In the early 1920s Otto Warburg observed that cancer cells have altered metabolism and from this, posited that mitochondrial dysfunction underpinned the aetiology of cancers. The more recent identification of mutations of mitochondrial metabolic enzymes in a wide range of human cancers has now provided a direct link between metabolic alterations and cancer. In this review we discuss the consequences of dysfunction of three metabolic enzymes involved in or associated with the tricarboxylic acid (TCA) cycle: succinate dehydrogenase (SDH), fumarate hydratase (FH) and isocitrate dehydrogenase (IDH) focusing on the similarity between the phenotypes of cancers harbouring these mutations.

Nrf2 (nuclear factor erytheroid-derived-2-like 2) transcriptional programmes are activated by a variety of cellular stress conditions to maintain cellular homoeostasis. Under non-stress conditions, Nrf2 is under tight regulation by the ubiquitin proteasome system (UPS). Detailed mechanistic investigations have shown the Kelch-like ECH-associated protein 1 (Keap1)–cullin3 (Cul3)–ring-box1 (Rbx1) E3-ligase to be the primary Nrf2 regulatory system. Recently, both beta-transducin repeat-containing E3 ubiquitin protein ligase (β-TrCP) and E3 ubiquitin-protein ligase synoviolin (Hrd1) have been identified as novel E3 ubiquitin ligases that negatively regulate Nrf2 through Keap1-independent mechanisms. In addition to UPS-mediated regulation of Nrf2, investigations have revealed a cross-talk between Nrf2 and the autophagic pathway resulting in activation of Nrf2 in a non-canonical manner. In addition to regulation at the protein level, Nrf2 was recently shown to be regulated at the transcriptional level by oncogenic K-rat sarcoma (Ras). A consequence of these differential regulatory mechanisms is the dual role of Nrf2 in cancer: the canonical, protective role and the non-canonical ‘dark-side’ of Nrf2. Based on the protective role of Nrf2, a vast effort has been dedicated towards identifying novel chemical inducers of Nrf2 for the purpose of chemoprevention. On the other hand, upon malignant transformation, some cancer cells have a constitutively high level of Nrf2 offering a growth advantage, as well as rendering cancer cells resistant to chemotherapeutics. This discovery has led to a new paradigm in cancer treatment; the initially counterintuitive use of Nrf2 inhibitors as adjuvants in chemotherapy. Herein, we will discuss the mechanisms of Nrf2 regulation and how this detailed molecular understanding can be leveraged to develop Nrf2 modulators to prevent diseases, mitigate disease progression or overcome chemoresistance.

The oxidoreductase Trx-1 (thioredoxin 1) is highly conserved and found intra- and extra-cellularly in mammalian systems. There is increasing interest in its capacity to regulate immune function based on observations of altered distribution and expression during ageing and disease. We have investigated previously whether extracellular T-cell or peripheral blood mononuclear cell Trx-1 levels serve as a robust marker of ageing. In a preliminary study of healthy older adults compared with younger adults, we showed that there was a significant, but weak, relationship with age. Interestingly, patients with rheumatoid arthritis and cancer have been described by others to secrete or express greater surface Trx-1 than predicted. It is interesting to speculate whether a decline in Trx-1 during ageing protects against such conditions, but correspondingly increases risk of disease associated with Trx-1 depletion such as cardiovascular disease. These hypotheses are being explored in the MARK-AGE study, and preliminary findings confirm an inverse correlation of surface Trx-1 with age. We review recent concepts around the role of Trx-1 and its partners in T-cell function on the cell surface and as an extracellular regulator of redox state in a secreted form. Further studies on the redox state and binding partners of surface and secreted Trx-1 in larger patient datasets are needed to improve our understanding of why Trx-1 is important for lifespan and immune function.

ROS (reactive oxygen species) have long been regarded as a series of destructive molecules that have a detrimental effect on cell homoeostasis. In support of this are the myriad antioxidant defence systems nearly all eukaryotic cells have that are designed to keep the levels of ROS in check. However, research data emerging over the last decade have demonstrated that ROS can influence a range of cellular events in a manner similar to that seen for traditional second messenger molecules such as cAMP. Hydrogen peroxide (H 2 O 2 ) appears to be the main ROS with such signalling properties, and this molecule has been shown to affect a wide range of cellular functions. Its localized synthesis by the Nox (NADPH oxidase) family of enzymes and how these enzymes are regulated is of particular interest to those who work in the field of tumour biology.

RTT (Rett syndrome) is a severe progressive neurodevelopmental disorder with a monogenetic cause, but complex and multifaceted clinical appearance. Compelling evidence suggests that mitochondrial alterations and aberrant redox homoeostasis result in oxidative challenge. Yet, compared with other severe neuropathologies, RTT is not associated with marked neurodegeneration, but rather a chemical imbalance and miscommunication of neuronal elements. Different pharmacotherapies mediate partial improvement of conditions in RTT, and also antioxidants or compounds improving mitochondrial function may be of potential merit. In the present paper, we summarize findings from patients and transgenic mice that point towards the nature of RTT as a mitochondrial disease. Also, open questions are addressed that require clarification to fully understand and successfully target the associated cellular redox imbalance.

Self-fertilization, which results in reduced fitness of offspring, is a common problem in hermaphrodite angiosperms. To prevent this, many plants utilize SI (self-incompatibility), which is determined by the multi-allelic S -locus, that allows discrimination between self (incompatible) and non-self (compatible) pollen by the pistil. In poppy ( Papaver rhoeas ), the pistil S- determinant ( PrsS ) is a small secreted protein which interacts with the pollen S -determinant PrpS , a ~20 kDa novel transmembrane protein. Interaction of matching pollen and pistil S -determinants results in self-recognition, initiating a Ca 2+ -dependent signalling network in incompatible pollen. This triggers several downstream events, including alterations to the cytoskeleton, phosphorylation of sPPases (soluble inorganic pyrophosphatases) and an MAPK (mitogen-activated protein kinase), increases in ROS (reactive oxygen species) and nitric oxide (NO), and activation of several caspase-like activities. This results in the inhibition of pollen tube growth, prevention of self-fertilization and ultimately PCD (programmed cell death) in incompatible pollen. The present review focuses on our current understanding of the integration of these signals with their targets in the SI/PCD network. We also discuss our recent functional expression of PrpS in Arabidopsis thaliana pollen.

Circadian rhythms are a hallmark of living organisms, observable in all walks of life from primitive bacteria to highly complex humans. They are believed to have evolved to co-ordinate the timing of biological and behavioural processes to the changing environmental needs brought on by the progression of day and night through the 24-h cycle. Most of the modern study of circadian rhythms has centred on so-called TTFLs (transcription–translation feedback loops), wherein a core group of ‘clock’ genes, capable of negatively regulating themselves, produce oscillations with a period of approximately 24 h. Recently, however, the prevalence of the TTFL paradigm has been challenged by a series of findings wherein circadian rhythms, in the form of redox reactions, persist in the absence of transcriptional cycles. We have found that circadian cycles of oxidation and reduction are conserved across all domains of life, strongly suggesting that non-TTFL mechanisms work in parallel with the canonical genetic processes of timekeeping to generate the cyclical cellular and behavioural phenotypes that we commonly recognize as circadian rhythms.

The events leading to the degradation of the endogenous PrP C (normal cellular prion protein) have been the subject of numerous studies. Two cleavage processes, α-cleavage and β-cleavage, are responsible for the main C- and N-terminal fragments produced from PrP C . Both cleavage processes occur within the N-terminus of PrP C , a region that is significant in terms of function. α-Cleavage, an enzymatic event that occurs at amino acid residues 110 and 111 on PrP C , interferes with the conversion of PrP C into the prion disease-associated isoform, PrP Sc (abnormal disease-specific conformation of prion protein). This processing is seen as a positive event in terms of disease development. The study of β-cleavage has taken some surprising turns. β-Cleavage is brought about by ROS (reactive oxygen species). The C-terminal fragment produced, C2, may provide the seed for the abnormal conversion process, as it resembles in size the fragments isolated from prion-infected brains. There is, however, strong evidence that β-cleavage provides an essential process to reduce oxidative stress. β-Cleavage may act as a double-edged sword. By β-cleavage, PrP C may try to balance the ROS levels produced during prion infection, but the C2 produced may provide a PrP Sc seed that maintains the prion conversion process.

Cell invasion plays a central role in a wide variety of biological phenomena and is the cause of tumour growth and metastasis. Understanding the biochemical mechanisms that control cell invasion is one of the major goals of our laboratory. Podosomes and invadopodia are specialized cellular structures present in cells with physiological or pathological invasive behaviours. These transient structures are localized at the ventral cell surface, contain an array of different proteins and facilitate cell–substrate adhesion, as well as the local proteolytic activity necessary for extracellular matrix remodelling and subsequent cellular invasion. We have shown previously that the adaptor proteins and Src substrates Tks4 and Tks5 are required for podosome and invadopodia formation, for cancer cell invasion in vitro , and for tumour growth in vivo . We have also defined a role for the Tks-mediated generation of ROS (reactive oxygen species) in both podosome and invadopodia formation, and invasive behaviour. Tks4 and Tks5 are also required for proper embryonic development, probably because of their roles in cell migration. Finally, we recently implicated podosome formation as part of the synthetic phenotype of vascular smooth muscle cells. Inhibitors of podosome and invadopodia formation might have utility in the treatment of vascular diseases and cancer. We have therefore developed a high-content cell-based high-throughput screening assay that allows us to identify inhibitors and activators of podosome/invadopodia formation. We have used this assay to screen for small-molecule inhibitors and defined novel regulators of invadopodia formation. In the present paper, I review these recent findings.

Progeroid laminopathies are characterized by the abnormal processing of lamin A, the appearance of misshapen nuclei, and the accumulation and persistence of DNA damage. In the present article, I consider the contribution of defective DNA damage pathways to the pathology of progeroid laminopathies. Defects in DNA repair pathways appear to be caused by a combination of factors. These include abnormal epigenetic modifications of chromatin that are required to recruit DNA repair pathways to sites of DNA damage, abnormal recruitment of DNA excision repair proteins to sites of DNA double-strand breaks, and unrepairable ROS (reactive oxygen species)-induced DNA damage. At least two of these defective processes offer the potential for novel therapeutic approaches.

The fifth U.K. meeting on nuclear envelope disease and chromatin brought together international experts from across the field of nuclear envelope biology to discuss the advancements in a class of tissue-specific degenerative diseases called the laminopathies. Clinically, these range from relatively mild fat-wasting disorders to the severe premature aging condition known as Hutchinson–Gilford progeria syndrome. Since the first association of the nuclear envelope with human inherited disease in 1994, there has been an exponential increase in an unexpected variety of functions associated with nuclear envelope proteins, ranging from mechanical support and nucleocytoskeletal connections to regulation of chromatin organization and gene expression. This Biochemical Society Focused Meeting reinforced the functional complexity of nuclear-associated diseases, revealed new avenues to be investigated and highlighted the signalling pathways suitable as therapeutic targets.

A comprehensive understanding of ROS (reactive oxygen species)-dependent cellular interaction requires the previously unmet ability to simultaneously monitor the intra- and extra-cellular environments. The present review assesses the potential of novel electrochemical and fluorescent-based nanosensor approaches to address the limitations of existing techniques for ROS analysis. Data generated by these new approaches have already contributed significantly to current understanding of the roles that these species play in various in vitro scenarios. However, integration of these novel approaches has the potential to offer, for the first time, the unparalleled ability to measure simultaneously and in real-time ROS flux in both the intra- and extra-cellular environments.

HE (hydroethidine), a widely used fluorescent dye for detecting intracellular superoxide, undergoes specific oxidation and hydroxylation reactions. The reaction between HE and O 2 •− (superoxide radical) yields a diagnostic marker product, 2-hydroxyethidium. This is contrary to the popular notion that O 2 •− oxidizes HE to form ethidium. HE, however, undergoes a non-specific oxidation to form ethidium in the presence of other oxidants (hydroxyl radical, peroxynitrite and perferryl iron) and other dimeric products. The mitochondria-targeted HE analogue Mito-SOX® undergoes the same type of oxidative chemistry to form products similar to those formed from HE. On the basis of the oxidative chemical mechanism of HE and Mito-SOX®, we conclude that flurorescence microscopy or related techniques are not sufficient to measure the superoxide-specific hydroxylated products. HPLC methodologies are required to separate and identify these products. Peroxynitrite reacts rapidly and stoichiometrically with boronates to form specific products. Assays using fluorescent-based boronate probes will be more reliable for peroxynitrite determination than those using either dichlorodihydrofluorescein or dihydrorhodamine.

During the last decade, the possibility that ‘mild’ uncoupling could be protective against oxidative damage by diminishing ROS (reactive oxygen species) production has attracted much interest. In the present paper, we briefly examine the evidence for this possibility. It is only ROS production from succinate under reverse electron-flow conditions that is sensitive to membrane potential fluctuations, and so only this type of ROS production could be affected; however, the conditions under which succinate-supported ROS production is observed include succinate concentrations that are supraphysiological. Any decrease in membrane potential, even ‘mild uncoupling’, must necessarily lead to large increases in respiration, i.e. it must be markedly thermogenic. Mitochondria within cells are normally ATP-producing and thus already have a diminished membrane potential, and treatment of cells, organs or animals with small amounts of artificial uncoupler does not seem to have beneficial effects that are explainable via reduced ROS production. Although it has been suggested that members of the uncoupling protein family (UCP1, UCP2 and UCP3) may mediate a mild uncoupling, present evidence does not unequivocally support such an effect, e.g. the absence of the truly uncoupling protein UCP1 is not associated with increased oxidative damage. Thus present evidence does not support mild uncoupling as a physiologically relevant alleviator of oxidative damage.

Yeast multicellular colonies possess several traits that are absent from individual yeasts. These include the ability to synchronize colony population development and adapt its metabolism to different environmental changes, such as nutrient depletion. This, together with cell diversification to cell variants with distinct metabolic and other properties, contributes to the main goal of the colony population: to achieve longevity. In this respect, a benefit to individual cells is subordinated to the benefit to the whole population, exhibiting a kind of altruistic behaviour. For example, some colony cells located at particular positions undergo regulated cell dying and provide components to other cells located in more propitious areas. The enhancement of techniques that enable the in vivo investigation of three-dimensional spatiotemporal colony development may lead to new discoveries on metabolic differentiation and regulation in the near future.

Reactive species of oxygen, nitrogen and sulfur play cell signalling roles in human health, e.g. recent studies have shown that increased dietary nitrate, which is a source of RNS (reactive nitrogen species), lowers resting blood pressure and the oxygen cost of exercise. In such studies, plasma nitrite and nitrate are readily determined by chemiluminescence. At sites of inflammation, such as the joints of RA (rheumatoid arthritis) patients, the generation of ROS (reactive oxygen species) and RNS overwhelms antioxidant defences and one consequence is oxidative/nitrative damage to proteins. For example, in the inflamed joint, increased RNS-mediated protein damage has been detected in the form of a biomarker, 3-nitrotyrosine, by immunohistochemistry, Western blotting, ELISAs and MS. In addition to NO • , another cell-signalling gas produced in the inflamed joint is H 2 S (hydrogen sulfide), an RSS (reactive sulfur species). This gas is generated by inflammatory induction of H 2 S-synthesizing enzymes. Using zinc-trap spectrophotometry, we detected high (micromolar) concentrations of H 2 S in RA synovial fluid and levels correlated with clinical scores of inflammation and disease activity. What might be the consequences of the inflammatory generation of reactive species? Effects on inflammatory cell-signalling pathways certainly appear to be crucial, but in the current review we highlight the concept that ROS/RNS-mediated protein damage creates neoepitopes, resulting in autoantibody formation against proteins, e.g. type-II collagen and the complement component, C1q. These autoantibodies have been detected in inflammatory autoimmune diseases.

During chronic inflammation and ageing, the increase in oxidative stress in both intracellular and extracellular compartments is likely to influence local cell functions. Redox changes alter the T-cell proteome in a quantitative and qualitative manner, and post-translational modifications to surface and cytoplasmic proteins by increased reactive species can influence T-cell function. Previously, we have shown that RA (rheumatoid arthritis) T-cells exhibit reduced ROS (reactive oxygen species) production in response to extracellular stimulation compared with age-matched controls, and basal ROS levels [measured as DCF (2′,7′-dichlorofluorescein) fluorescence] are lower in RA T-cells. In contrast, exposing T-cells in vitro to different extracellular redox environments modulates intracellular signalling and enhances cytokine secretion. Together, these data suggest that a complex relationship exists between intra- and extra-cellular redox compartments which contribute to the T-cell phenotype.

The use of non-mammalian model organisms, including yeast Saccharomyces cerevisiae , can provide new insights into eukaryotic PCD (programmed cell death) pathways. In the present paper, we report recent achievements in the elucidation of the events leading to PCD that occur as a response to yeast treatment with AA (acetic acid). In particular, ROS (reactive oxygen species) generation, cyt c (cytochrome c ) release and mitochondrial function and proteolytic activity will be dealt with as they vary along the AA-PCD time course by using both wild-type and mutant yeast cells. Two AA-PCD pathways are described sharing common features, but distinct from one another with respect to the role of ROS and mitochondria, the former in which YCA1 acts upstream of cyt c release and caspase-like activation in a ROS-dependent manner and the latter in which cyt c release does not occur, but caspase-like activity increases, in a ROS-independent manner.

FOH (farnesol), a non-sterol isoprenoid produced by dephosphorylation of farnesyl pyrophosphate, has been shown to inhibit proliferation and induce apoptosis. We have been using Aspergillus nidulans and FOH as a model system and cell death stimulus, respectively, aiming to understand by which means filamentous fungi are driven towards cell death. Here, we review some of our findings about FOH-induced cell death in A. nidulans .

ROS (reactive oxygen species) are considered to be a major cause of cellular oxidative stress, linked to neuromuscular diseases and aging. Complex I (NADH:ubiquinone oxidoreductase) is one of the main contributors to superoxide production by mitochondria, and knowledge of its mechanism of O 2 reduction is required for the formulation of causative connections between complex I defects and pathological effects. There is evidence for two distinct (but not mutually exclusive) sites of O 2 reduction by complex I. Studies of the isolated enzyme largely support the participation of the reduced flavin mononucleotide in the active site for NADH oxidation, and this mechanism is supported in mitochondria by correlations between the NAD(P) + potential and O 2 reduction. In addition, studies of intact mitochondria or submitochondrial particles have suggested a mechanism involving the quinone-binding site, supported by observations during reverse electron transport and the use of ‘Q-site’ inhibitors. Here, we discuss extant data and models for O 2 reduction by complex I. We compare results from the isolated enzyme with results from intact mitochondria, aiming to identify similarities and differences between them and progress towards combining them to form a single, unified picture.

Pancreatic β-cells are essential for the maintenance of glucose homoeostasis, and dysfunction of these insulin-secreting cells results in the development of diabetes. In the course of events leading from obesity to Type 2 diabetes, several mechanisms are currently envisaged. Among them, lipids and oxidative stress are considered as toxic candidates for the β-cell. The cellular link between fatty acids and ROS (reactive oxygen species) is essentially the mitochondrion, a key organelle for the control of insulin secretion. Mitochondria are the main source of ROS and are also the primary target of oxidative attacks. The present review presents the current knowledge of lipotoxicity related to oxidative stress in the context of mitochondrial function in the β-cell.

This review presents a broad overview of phosphorylation and signalling in plants. Much of the work of my group in plants focuses on understanding the mechanisms that regulate the production of carbon and nitrogen metabolites in leaves; in this review, I will discuss nitrate, which is one of the most important of these inorganic nutrients. I also detail how protein phosphorylation in plant cells is altered in response to the presence of reactive oxygen species.

The controlled formation of ROS (reactive oxygen species) and RNS (reactive nitrogen species) is now known to be critical in cellular redox signalling. As with the more familiar phosphorylation-dependent signal transduction pathways, control of protein function is mediated by the post-translational modification at specific amino acid residues, notably thiols. Two important classes of oxidant-derived signalling molecules are the lipid oxidation products, including those with electrophilic reactive centres, and decomposition products such as lysoPC (lysophosphatidylcholine). The mechanisms can be direct in the case of electrophiles, as they can modify signalling proteins by post-translational modification of thiols. In the case of lysoPC, it appears that secondary generation of ROS/RNS, dependent on intracellular calcium fluxes, can cause the secondary induction of H 2 O 2 in the cell. In either case, the intracellular source of ROS/RNS has not been defined. In this respect, the mitochondrion is particularly interesting since it is now becoming apparent that the formation of superoxide from the respiratory chain can play an important role in cell signalling, and oxidized lipids can stimulate ROS formation from an undefined source. In this short overview, we describe recent experiments that suggest that the cell signalling mediated by lipid oxidation products involves their interaction with mitochondria. The implications of these results for our understanding of adaptation and the response to stress in cardiovascular disease are discussed.

Nitric oxide (NO) can induce cell death; however, NO-induced cell death may be dependent/conditional on factors other than NO itself. Whether NO kills a particular cell depends on the amount of NO, source of NO, time of exposure to NO, cell type and the levels of other factors including, particularly oxygen, superoxide, H 2 O 2 , antioxidants, thiols and glycolysis.

The PrP C [cellular isoform of PrP (prion protein)] can undergo a conformational conversion to produce a proteinase-resistant form PrP Sc (scrapie isoform of PrP), a step critical for the development of prion disease. Although essential for disease progression, the normal cellular function of PrP C remains unknown. Suggestions to date have centred on a protective role against oxidative stress. We have demonstrated that ROS (reactive oxygen species)-mediated β-cleavage of PrP C occurs at the cell surface, can be inhibited following hydroxyl radical quenching and has a prerequisite for the octarepeat region in the N-terminus of the protein. Significantly, two disease-associated mutants of PrP, namely PG14 and A116V (Ala 116 →Val), were unable to undergo β-cleavage and this lack of proteolysis was accompanied by functional consequences in cells expressing these mutant proteins. The cells were found to be less viable following exposure to copper and H 2 O 2 , had reduced levels of glutathione peroxidase and increased amounts of intracellular oxygen radicals. These results suggest that β-cleavage of PrP C is an initial consequence following exposure to ROS in the extracellular environment contributing to a pathway involved in antioxidant protection of neuronal cells.

Sensing and signalling events that detect abiotic stress-induced changes in plant water status and initiate downstream stress responses such as ABA (abscisic acid) accumulation and osmoregulation remain uncharacterized in plants. Although conclusive results are lacking, recent results from plants, and analogies to signalling in other organisms, suggest possible mechanisms for sensing altered water status and initial transduction of that signal. Internal signals that act downstream of ABA and modulate stress responses to reflect the type and severity of the stress and the metabolic status of the plant are also not well understood. Two specific types of signalling, sugar sensing and reactive oxygen signalling, are likely to be modulators of ABA response under stress. For both upstream sensing and signalling of plant water status as well as downstream modulation of ABA response, present results suggest several genetic strategies with high potential to increase our understanding of the molecular basis by which plants sense and respond to altered water status.

Although reactive oxygen species play important roles in cellular physiology as signalling molecules, their molecular targets are largely unknown. A probable group of targets for mediating many of the effects of reactive oxygen species on cell signalling is the large diverse family of cysteine-dependent phosphatases, which includes the protein tyrosine phosphatases. Our work and that of others suggest that the oxidative inactivation of protein and lipid phosphatases plays an important part in signalling, downstream of many cellular stimuli. Future studies should give us a clearer picture of the role of phosphatase inactivation in cellular behaviour and explain how specificity is achieved in redox signalling.

The inhibitors of oxidative phosphorylation induced fragmentation of mitochondria without any signs of apoptosis in CV-1 and HeLa cells. Prolonged treatment with the uncouplers (alone or in combination with the inhibitors of respiration) caused perinuclear clusterization of mitochondria, followed by their selective elimination. The fraction of mitochondria-depleted cells remained viable.

Ever since ROS (reactive oxygen species) were shown to meet the criteria of true signalling molecules, such as regulated production and a specific biological function, many efforts have been made to understand the precise role of ROS. The function of ROS in pathological mechanisms is taking a more and more central role in various fields of biomedical research, including neurobiology, cardiology and cancer. An elevated oxidative status has been found in many types of cancer cells, and the introduction of chemical and enzymological antioxidants can inhibit tumour cell proliferation, pointing to a critical role of ROS in mediating loss of growth control. The present review describes ROS-regulated mechanisms that are associated with cancer and tumour invasiveness. The cellular processes that are linked to these ROS functions are mitogenic signalling and cell motility, while ROS have also been implicated in apoptosis and cellular senescence, two mechanisms regarded as being anti-tumorigenic. This ‘two-faced’ character of free radicals will be discussed and placed in the context of the physiological conditions of the tumour cell, the different molecular backgrounds, and the specific ROS. More detailed understanding of the signalling pathways regulated by ROS in tumour cells will open up new prospects for chemo- or gene-therapeutic interventions.