Direct oxidative modifications of signalling proteins in mammalian cells and their effects on apoptosis

The production of ROS is an inevitable consequence of metabolism. However, high levels of ROS within a cell can be lethal and so the cell has a number of defences against oxidative cell stress. Occasionally the cell's antioxidant mechanisms fail and oxidative stress occurs. High levels of ROS within a cell have a number of direct and indirect consequences on cell signalling pathways and may result in apoptosis or necrosis. Although some of the indirect effects of ROS are well known, limitations in technology mean that the direct effects of the cell's redox environment upon proteins are less understood. Recent work by a number of groups has demonstrated that ROS can directly modify signalling proteins through different modifications, for example by nitrosylation, carbonylation, di-sulphide bond formation and glutathionylation. These modifications modulate a protein's activity and several recent papers have demonstrated their importance in cell signalling events, especially those involved in cell death/survival. Redox modification of proteins allows for further regulation of cell signalling pathways in response to the cellular environment. Understanding them may be critical for us to modulate cell pathways for our own means, such as in cytotoxic drug treatments of cancer cells. Protein modifications mediated by oxidative stress can modulate apoptosis, either through specific protein modifications resulting in regulation of signalling pathways, or through a general increase in oxidised proteins resulting in reduced cellular function. This review discusses direct oxidative protein modifications and their effects on apoptosis.     

Lipid peroxidation and apoptotic response in rat brain areas induced by long‐term administration of nandrolone: the mutual crosstalk between ROS and NF‐kB

The aim of this study was to evaluate the played by oxidative stress in the apoptotic response in different brain areas of rats chronically treated with supra‐physiological doses of nandrolone decanoate (ND). Immunohistochemical study and Western blot analysis were performed to evaluate cells' apoptosis and to measure the effects of expression of specific mediators, such as NF‐κB (nuclear factor kappa‐light‐chain‐enhancer of activated B cells), Bcl‐2 (B‐cell lymphoma 2), SMAC/DIABLO (second mitochondria‐derived activator of caspases/direct IAP‐binding protein with low PI) and VMAT2 (vesicular monoamine transporter 2) on apoptosis. The results of the present study indicate that a long‐term administration of ND promotes oxidative injury in rat brain specific areas. A link between oxidative stress and NF‐κB signalling pathways is supported by our results. In addition to high levels of oxidative stress, we consistently observed a strong immunopositivity to NF‐κB. It has been argued that one of the pathways leading to the activation of NF‐κB could be under reactive oxygen species (ROS)‐mediated control. In fact, growing evidence suggests that although in limited doses, endogenous ROS may play an activating role in NF‐κB signalling, while above a certain threshold, they may negatively impact upon this signalling. However, a mutual crosstalk between ROS and NF‐κB exists and recent studies have shown that ROS activity is subject to negative feedback regulation by NF‐κB, and that this negative regulation of ROS is the means through which NF‐κB counters programmed cells.     

Iron homeostasis and iron-regulated ROS in cell death,senescence and human diseases

BackgroundIron is essential for many types of biological processes. However, excessive iron can be cytotoxic and can lead to many diseases. Since ferroptosis, which is an iron-dependent regulated form of necrosis, was recently discovered, iron and iron-catalysed oxidative stress have attracted much interest because of their sophisticated mechanism of cellular signalling leading to cell death and associated with various diseases.Scope of reviewIn this review, we first focus on how iron catalyses reactive oxygen species (ROS). Next, we discuss the roles of iron in cell death and senescence and, in particular, the downstream signalling pathways of ROS. Finally, we discuss the potential regulation mechanism of iron as a therapeutic target for various iron-related diseases.Major conclusionsBoth labile iron released from organelles upon various stresses and iron incorporated in enzymes produce ROS, including lipid ROS. ROS produced by iron activates various signalling pathways, including mitogen-activated protein kinase (MAPK) signalling pathways such as the apoptosis signal-regulating kinase 1 (ASK1)-p38/JNK pathway. These ROS-activated signalling pathways regulate senescence or cell death and are linked to cancer, ischaemia-reperfusion injury during transplantation and ageing-related neurodegenerative diseases.General significanceIron overload damages cells and causes harmful effects on the body through oxidative stress. Thus, understanding the spatiotemporal availability of iron and the role of iron in generating ROS will provide clues for the suppression of ROS and cytotoxic redox-active iron. Moreover, elucidating the molecular mechanisms and signalling pathways of iron-dependent cytotoxicity will enable us to find novel therapeutic targets for various diseases.     

Multifaceted deaths orchestrated by mitochondria in neurones

Neurones undergo diverse forms of cell death depending on the nature and severity of the stress. These death outcomes are now classified into various types of programmed cell death, including apoptosis, autophagy and necrosis. Each of these pathways can run in parallel and all have mitochondria as a central feature. Recruitment of mitochondria into cell death signalling involves either (or both) induction of specific death responses through release of apoptogenic proteins into the cytosol, or perturbation in function leading to loss of mitochondrial energisation and ATP synthesis. Cross-talk between these signalling pathways, particularly downstream of mitochondria, determines the resultant pattern of cell death. The differential recruitment of specific death pathways depends on the timing of engagement of mitochondrial signalling. Other influences on programmed cell death pathways occur through stress of the endoplasmic reticulum and the associated ubiquitin-proteasome system normally handling potentially neurotoxic protein aggregates. Based upon contemporary evidence apoptosis is a relatively rare in the mature brain whereas the contribution of programmed necrosis to various neuropathologies has been underestimated. The death outcomes that neurones exhibit during acute or chronic injury or pathological conditions considered here (oxidative stress, hypoxic-ischaemic injury, amyotrophic lateral sclerosis, Parkinson's and Huntington's diseases) fall within a spectrum of the diverse death types across the apoptosis-necrosis continuum. Indeed, dying or dead neurones may simultaneously manifest characteristics of more than one type of death pathway. Understanding neuronal death pathways and their cross-talk not only informs the detailed pathobiology but also suggests novel therapeutic strategies.     

Clostridium perfringens Phospholipase C Induced ROS Production and Cytotoxicity Require PKC,MEK1 and NFκB Activation

Clostridium perfringens phospholipase C (CpPLC), also called α-toxin, is the most toxic extracellular enzyme produced by this bacteria and is essential for virulence in gas gangrene. At lytic concentrations, CpPLC causes membrane disruption, whereas at sublytic concentrations this toxin causes oxidative stress and activates the MEK/ERK pathway, which contributes to its cytotoxic and myotoxic effects. In the present work, the role of PKC, ERK 1/2 and NFκB signalling pathways in ROS generation induced by CpPLC and their contribution to CpPLC-induced cytotoxicity was evaluated. The results demonstrate that CpPLC induces ROS production through PKC, MEK/ERK and NFκB pathways, the latter being activated by the MEK/ERK signalling cascade. Inhibition of either of these signalling pathways prevents CpPLC''s cytotoxic effect. In addition, it was demonstrated that NFκB inhibition leads to a significant reduction in the myotoxicity induced by intramuscular injection of CpPLC in mice. Understanding the role of these signalling pathways could lead towards developing rational therapeutic strategies aimed to reduce cell death during a clostridialmyonecrosis.     

ER-stress and apoptosis: molecular mechanisms and potential relevance in infection

During ER-stress, one of the responses a cell can choose is apoptosis. Apoptosis generally is a cell's preferred response when other control mechanisms are overwhelmed. We now have a reasonably clear molecular picture what is happening once the apoptotic apparatus has been started. Unclear however are the majority of the upstream pathways that connect other signalling to apoptosis. During ER-stress, confirmed apoptosis-regulating targets are pro- and anti-apoptotic proteins of the Bcl-2-family, whose concerted action induces apoptosis. I will here discuss how mitochondrial apoptosis is triggered, how this is linked to the ER-stress response and in what way this may be relevant during microbial infections.     

Reactive oxygen species (ROS) homeostasis and redox regulation in cellular signaling

Reactive oxygen species (ROS) are generated during mitochondrial oxidative metabolism as well as in cellular response to xenobiotics, cytokines, and bacterial invasion. Oxidative stress refers to the imbalance due to excess ROS or oxidants over the capability of the cell to mount an effective antioxidant response. Oxidative stress results in macromolecular damage and is implicated in various disease states such as atherosclerosis, diabetes, cancer, neurodegeneration, and aging. Paradoxically, accumulating evidence indicates that ROS also serve as critical signaling molecules in cell proliferation and survival. While there is a large body of research demonstrating the general effect of oxidative stress on signaling pathways, less is known about the initial and direct regulation of signaling molecules by ROS, or what we term the "oxidative interface." Cellular ROS sensing and metabolism are tightly regulated by a variety of proteins involved in the redox (reduction/oxidation) mechanism. This review focuses on the molecular mechanisms through which ROS directly interact with critical signaling molecules to initiate signaling in a broad variety of cellular processes, such as proliferation and survival (MAP kinases, PI3 kinase, PTEN, and protein tyrosine phosphatases), ROS homeostasis and antioxidant gene regulation (thioredoxin, peroxiredoxin, Ref-1, and Nrf-2), mitochondrial oxidative stress, apoptosis, and aging (p66Shc), iron homeostasis through iron-sulfur cluster proteins (IRE-IRP), and ATM-regulated DNA damage response.     

Peroxisomes sense and respond to environmental cues by regulating ROS and RNS signalling networks

Background Peroxisomes are highly dynamic, metabolically active organelles that used to be regarded as a sink for H₂O₂ generated in different organelles. However, peroxisomes are now considered to have a more complex function, containing different metabolic pathways, and they are an important source of reactive oxygen species (ROS), nitric oxide (NO) and reactive nitrogen species (RNS). Over-accumulation of ROS and RNS can give rise oxidative and nitrosative stress, but when produced at low concentrations they can act as signalling molecules.Scope This review focuses on the production of ROS and RNS in peroxisomes and their regulation by antioxidants. ROS production is associated with metabolic pathways such as photorespiration and fatty acid β-oxidation, and disturbances in any of these processes can be perceived by the cell as an alarm that triggers defence responses. Genetic and pharmacological studies have shown that photorespiratory H₂O₂ can affect nuclear gene expression, regulating the response to pathogen infection and light intensity. Proteomic studies have shown that peroxisomal proteins are targets for oxidative modification, S-nitrosylation and nitration and have highlighted the importance of these modifications in regulating peroxisomal metabolism and signalling networks. The morphology, size, number and speed of movement of peroxisomes can also change in response to oxidative stress, meaning that an ROS/redox receptor is required. Information available on the production and detection of NO/RNS in peroxisomes is more limited. Peroxisomal homeostasis is critical for maintaining the cellular redox balance and is regulated by ROS, peroxisomal proteases and autophagic processes.Conclusions Peroxisomes play a key role in many aspects of plant development and acclimation to stress conditions. These organelles can sense ROS/redox changes in the cell and thus trigger rapid and specific responses to environmental cues involving changes in peroxisomal dynamics as well as ROS- and NO-dependent signalling networks, although the mechanisms involved have not yet been established. Peroxisomes can therefore be regarded as a highly important decision-making platform in the cell, where ROS and RNS play a determining role.     

Oxidative stress and autophagy: the clash between damage and metabolic needs

Autophagy is a catabolic process aimed at recycling cellular components and damaged organelles in response to diverse conditions of stress, such as nutrient deprivation, viral infection and genotoxic stress. A growing amount of evidence in recent years argues for oxidative stress acting as the converging point of these stimuli, with reactive oxygen species (ROS) and reactive nitrogen species (RNS) being among the main intracellular signal transducers sustaining autophagy. This review aims at providing novel insight into the regulatory pathways of autophagy in response to glucose and amino acid deprivation, as well as their tight interconnection with metabolic networks and redox homeostasis. The role of oxidative and nitrosative stress in autophagy is also discussed in the light of its being harmful for both cellular biomolecules and signal mediator through reversible posttranslational modifications of thiol-containing proteins. The redox-independent relationship between autophagy and antioxidant response, occurring through the p62/Keap1/Nrf2 pathway, is also addressed in order to provide a wide perspective upon the interconnection between autophagy and oxidative stress. Herein, we also attempt to afford an overview of the complex crosstalk between autophagy and DNA damage response (DDR), focusing on the main pathways activated upon ROS and RNS overproduction. Along these lines, the direct and indirect role of autophagy in DDR is dissected in depth.     

Astaxanthin inhibits apoptosis in alveolar epithelial cells type II in vivo and in vitro through the ROS‐dependent mitochondrial signalling pathway

Oxidative stress is an important molecular mechanism underlying lung fibrosis. The mitochondrion is a major organelle for oxidative stress in cells. Therefore, blocking the mitochondrial signalling pathway may be the best therapeutic manoeuver to ameliorate lung fibrosis. Astaxanthin (AST) is an excellent antioxidant, but no study has addressed the pathway of AST against pulmonary oxidative stress and free radicals by the mitochondrion‐mediated signalling pathway. In this study, we investigated the antioxidative effects of AST against H₂O₂‐ or bleomycin (BLM)‐induced mitochondrial dysfunction and reactive oxygen species (ROS) production in alveolar epithelial cells type II (AECs‐II) in vivo and in vitro. Our data show that AST blocks H₂O₂‐ or BLM‐induced ROS generation and dose‐dependent apoptosis in AECs‐II, as characterized by changes in cell and mitochondria morphology, translocation of apoptotic proteins, inhibition of cytochrome c (Cyt c) release, and the activation of caspase‐9, caspase‐3, Nrf‐2 and other cytoprotective genes. These data suggest that AST inhibits apoptosis in AECs‐II cells through the ROS‐dependent mitochondrial signalling pathway and may be of potential therapeutic value in lung fibrosis treatment.     

Pterostilbene protects against H2O2-induced oxidative stress by regulating GAS6/Axl signaling in HL-1 cells

Pterostilbene (PTE, trans-3,5-dimethoxy-4′-hydroxystilbene), a natural plant polyphenol, possesses numerous pharmacological effects, including antioxidant, antidiabetic, antiatherosclerotic, and neuroprotective aspects. This study aims to investigate whether PTE plays a protective role against oxidative stress injury by GAS6/Axl signaling pathway in cardiomyocytes. Hydrogen peroxide (H₂O₂)-induced oxidative stress HL-1 cells were used as models. The mechanism by which PTE protected oxidative stress is investigated by combining cell viability, cell ROS levels, apoptosis assay, molecular docking, quantitative real-time PCR, and western blot analysis. GAS6 shRNA was performed to investigate the involvement of GAS6/Axl pathways in PTE's protective role. The results showed that PTE treatment improved the cell morphology and viability, and inhibited the apoptosis rate and ROS levels in H₂O₂-injured HL-1 cells. Particularly, PTE treatment upregulated the levels of GAS6, Axl, and markers related to oxidative stress, apoptosis, and mitochondrial function related. Molecular docking showed that PTE and GAS6 have good binding ability. Taken together, PTE plays a protective role against oxidative stress injury through inhibiting oxidative stress and apoptosis and improving mitochondrial function. Particularly, GAS6/Axl axis is the surprisingly prominent in the PTE-mediated pleiotropic effects.     

Activation of Autophagic Flux against Xenoestrogen Bisphenol-A-induced Hippocampal Neurodegeneration via AMP kinase (AMPK)/Mammalian Target of Rapamycin (mTOR) Pathways

The human health hazards related to persisting use of bisphenol-A (BPA) are well documented. BPA-induced neurotoxicity occurs with the generation of oxidative stress, neurodegeneration, and cognitive dysfunctions. However, the cellular and molecular mechanism(s) of the effects of BPA on autophagy and association with oxidative stress and apoptosis are still elusive. We observed that BPA exposure during the early postnatal period enhanced the expression and the levels of autophagy genes/proteins. BPA treatment in the presence of bafilomycin A₁ increased the levels of LC3-II and SQSTM1 and also potentiated GFP-LC3 puncta index in GFP-LC3-transfected hippocampal neural stem cell-derived neurons. BPA-induced generation of reactive oxygen species and apoptosis were mitigated by a pharmacological activator of autophagy (rapamycin). Pharmacological (wortmannin and bafilomycin A₁) and genetic (beclin siRNA) inhibition of autophagy aggravated BPA neurotoxicity. Activation of autophagy against BPA resulted in intracellular energy sensor AMP kinase (AMPK) activation, increased phosphorylation of raptor and acetyl-CoA carboxylase, and decreased phosphorylation of ULK1 (Ser-757), and silencing of AMPK exacerbated BPA neurotoxicity. Conversely, BPA exposure down-regulated the mammalian target of rapamycin (mTOR) pathway by phosphorylation of raptor as a transient cell''s compensatory mechanism to preserve cellular energy pool. Moreover, silencing of mTOR enhanced autophagy, which further alleviated BPA-induced reactive oxygen species generation and apoptosis. BPA-mediated neurotoxicity also resulted in mitochondrial loss, bioenergetic deficits, and increased PARKIN mitochondrial translocation, suggesting enhanced mitophagy. These results suggest implication of autophagy against BPA-mediated neurodegeneration through involvement of AMPK and mTOR pathways. Hence, autophagy, which arbitrates cell survival and demise during stress conditions, requires further assessment to be established as a biomarker of xenoestrogen exposure.     

The tyrphostin AG1478 augments oridonin-induced A431 cell apoptosis by blockage of JNK MAPK and enhancement of oxidative stress

Abstract

Oridonin, a diterpenoid compound, extracted and purified from Rabdosia rubescen has been reported to have cytotoxic effect on tumour cells through apoptosis, and tyrosine kinase pathways are involved in these processes. A specific epidermal growth factor receptor (EGFR) inhibitor AG1478 was used to examine the relationship between EGFR signal pathways and oridonin-induced apoptosis and autophagy in EGFR abundant human epidermoid carcinoma A431 cells. Inhibition of EGFRaugmented oridonin-induced A431 cell apoptosis, while the changes of expression of downstream proteins, Bcl-2, Bcl-xL, Bax, cytochrome c, pro-caspase-3, Fas, FADD and pro-caspase-8 suggested that both the intrinsic and extrinsic apoptotic pathways are involved in these processes. Pretreatment with AG1478 aggravated oridonin-induced loss of mitochondrial membrane potential (MMP) and increased ROS generation in A431 cells, while a ROS scavenger, N-acetylcysteine (NAC) completely reversed oridonin- and AG1478-induced ROS generation and apoptosis. Therefore, AG1478 augmented oridonin-induced apoptosis by enhancing oxidative stress. Pretreatment with AG1478 decreased the expression of downstream MAPK proteins ERK, JNK and P38 and their phosphorylated forms to varying degrees compared with oridonin alone treatment. Then after administration of ERK, JNK and P38 inhibitors, only JNK inhibitor SP600125 effectively augmented oridonin-induced apoptosis and ROS generation. Therefore, in EGFR downstream pathways, JNK played a major role in preventing oridonin-induced apoptosis. Autophagy antagonised apoptosis and exerted a protective effect in A431 cells, and both AG1478 and SP600125 decreased oridonin-induced autophagy. Inhibition of EGFR augmented oridonin-induced apoptosis and this was caused by enhanced oxidative stress, and JNK played a major protective role by increasing autophagy, leading to antagonising apoptosis and ROS generation.