formed by heme peroxidase family members are important in tissue development (e.g. hypobromous acid, HOBr, formation by peroxidasin) and in the synthesis of thyroid hormones (hypoiodous acid, HOI, synthesized by thyroid peroxidase). However, inadvertent, misplaced or poorly-controlled production of these species can result in host tissue damage, and this underlies the strong association between high levels of some of these enzymes and multiple inflammatory pathologies. As a consequence, there is widespread interest in understanding the kinetics and mechanisms of biomolecule modification by these species, which differ dramatically in their actions, the nature of the products formed (as some of these are specific biomarkers of enzyme activity), and the biological consequences of these reactions in a wide range of diseases associated with acute or chronic inflammation. Increased knowledge of these processes, has allowed the development of a number of alternative and complementary strategies that allow modulation of oxidant formation and subsequent damage. This review discusses developments in these fields and the prospects for tailored inhibition of specific members of this enzyme family.
Multiple roles of reactive oxygen species (ROS) and their consequences for health and disease are emerging throughout biological sciences. This development has led researchers unfamiliar with the ...complexities of ROS and their reactions to employ commercial kits and probes to measure ROS and oxidative damage inappropriately, treating ROS (a generic abbreviation) as if it were a discrete molecular entity. Unfortunately, the application and interpretation of these measurements are fraught with challenges and limitations. This can lead to misleading claims entering the literature and impeding progress, despite a well-established body of knowledge on how best to assess individual ROS, their reactions, role as signalling molecules and the oxidative damage that they can cause. In this consensus statement we illuminate problems that can arise with many commonly used approaches for measurement of ROS and oxidative damage, and propose guidelines for best practice. We hope that these strategies will be useful to those who find their research requiring assessment of ROS, oxidative damage and redox signalling in cells and in vivo.
There is considerable interest in the role that mammalian heme peroxidase enzymes, primarily myeloperoxidase, eosinophil peroxidase and lactoperoxidase, may play in a wide range of human pathologies. ...This has been sparked by rapid developments in our understanding of the basic biochemistry of these enzymes, a greater understanding of the basic chemistry and biochemistry of the oxidants formed by these species, the development of biomarkers that can be used damage induced by these oxidants in vivo, and the recent identification of a number of compounds that show promise as inhibitors of these enzymes. Such compounds offer the possibility of modulating damage in a number of human pathologies. This reviews recent developments in our understanding of the biochemistry of myeloperoxidase, the oxidants that this enzyme generates, and the use of inhibitors to inhibit such damage.
γ-aminobutyric acid-mediated (GABAergic) inhibition plays a critical role in shaping neuronal activity in the neocortex. Numerous experimental investigations have examined perisomatic inhibitory ...synapses, which control action potential output from pyramidal neurons. However, most inhibitory synapses in the neocortex are formed onto pyramidal cell dendrites, where theoretical studies suggest they may focally regulate cellular activity. The precision of GABAergic control over dendritic electrical and biochemical signaling is unknown. By using cell type-specific optical stimulation in combination with two-photon calcium (Ca²⁺) imaging, we show that somatostatin-expressing interneurons exert compartmentalized control over postsynaptic Ca²⁺ signals within individual dendritic spines. This highly focal inhibitory action is mediated by a subset of GABAergic synapses that directly target spine heads. GABAergic inhibition thus participates in localized control of dendritic electrical and biochemical signaling.
The heme peroxidase family generates a battery of oxidants both for synthetic purposes, and in the innate immune defence against pathogens. Myeloperoxidase (MPO) is the most promiscuous family ...member, generating powerful oxidizing species including hypochlorous acid (HOCl). Whilst HOCl formation is important in pathogen removal, this species is also implicated in host tissue damage and multiple inflammatory diseases. Significant oxidant formation and damage occurs extracellularly as a result of MPO release via phagolysosomal leakage, cell lysis, extracellular trap formation, and inappropriate trafficking. MPO binds strongly to extracellular biomolecules including polyanionic glycosaminoglycans, proteoglycans, proteins, and DNA. This localizes MPO and subsequent damage, at least partly, to specific sites and species, including extracellular matrix (ECM) components and plasma proteins/lipoproteins. Biopolymer-bound MPO retains, or has enhanced, catalytic activity, though evidence is also available for non-catalytic effects. These interactions, particularly at cell surfaces and with the ECM/glycocalyx induce cellular dysfunction and altered gene expression. MPO binds with higher affinity to some damaged ECM components, rationalizing its accumulation at sites of inflammation. MPO-damaged biomolecules and fragments act as chemo-attractants and cell activators, and can modulate gene and protein expression in naïve cells, consistent with an increasing cycle of MPO adhesion, activity, damage, and altered cell function at sites of leukocyte infiltration and activation, with subsequent tissue damage and dysfunction. MPO levels are used clinically both diagnostically and prognostically, and there is increasing interest in strategies to prevent MPO-mediated damage; therapeutic aspects are not discussed as these have been reviewed elsewhere.
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•Myeloperoxidase (MPO) is released extracellularly from leukocytes at sites of inflammation.•MPO generates multiple oxidants to kill pathogens, but also induces host tissue damage.•MPO binds to extracellular matrix proteins, glycosaminoglycans and plasma (lipo)proteins.•Binding via ionic interactions results in site-specific effects and localized modifications.•Biological effects are induced by both oxidant formation and ionic interactions.
Proteins are a major target for oxidants as a result of their abundance in biological systems, and their high rate constants for reaction. Kinetic data for a number of radicals and non-radical ...oxidants (e.g. singlet oxygen and hypochlorous acid) are consistent with proteins consuming the majority of these species generated within cells. Oxidation can occur at both the protein backbone and on the amino acid side-chains, with the ratio of attack dependent on a number of factors. With some oxidants, damage is limited and specific to certain residues, whereas other species, such as the hydroxyl radical, give rise to widespread, relatively non-specific damage. Some of the major oxidation pathways, and products formed, are reviewed. The latter include reactive species, such as peroxides, which can induce further oxidation and chain reactions (within proteins, and via damage transfer to other molecules) and stable products. Particular emphasis is given to the oxidation of methionine residues, as this species is readily oxidised by a wide range of oxidants. Some side-chain oxidation products, including methionine sulfoxide, can be employed as sensitive, specific, markers of oxidative damage. The product profile can, in some cases, provide valuable information on the species involved; selected examples of this approach are discussed. Most protein damage is non-repairable, and has deleterious consequences on protein structure and function; methionine sulfoxide formation can however be reversed in some circumstances. The major fate of oxidised proteins is catabolism by proteosomal and lysosomal pathways, but some materials appear to be poorly degraded and accumulate within cells. The accumulation of such damaged material may contribute to a range of human pathologies.
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