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Vascular dysfunctions such as vascular hyporeactivity following ischemic/hypoxic injury are a major cause of death in injured patients. In this study, we showed that treatment with mitochondrial division inhibitor 1 (Mdivi-1), a selective inhibitor of dynamin-related protein 1 (Drp1), significantly improved vascular reactivity in ischemic rats by attenuating oxidative stress. The antioxidative effects of Mdivi-1 were relatively Drp1-independent, and possibly due to an increase in the levels of the antioxidant enzymes, SOD1 and catalase, as well as to enhanced Nrf2 expression. In addition, we found that while Mdivi-1 had little effect on Drp1 GTPase activity in vascular smooth muscle cells, it inhibited hypoxia-induced Drp1 phosphorylation at Ser-616, reducing excessive mitochondrial fission and slightly enhancing mitochondrial fusion. These effects possibly contributed to vascular protection at an early stage of ischemic/hypoxic injury. Finally, Mdivi-1 stabilized hemodynamics, increased vital organ perfusion, and improved rat survival after ischemic/hypoxic injury, proving a promising therapeutic agent for ischemic/hypoxic injury., Highlights • Mdivi-1 improved vascular contractility in ischemic rats. • Mdivi-1 attenuated hypoxia-induced oxidative stress and mitochondrial changes. • Drp1 recruitment to mitochondria, not GTPase activity, involved in Mdivi-1 effects. • Mdivi-1 has therapeutic potential against ischemic injury.

Enzymatic and non-enzymatic oxidation of unsaturated fatty acids gives rise to reactive species that covalently modify nucleophilic residues within redox sensitive protein sensors in a process called lipoxidation. This triggers adaptive signaling pathways that ultimately lead to increased resistance to stress. In this graphical review, we will provide an overview of pathways affected by protein lipoxidation and the key signaling proteins being altered, focusing on the KEAP1-NRF2 and heat shock response pathways. We review the mechanisms by which lipid peroxidation products can serve as second messengers and evoke cellular responses via covalent modification of key sensors of altered cellular environment, ultimately leading to adaptation to stress., Graphical abstract fx1

Nrf2 pathway has been known to be protective against cancer progression however recent studies have revealed that the antioxidant activity of Nrf2 contributes to chemotherapy resistance. For many years, hyperthermia has been used as an additional therapy to increase the efficiency of chemotherapy and radiotherapy. Besides the positive effects of hyperthermia during treatment procedure, thermotolerance has been found to develop against heat treatment. Although the involved molecular mechanisms have not been fully clarified, heat shock proteins (HSP) and proteasome activity are known to be involved in the acquisition of thermotolerance. The aim of this study was to investigate the potential beneficial effects of combining hyperthermia with Nrf2 silencing to inhibit molecular mechanisms leading to induction of defense mechanisms in transcription level. Following heat treatment of HT22 cells, HSP70 and the proteasome levels and as well as proteasome activity were found to be elevated in the nucleus. Our results demonstrated that Nrf2 silencing reduced defense mechanisms against heat treatment both in antioxidant and proteolytic manner and Nrf2 may be a potential target for therapeutic approach in order to improve the beneficial effects of hyperthermia in cancer therapy., Graphical abstract fx1, Highlights • Hyperthermia increases HSP70, β5 levels and Proteasome activity in the nucleus. • Nrf2 pathway contributes to thermotolerance by HO-1 and GSTα. • Nrf2 contributes to thermotolerance via proteasome and HSP related degradation. • Nrf2 Inhibition may be a useful approach to overcome the thermotolerance.

Reactive oxygen species (ROS) are integral components of multiple cellular pathways even though excessive or inappropriately localized ROS damage cells. ROS function as anti-microbial effector molecules and as signaling molecules that regulate such processes as NF-kB transcriptional activity, the production of DNA-based neutrophil extracellular traps (NETs), and autophagy. The main sources of cellular ROS are mitochondria and NADPH oxidases (NOXs). In contrast to NOX-generated ROS, ROS produced in the mitochondria (mtROS) were initially considered to be unwanted by-products of oxidative metabolism. Increasing evidence indicates that mtROS have been incorporated into signaling pathways including those regulating immune responses and autophagy. As metabolic hubs, mitochondria facilitate crosstalk between the metabolic state of the cell with these pathways. Mitochondria and ROS are thus a nexus of multiple pathways that determine the response of cells to disruptions in cellular homeostasis such as infection, sterile damage, and metabolic imbalance. In this review, we discuss the roles of mitochondria in the generation of ROS-derived anti-microbial effectors, the interplay of mitochondria and ROS with autophagy and the formation of DNA extracellular traps, and activation of the NLRP3 inflammasome by ROS and mitochondria., Graphical abstract fx1, Highlights • Mitochondrial ROS production is regulated by and incorporated into immunity pathways. • ROS regulate mitophagy and xenophagy and neutrophil extracellular trap formation. • Mitochondria and mitochondrial ROS regulate NLRP3 inflammasome activation.

Free radicals originate from both exogenous environmental sources and as by-products of the respiratory chain and cellular oxygen metabolism. Sustained accumulation of free radicals, beyond a physiological level, induces oxidative stress that is harmful for the cellular homeodynamics as it promotes the oxidative damage and stochastic modification of all cellular biomolecules including proteins. In relation to proteome stability and maintenance, the increased concentration of oxidants disrupts the functionality of cellular protein machines resulting eventually in proteotoxic stress and the deregulation of the proteostasis (homeostasis of the proteome) network (PN). PN curates the proteome in the various cellular compartments and the extracellular milieu by modulating protein synthesis and protein machines assembly, protein recycling and stress responses, as well as refolding or degradation of damaged proteins. Molecular chaperones are key players of the PN since they facilitate folding of nascent polypeptides, as well as holding, folding, and/or degradation of unfolded, misfolded, or non-native proteins. Therefore, the expression and the activity of the molecular chaperones are tightly regulated at both the transcriptional and post-translational level at organismal states of increased oxidative and, consequently, proteotoxic stress, including ageing and various age-related diseases (e.g. degenerative diseases and cancer). In the current review we present a synopsis of the various classes of intra- and extracellular chaperones, the effects of oxidants on cellular homeodynamics and diseases and the redox regulation of chaperones., Graphical abstract Differential regulation of chaperones activity, under physiological conditions or during oxidative stress-mediated proteome instability., Highlights • Free radicals originate from various sources and at physiological concentrations are essential for the modulation of cell signalling pathways. • Abnormally high levels of free radicals induce oxidative stress and damage all cellular biomolecules, including proteins. • Molecular chaperones facilitate folding of nascent polypeptides, as well as holding, folding, and/or degradation of damaged proteins. • The expression and the activity of chaperones during oxidative stress are regulated at both the transcriptional and post-translational level.

The WNK (with no lysine kinase)–SPAK (SPS1-related proline/alanine-rich kinase)/OSR1 (oxidative stress-responsive kinase 1) signalling pathway plays an important role in controlling mammalian blood pressure by modulating the activity of ion co-transporters in the kidney. Recent studies have identified Gordon's hypertension syndrome patients with mutations in either CUL3 (Cullin-3) or the BTB protein KLHL3 (Kelch-like 3). CUL3 assembles with BTB proteins to form Cullin–RING E3 ubiquitin ligase complexes. To explore how a CUL3–KLHL3 complex might operate, we immunoprecipitated KLHL3 and found that it associated strongly with WNK isoforms and CUL3, but not with other components of the pathway [SPAK/OSR1 or NCC (Na+/Cl− co-transporter)/NKCC1 (Na+/K+/2Cl− co-transporter 1)]. Strikingly, 13 out of the 15 dominant KLHL3 disease mutations analysed inhibited binding to WNK1 or CUL3. The recombinant wild-type CUL3–KLHL3 E3 ligase complex, but not a disease-causing CUL3–KLHL3[R528H] mutant complex, ubiquitylated WNK1 in vitro. Moreover, siRNA (small interfering RNA)-mediated knockdown of CUL3 increased WNK1 protein levels and kinase activity in HeLa cells. We mapped the KLHL3 interaction site in WNK1 to a non-catalytic region (residues 479–667). Interestingly, the equivalent region in WNK4 encompasses residues that are mutated in Gordon's syndrome patients. Strikingly, we found that the Gordon's disease-causing WNK4[E562K] and WNK4[Q565E] mutations, as well as the equivalent mutation in the WNK1[479–667] fragment, abolished the ability to interact with KLHL3. These results suggest that the CUL3–KLHL3 E3 ligase complex regulates blood pressure via its ability to interact with and ubiquitylate WNK isoforms. The findings of the present study also emphasize that the missense mutations in WNK4 that cause Gordon's syndrome strongly inhibit interaction with KLHL3. This could elevate blood pressure by increasing the expression of WNK4 thereby stimulating inappropriate salt retention in the kidney by promoting activation of the NCC/NKCC2 ion co-transporters. The present study reveals how mutations that disrupt the ability of an E3 ligase to interact with and ubiquitylate a critical cellular substrate such as WNK isoforms can trigger a chronic disease such as hypertension.

Hepcidin is a liver-derived antimicrobial peptide that regulates iron absorption and is also an integral part of the acute phase response. In a previous report, we found evidence that this peptide could also be induced by toxic heavy metals and xenobiotics, thus broadening its teleological role as a defensin. However it remained unclear how its sensing of disparate biotic and abiotic stressors might be integrated at the transcriptional level. We hypothesized that its function in cytoprotection may be regulated by NFE2-related factor 2 (Nrf2), the master transcriptional controller of cellular stress defenses. In this report, we show that hepcidin regulation is inextricably linked to the acute stress response through Nrf2 signaling. Nrf2 regulates hepcidin expression from a prototypical antioxidant response element in its promoter, and by synergizing with other basic leucine-zipper transcription factors. We also show that polyphenolic small molecules or phytoestrogens commonly found in fruits and vegetables including the red wine constituent resveratrol can induce hepcidin expression in vitro and post-prandially, with concomitant reductions in circulating iron levels and transferrin saturation by one such polyphenol quercetin. Furthermore, these molecules derepress hepcidin promoter activity when its transcription by Nrf2 is repressed by Keap1. Taken together, the data show that hepcidin is a prototypical antioxidant response or cytoprotective gene within the Nrf2 transcriptional circuitry. The ability of phytoestrogens to modulate hepcidin expression in vivo suggests a novel mechanism by which diet may impact iron homeostasis., Graphical abstract fx1, Highlights • Nrf2 regulates HAMP transcription. • Phytoestrogens differentially induce HAMP transcription by Nrf2. • Post-prandial hepcidin induction reduces serum iron and transferrin saturation. • Nrf2 knockout results in iron-overload in mouse embryonic fibroblasts.

Despite the fact that several natural products (e.g. crude extracts or purified compounds) have been found to activate cell antioxidant responses and/or delay cellular senescence the effect(s) of small peptides on cell viability and/or modulation of protective mechanisms (e.g. the proteostasis network) remain largely elusive. We have thus studied a hexapeptide (Hexapeptide-11) of structure Phe–Val–Ala–Pro–Phe–Pro (FVAPFP) originally isolated from yeast extracts and later synthesized by solid state synthesis to high purity. We show herein that Hexapeptide-11 exhibits no significant toxicity in normal human diploid lung or skin fibroblasts. Exposure of fibroblasts to Hexapeptide-11 promoted dose and time-dependent activation of proteasome, autophagy, chaperones and antioxidant responses related genes. Moreover, it promoted increased nuclear accumulation of Nrf2; higher expression levels of proteasomal protein subunits and increased proteasome peptidase activities. In line with these findings we noted that Hexapeptide-11 conferred significant protection in fibroblasts against oxidative-stress-mediated premature cellular senescence, while at in vivo skin deformation assays in human subjects it improved skin elasticity. Finally, Hexapeptide-11 was found to induce the activity of extracellular MMPs and it also suppressed cell migration. Our presented findings indicate that Hexapeptide-11 is a promising anti-ageing agent., Graphical abstract, Highlights • Hexapeptide-11 shows no toxicity and promotes dose and time-dependent activation of proteasome, autophagy, chaperones and antioxidant responses related genes. • Hexapeptide-11 enhances nuclear accumulation of Nrf2; induces higher expression levels of proteasomal protein subunits and increases proteasome peptidase activities. • Treatment of normal human fibroblasts with Hexapeptide-11 suppresses oxidative-stress-mediated premature cellular senescence. • Hexapeptide-11 activates extracellular matrix metalloproteinases and suppresses cell migration. • At in vivo skin deformation assays Hexapeptide-11 improves skin elasticity.