Reactive oxygen species (ROS) have been shown to be toxic but also function as signalling molecules. This biological paradox underlies mechanisms that are important for the integrity and fitness of living organisms and their ageing. The pathways that regulate ROS homeostasis are crucial for mitigating the toxicity of ROS and provide strong evidence about specificity in ROS signalling. By taking advantage of the chemistry of ROS, highly specific mechanisms have evolved that form the basis of oxidant scavenging and ROS signalling systems.

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ROS as signalling molecules: mechanisms that generate specificity in ROS homeostasis

There is a vast literature on the generation and effects of reactive oxygen species in biological systems, both in relation to damage they cause and their involvement in cell regulatory and signaling pathways. The biological chemistry of different oxidants is becoming well understood, but it is often unclear how this translates into cellular mechanisms where redox changes have been demonstrated. This review addresses this gap. It examines how target selectivity and antioxidant effectiveness vary for different oxidants. Kinetic considerations of reactivity are used to assess likely targets in cells and how reactions might be influenced by restricted diffusion and compartmentalization. It also highlights areas where greater understanding is required on the fate of oxidants generated by cellular NADPH oxidases and on the identification of oxidant sensors in cell signaling.

Reconciling the chemistry and biology of reactive oxygen species

'Reactive oxygen species' (ROS) is an umbrella term for an array of derivatives of molecular oxygen that occur as a normal attribute of aerobic life. Elevated formation of the different ROS leads to molecular damage, denoted as 'oxidative distress'. Here we focus on ROS at physiological levels and their central role in redox signalling via different post-translational modifications, denoted as 'oxidative eustress'. Two species, hydrogen peroxide (H2O2) and the superoxide anion radical (O2·-), are key redox signalling agents generated under the control of growth factors and cytokines by more than 40 enzymes, prominently including NADPH oxidases and the mitochondrial electron transport chain. At the low physiological levels in the nanomolar range, H2O2 is the major agent signalling through specific protein targets, which engage in metabolic regulation and stress responses to support cellular adaptation to a changing environment and stress. In addition, several other reactive species are involved in redox signalling, for instance nitric oxide, hydrogen sulfide and oxidized lipids. Recent methodological advances permit the assessment of molecular interactions of specific ROS molecules with specific targets in redox signalling pathways. Accordingly, major advances have occurred in understanding the role of these oxidants in physiology and disease, including the nervous, cardiovascular and immune systems, skeletal muscle and metabolic regulation as well as ageing and cancer. In the past, unspecific elimination of ROS by use of low molecular mass antioxidant compounds was not successful in counteracting disease initiation and progression in clinical trials. However, controlling specific ROS-mediated signalling pathways by selective targeting offers a perspective for a future of more refined redox medicine. This includes enzymatic defence systems such as those controlled by the stress-response transcription factors NRF2 and nuclear factor-κB, the role of trace elements such as selenium, the use of redox drugs and the modulation of environmental factors collectively known as the exposome (for example, nutrition, lifestyle and irradiation).

Reactive oxygen species (ROS) as pleiotropic physiological signalling agents.

(Macro)autophagy is a bulk degradation process that mediates the clearance of long-lived proteins and organelles. Autophagy is initiated by double-membraned structures, which engulf portions of cytoplasm. The resulting autophagosomes ultimately fuse with lysosomes, where their contents are degraded. Although the term autophagy was first used in 1963, the field has witnessed dramatic growth in the last 5 years, partly as a consequence of the discovery of key components of its cellular machinery. In this review we focus on mammalian autophagy, and we give an overview of the understanding of its machinery and the signaling cascades that regulate it. As recent studies have also shown that autophagy is critical in a range of normal human physiological processes, and defective autophagy is associated with diverse diseases, including neurodegeneration, lysosomal storage diseases, cancers, and Crohn's disease, we discuss the roles of autophagy in health and disease, while trying to critically evaluate if the coincidence between autophagy and these conditions is causal or an epiphenomenon. Finally, we consider the possibility of autophagy upregulation as a therapeutic approach for various conditions.

https://www.physiology.org/doi/pdf/10.1152/physrev.00030.2009

Regulation of Mammalian Autophagy in Physiology and Pathophysiology

Disorders characterized by ischemia/reperfusion (I/R), such as myocardial infarction, stroke, and peripheral vascular disease, continue to be among the most frequent causes of debilitating disease and death. Tissue injury and/or death occur as a result of the initial ischemic insult, which is determined primarily by the magnitude and duration of the interruption in the blood supply, and then subsequent damage induced by reperfusion. During prolonged ischemia, ATP levels and intracellular pH decrease as a result of anaerobic metabolism and lactate accumulation. As a consequence, ATPase-dependent ion transport mechanisms become dysfunctional, contributing to increased intracellular and mitochondrial calcium levels (calcium overload), cell swelling and rupture, and cell death by necrotic, necroptotic, apoptotic, and autophagic mechanisms. Although oxygen levels are restored upon reperfusion, a surge in the generation of reactive oxygen species occurs and proinflammatory neutrophils infiltrate ischemic tissues to exacerbate ischemic injury. The pathologic events induced by I/R orchestrate the opening of the mitochondrial permeability transition pore, which appears to represent a common end-effector of the pathologic events initiated by I/R. The aim of this treatise is to provide a comprehensive review of the mechanisms underlying the development of I/R injury, from which it should be apparent that a combination of molecular and cellular approaches targeting multiple pathologic processes to limit the extent of I/R injury must be adopted to enhance resistance to cell death and increase regenerative capacity in order to effect long-lasting repair of ischemic tissues.

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Cell Biology of Ischemia/Reperfusion Injury

Nitric oxide (NO), synthesized in eukaryotes by the NO synthases, has multiple roles in signalling pathways and in protection against pathogens. Pathogenic microorganisms have apparently evolved defence mechanisms that counteract the effects of NO and related reactive nitrogen species. Regulatory proteins that sense NO mediate the primary response to NO and nitrosative stress. The only regulatory protein in enteric bacteria known to serve exclusively as an NO-responsive transcription factor is the enhancer binding protein NorR (refs 9, 10-11). In Escherichia coli, NorR activates the transcription of the norVW genes encoding a flavorubredoxin (FlRd) and an associated flavoprotein, respectively, which together have NADH-dependent NO reductase activity. The NO-responsive activity of NorR raises important questions concerning the mechanism of NO sensing. Here we show that the regulatory domain of NorR contains a mononuclear non-haem iron centre, which reversibly binds NO. Binding of NO stimulates the ATPase activity of NorR, enabling the activation of transcription by RNA polymerase. The mechanism of NorR reveals an unprecedented biological role for a non-haem mononitrosyl-iron complex in NO sensing.

A non-haem iron centre in the transcription factor NorR senses nitric oxide

Glutathione contributes to thiol-redox control and to extra-mitochondrial iron–sulphur cluster (ISC) maturation. To determine the physiological importance of these functions and sort out those that account for the GSH requirement for viability, we performed a comprehensive analysis of yeast cells depleted of or containing toxic levels of GSH. Both conditions triggered an intense iron starvation-like response and impaired the activity of extra-mitochondrial ISC enzymes but did not impact thiol-redox maintenance, except for high glutathione levels that altered oxidative protein folding in the endoplasmic reticulum. While iron partially rescued the ISC maturation and growth defects of GSH-depleted cells, genetic experiments indicated that unlike thioredoxin, glutathione could not support by itself the thiol-redox duties of the cell. We propose that glutathione is essential by its requirement in ISC assembly, but only serves as a thioredoxin backup in cytosolic thiol-redox maintenance. Glutathione-high physiological levels are thus meant to insulate its cytosolic function in iron metabolism from variations of its concentration during redox stresses, a model challenging the traditional view of it as prime actor in thiol-redox control.

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Glutathione revisited: a vital function in iron metabolism and ancillary role in thiol-redox control.

Ferric uptake regulation protein (Fur) is a bacterial global regulator that uses iron as a cofactor to bind to specific DNA sequences. The function of Fur is not limited to iron homeostasis. A wide variety of genes involved in various mechanisms such as oxidative and acid stresses are under Fur control. Flavohemoglobin (Hmp) is an NO-detoxifying enzyme induced by NO and nitrosothiol compounds. Fur recently was found to regulate hmp in Salmonella typhimurium, and in Escherichia coli, the iron-chelating agent 2,2'-dipyridyl induces hmp expression. We now establish direct inhibition of E. coli Fur activity by NO. By using chromosomal Fur-regulated lacZ reporter fusion in E. coli, Fur activity is switched off by NO at micromolar concentration. In vitro Fur DNA-binding activity, as measured by protection of restriction site in aerobactin promoter, is directly sensitive to NO. NO reacts with Fe(II) in purified FeFur protein to form a S = 12 low-spin FeFur-NO complex with a g = 2.03 EPR signal. Appearance of the same EPR signal in NO-treated cells links nitrosylation of the iron with Fur inhibition. The nitrosylated Fur protein is still a dimer and is stable in anaerobiosis but slowly decays in air. This inhibition probably arises from a conformational switch, leading to an inactive dimeric protein. These data establish a link between control of iron metabolism and the response to NO effects.

https://www.pnas.org/content/pnas/99/26/16619.full.pdf

Direct inhibition by nitric oxide of the transcriptional ferric uptake regulation protein via nitrosylation of the iron

Thiol-based peroxidases consist of the peroxiredoxins (Prx) and the related glutathione peroxidase (GPx)-like enzymes. Their catalytic function is to reduce peroxides by using the reactivity of the cysteine residue, and their presumed primary physiologic role is to protect living organisms from peroxide toxicity. However, as peroxide-metabolizing enzymes, they also regulate hydrogen peroxide (H2O2) signaling. We review here enzymatic and biochemical attributes of thiol peroxidases that specify both distinctive peroxide-scavenging functions and the property of regulating H2O2 signaling. We then discuss possible thiol peroxidase physiologic functions, based on selected observations made in microorganisms and mammals.

Benoît D'Autréaux

Papers

ROS as signalling molecules: mechanisms that generate specificity in ROS homeostasis

A non-haem iron centre in the transcription factor NorR senses nitric oxide

Glutathione revisited: a vital function in iron metabolism and ancillary role in thiol-redox control.

Direct inhibition by nitric oxide of the transcriptional ferric uptake regulation protein via nitrosylation of the iron

The dual functions of thiol-based peroxidases in H2O2 scavenging and signaling.