Antioxidants and HNE in redox homeostasis

Author’s Accepted Manuscript Antioxidants and HNE in redox homeostasis Wojciech Łuczaj, Agnieszka Gęgotek, Elżbieta Skrzydlewska

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To appear in: Free Radical Biology and Medicine Received date: 30 September 2016 Revised date: 16 November 2016 Accepted date: 17 November 2016 Cite this article as: Wojciech Łuczaj, Agnieszka Gęgotek and Elżbieta Skrzydlewska, Antioxidants and HNE in redox homeostasis, Free Radical Biology and Medicine, http://dx.doi.org/10.1016/j.freeradbiomed.2016.11.033 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Antioxidants and HNE in redox homeostasis. Wojciech Łuczaj, Agnieszka Gęgotek, Elżbieta Skrzydlewska* Department of Analytical Chemistry, Medical University of Bialystok, Mickiewicza 2d, 15-222 Bialystok, Poland *

Corresponding author: Department of Analytical Chemistry, Medical University of Bialystok, Mickiewicza 2D, 15-222 Bialystok, Poland. Tel/Fax: +48 857485707. [email protected]

Abstract Under physiological conditions, cells are in a stable state known as redox homeostasis, which is maintained by the balance between continuous ROS/RNS generation and several mechanisms involved in antioxidant activity. ROS overproduction results in alterations in the redox homeostasis that promote oxidative damage to major components of the cell, including the biomembrane phospholipids. Lipid peroxidation subsequently generates a diverse set of products, including -unsaturated aldehydes. Of these products, 4-hydroxy-2-nonenal (HNE) is the most studied aldehyde on the basis of its involvement in cellular physiology and pathology. This review summarizes the current knowledge in the field of HNE generation, metabolism, and detoxification, as well as its interactions with various cellular macromolecules (protein, phospholipid, and nucleic acid). The formation of HNE-protein adducts enables HNE to participate in multi-step regulation of cellular metabolic pathways that include signaling and transcription of antioxidant enzymes, pro-inflammatory factors, and anti-apoptotic proteins. The most widely described roles for HNE in the signaling pathways are associated with its activation of kinases, as well as transcription factors that are responsible for redox homeostasis (Ref-1, Nrf2, p53, NFκB, and Hsf1). Depending on its level, HNE exerts harmful or protective effects associated with the induction of antioxidant defense mechanisms. These effects make HNE a key player in maintaining redox homeostasis, as well as producing imbalances in this system that participate in aging and the development of pathological conditions.

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Abbreviations: 15-LOX, 15-lipoxygenase; 4–HNE, 4-hydroxynonenal; AKRs, aldo-keto reductases; ALDHs, aldehyde dehydrogenases; AP-1, activator protein 1; ARE, antioxidant responsive element; Bcl-2, B-cell lymphoma 2; COX-2, cyclooxygenase-2; DHN, 1,4-dihydroxy-2-nonene; DAG, diacylglicerol; ELISA, enzymelinked immunosorbent assay; ERK, extracellular signal-regulated kinase; Gadd45, Growth arrest and DNAdamage-inducible protein; GCL, glutamate cysteine ligase; GSH-Px, glutathione peroxidase; GSSG, glutathione disulphide; GSTs, glutathione S-transferases; HIF-1, hypoxia-inducible factor 1; HPETE, 11/15 hydroperoxyeicosatetraenoate; HPODE, 9/13 hydroperoxyoctadecadienoate; Hsf1, Heat shock factor protein 1; IKK, IκB kinase; IL, interleukin; IP3, inositol trisphosphate; IRES, internal ribosomal entry sites; JNK, Jun Nterminal kinase; Keap1, Kelch-like ECH-associated protein 1; L4CL, tetralinoleoylcardiolipin; LOOH, lipid hydroperoxides; MAPKs, mitogen-activated protein kinases; Mdm2, Mouse double minute 2 homolog; MRP, multidrug resistance proteins; NADPH, reduced nicotinamide adenine dinucleotide phosphate; NFκB, nuclear factor kappa-light-chain-enhancer of activated B cells; Nrf2, nuclear factor (erythroid-derived 2)-like 2; PAQR3, Progestin And AdipoQ Receptor Family Member 3; PE, phosphatidylethanolamines; PKC, protein kinase C; PLA2, phospholipase A2; PLC, phospholipase C; PPARs, peroxisome proliferator-activated receptors; PS, phosphatidylserines; PUFAs, polyunsaturated fatty acids; Ref-1, Redox effector factor-1; RLIP76, Ralinteracting GTPase-activating protein; RNS reactive nitrogen species; ROS, reactive oxygen species; SOD, superoxide dismutase; TNFα, tumor necrosis factor-α; Trx, thioredoxin; TrxR, thioredoxin reductase;

Keywords: 4-hydroxy-2-nonenal; Antioxidants; Lipid peroxidation; Redox homeostasis, Transcription factors.

Introduction A basic feature of cell physiology, specifically respiration and cell signaling, is the generation of reactive oxygen and nitrogen species (ROS/RNS). Cells are in a stable state when the rate of ROS/RNS generation is balanced by the scavenging capacity of various antioxidant compounds. These compounds, which are present at relatively low levels, are able to compete with other oxidizable substrates and thus significantly delay or inhibit the oxidation of the other substrates. On the other hand, the antioxidant defense must permit the ROS-mediated roles in cell signaling and redox regulation that subsequently lead to increased expression of 2

the genes for various antioxidant proteins [1]. The human antioxidant defense includes endogenous antioxidant enzymes including superoxide dismutase, catalase, and glutathione peroxidase, as well as other proteins and low molecular-weight compounds such as glutathione, uric acid, coenzyme Q, and lipoic acid. Moreover, the classical antioxidant system is supported by additional defense systems that remove or repair damaged biomolecules before their accumulation can result in alterations in the cellular metabolism. The interactions between the ROS/RNS and these antioxidants in the redox homeostatic balance produce metabolic responses to endogenous as well as exogenous signals. These signals modulate the appropriate induction of adaptation processes or alternatively, the activation of cell death mechanisms. Therefore, cellular redox homeostasis plays a key role in physiological as well as most pathophysiological processes. Elevated ROS levels that cannot be counteracted by the cellular antioxidant abilities induce redox imbalance that results in uncontrolled oxidative stress. This situation causes oxidative modifications in the structure and function of cellular components and consequently leads to cell, tissue and organ injury. Thus, redox imbalance plays a significant role in development of numerous disease conditions, which demonstrates the biological relevance of redox regulation. Lipid peroxidation Among the cellular components, the most sensitive to oxidative modifications are the membrane phospholipids. Specifically, these lipids exhibit elevated susceptibility to oxidation by various endogenous ROS/RNS as well as exogenous inducers such as some transition metal ions, high energy irradiation and xenobiotics that are capable of initiating lipid peroxidation in the biomembranes. Lipid peroxidation encompasses various mechanisms that can be classified as free radical-mediated oxidation, free radical-independent non-enzymatic oxidation and enzymatic oxidation [2]. The main pathway resulted from oxidative stress is free radical peroxidation proceeding by chain reactions in the biomembranes. These reactions are initiated by various types of active species and are involved in development of pathologies. The solubility of molecular oxygen and therefore the ROS/RNS generation in the biomembranes is high. Thus, the membrane phospholipids, which containing high levels of polyunsaturated

fatty

acids

(PUFAs)

including

arachidonic,

linoleic,

linolenic,

docosahexaenoic, and eicosapentaenoic acids, are extremely sensitive to attack by ROS/RNS and other electrophiles [3]. Moreover, after reacting with the free radicals, the PUFAs themselves become reactive free radicals and are capable of propagating lipid peroxidation chain reactions [4]. 3

The most powerful initiators of lipid peroxidation are the hydroxyl and hydroperoxyl radicals. Hydroxyl radicals are generated from hydrogen peroxide during cellular oxygen metabolism via the Fenton and Haber–Weiss reactions, in the presence of free iron or copper ions, and from peroxynitrite or as a result of high energy irradiation [5]. Superoxide and nitric oxide, which are generated by the activities of NADPH oxidase, xanthine oxidase, and nitric oxide synthase, are not able to initiate lipid peroxidation, but they react to give peroxynitrite, which may initiate chain reactions [3]. However, the protonated form of superoxide, the hydroperoxyl radical, is a sufficiently strong oxidant that it can initiate oxidation of PUFAs and causes the transformation of hydrogen peroxide to the hydroxyl radical [6]. HNE generation As a result of the ROS/RNS reactions with PUFAs, hydrogen is abstracted from the alpha carbon with the insertion of oxygen, which results in the generation of lipid peroxyl radicals that are subsequently oxidized to lipid hydroperoxides (Fig. 1). As the result of LOOH (lipid hydroperoxides) decomposition, diverse products are generated. Among these, the -unsaturated aldehydes deserve special attention, particularly 4-hydroxy-2-nonenal (4hydroxy-2,3-trans-nonenal, HNE), which is a major product of the decomposition of hydroperoxides of -6 fatty acids such as linoleic and arachidonic [7]. The first mechanism that was proposed for the formation of HNE involved the generation of hydroperoxyperoxides (9/13 hydroperoxyoctadecadienoate (HPODE) and 11/15 hydroperoxyeicosatetraenoate (HPETE)) as derivatives of linoleic and arachidonic acid [8, 9]. HNE was obtained from these intermediates as a result of Hock cleavage (in the presence of Fe2+) in the mechanism proposed by Pryor and as -scission in another mechanism [10, 11]. Two alternative nonenzymatic mechanisms of HNE generation were reported later. One of these involves cyclization of peroxyl radicals to form a dioxetane that can rearrange resulting in chain cleavage, and the second, similar to that for hydroperoxide decomposition, involves Hock rearrangement and Hock cleavage [12, 13]. It should also be noted that HPODE and HPETE were considered to be intermediates that commonly resulted from free radical mechanisms in which hydrogen was abstracted at bis-allylic sites and molecular oxygen was added by an enzymatic process that involved the 15-lipoxygenases (15-LOX). In this process, 13-HPODE is produced by the oxidation of linoleic acid by 15-LOX-1, whereas 15-HPETE is generated during oxidation of arachidonic acid by 15-LOX-2 [13, 14]. Thus, HNE may be an end-product of free radical-mediated as well as enzymatic oxidation of the -6 PUFAs. Moreover, cyclooxygenase-2 (COX-2) was also suggested to be involved in the HNE 4

formation triggered by Escherichia faecalis, a human intestinal commensal [15]. More recently, a novel mechanism of HNE formation, specific for linoleic acid, was demonstrated to occur, which represents an alternative for the bis-allylic hydrogen abstraction. This mechanism involves oxidation of a mitochondrial cardiolipin, tetralinoleoylcardiolipin (L4CL). On the basis of its four linoleic acid chains, this molecule is very prone to free radical oxidation and results in cross-chain peroxyl radical addition and decomposition [16]. The level of HNE generated during lipid peroxidation primarily depends on the availability of endogenous PUFAs as well as exogenously provided with the food. This statement refers especially to -6 PUFAs, which have been shown to be major precursors of HNE [7]. It makes the content of -6 PUFAs versus -3 PUFAs in the food is crucial in this matter. Therefore the ratio of -6/-3 fatty acids, increased in Western diet during the past few decades up to 17:1, is very important factor pivotal for oxidative stress intensification. Thus delivering of -6 PUFAs with the diet results in an increase of HNE level what leads to development of many diseases including rheumatoid arthritis, inflammatory bowel disease, nonalcoholic fatty liver disease, obesity, cardiovascular diseases, neurodegenerative diseases and cancer [17]. Increased content of -3 PUFA in the diet is strictly associated with prevention, thus the decrease of the ratio of -6/-3 fatty acids significantly reduces the risk of chronic diseases. However, recent studies showed possible dual implications of PUFAs intake. It has been shown that ketogenic diet high in linoleic and arachidonic acids enhanced oxidative stress with significant increase of 4-HNE-modified proteins level [18]. Another study indicated for upregulation of ω-1 hydroxylation of 4-hydroxynonenoate leading to decrease of the HNE level as consequence of ketogenic diet [19]. The correlation between high content of -6 PUFAs in diet and enhanced oxidative stress and HNE pathological action remains unquestionable.

Antioxidants versus lipid peroxidation Lipid peroxidation is regulated by antioxidants including proteins and low molecular compounds that participate in biomembrane protection. The most relevant anti-peroxidative antioxidant is GSH-Px, which catalyzes the continuous reduction of lipid peroxides at the expense of reduced glutathione. This reaction depends on the presence of selenium. Most isoforms of glutathione peroxidase contain a selenocysteine residue and have a Lys-92 residue at their active sites. The lysine can easily form a Schiff base with HNE that is reversible in an aqueous environment [20]. Formation of this adducts leads to inhibition of the glutathione 5

peroxidase activity, which has been observed in aging and during oxidative stress in neurons [21]. Moreover, an increased HNE level is accompanied by a decrease in the glutathione peroxidase activity in various pathologies such as cancer, hypertension, and Lyme disease [22-24]. However, an elevated activity of this antioxidant is observed in skin cells after UV irradiation [25]. GSH-Px activity also depends on the level of its co-substrate, GSH, which is oxidized in stress conditions and can react with HNE [26]. The oxidized form of GSH, glutathione disulphide (GSSG), can be reduced by glutathione reductase using NADPH as an electron donor. However, HNE decreases the glutathione reductase activity [27]. The reaction of GSH with HNE, which is catalyzed by the glutathione S-transferases (GSTs), leads to the formation of GS-HNE adducts that cause decreases in the cellular GSH level and GSH-Px activity [28]. This situation promotes lipid peroxidation and the production of signalling molecules [29-31]. HNE regulates the GSH level by stimulating γ-glutamyl transpeptidase through the MAPK signaling pathways and enhancing the expression of the glutathione S-transferases [32]. HNE not only acts as a substrate for the GSTs but may also cause oxidative modification of this enzyme by covalent binding, which has been observed in Alzheimer’s disease [33]. Therefore, HNE may be considered to be an important mediator of oxidation-induced impairment of the GST-mediated detoxification system. Another antioxidant enzyme, catalase, is mainly responsible for the decomposition of hydrogen peroxide but also exhibits other peroxidase activities. This enzyme is also sensitive to HNE. Under stress conditions, HNE forms adducts with the membrane-associated catalase, what leads to its inactivation. However, catalase is protected against the formation of adducts with HNE by presence of lysine or cysteine in cytosol, that prevents the HNEmediated apoptosis [34]. At low levels, HNE inactivates catalase via autoamplificatory mechanisms based on singlet oxygen formation as has been observed in tumor cells [34, 35]. HNE also interferes with the thioredoxin system, which consists of thioredoxin (Trx) and thioredoxin reductase (TrxR) and is the major NADPH-dependent system responsible for the formation of reduced disulfide bonds in cell. Trx is a selenium-dependent enzyme with a broad substrate specificity that includes hydroperoxides. HNE can inhibit the activity of TrxR by binding covalently to its cysteine and selenocysteine residues (Cys-496 and Sec-497) [36]. The second component of the thioredoxin system is Trx, which contains two vicinal cysteine residues in a CxxC motif. These are the main residues that are exposed to HNE adduction. These residues are responsible for the ability of Trx to reduce other proteins by cysteine thioldisulfide exchange, and HNE bound to Trx inhibits its reduction abilities and precludes the 6

interaction between Trx and thioredoxin reductase [37]. Moreover, the HNE-Trx adducts decrease the mitochondrial potential and disrupt ATP synthesis [38]. The disordered energy metabolism caused by the formation of HNE-Trx adducts also leads to decrease in the cell viability through the activation of caspase-3 [39]. Furthermore, these adducts influence the levels of reduced thiol groups and lead to direct activation of various transcription factors including NFκB or indirect activation through the Ref-1/Hsp70 pathway [40, 41]. Independent of the enzymatic antioxidant defense, both exogenous and endogenous non-enzymatic antioxidants including -tocopherol, ascorbic acid and GSH protect the cells as well as the organism by inhibiting the propagation of lipid peroxidation at the early stage of the free radical attack. [42]. -Tocopherol is a lipophilic antioxidant that acts as a ”chainbreaking” antioxidant that can terminate the propagation steps of lipid peroxidation. This was confirmed by the fact that the use of -tocopherol-coated filters for hemodialysis caused a decrease in the urine HNE levels in uraemic subjects [43]. The interactions among those low molecular antioxidants allow for their continuous regeneration and the protection of the biomembrane. It is believed that dietary supplementation with α-tocopherol, ascorbic acid and several natural polyphenols not only prevent but may also act therapeutically on many chronic diseases associated with oxidative stress, especially cancer, in which increased level of HNE protein modification was observed [44-46]. The most abundant group of plant polyphenols are flavonoids, which are divided into number subclasses, primarily flavanols, known also as catechins (e.g. EC, EGC, EGC and EGCG), flavonols (e.g. quercetin) and anthocyanins [47]. Due to the presence of hydroxyl groups and hydrophobic core in chemical structure of flavonoids, they are partially water-soluble compounds which are able to penetrate the phospholipid bilayer. Therefore, among natural antioxidants, these compounds are the most studied in respective to their antioxidative properties particularly towards phospholipid. Their antioxidant activity is based on different mechanisms including scavenging of free radicals, chelating of transition metal ions or inhibition of activity of enzymes participating in free radicals generation [48]. It has been shown that catechins contained in green or black tea, as well as administrated alone prevent oxidative stress, exhibit anti-aging activity and protect phospholipid against oxidation during alcohol intoxication thus prevent HNE generation [49,50]. However, anthocyanins present in berry fruits, inhibit enzymatic and ROS dependent lipid peroxidation thus act as anti-inflammatory agents [51]. Additionally, they prevent increase in HNE level in rat tissues resulted from oxidative stress induced by ethanol intoxication [52]. Moreover anthocyanins through reducing of oxidative conditions prevent

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protein modifications and accumulation of advanced glycation end products and consequently the neurodegeneration [53]. Another polyphenol – resveratrol inhibits the oxidation of the LDL via chelating copper ions or by direct scavenging of the free radicals and in this way the risk of cardiovascular diseases is decreased [54]. In recent years many examinations were concerned antioxidant activity of flavonol quercetin increased cells resistance to 4-HNE-mediated pro-inflammatory mediators production [55] and prevented coronary heart disease through inhibition of metalloproteinases expression [56]. These evidences indicated on decreased HNE generation and suggested that therapeutic action of quercetin is due to the NFκB, and Nrf2/ARE pathways modulation [57]. Independently from the number of data confirming therapeutic effects of natural antioxidants against lipid peroxidation further research studies are still needed to establish them as therapeutic target. Additionally, some antioxidants on one side exert therapeutic action in respect to oxidative damages prevention, but on the other hand, they cause negative outcomes during clinical trials. For this reason therapeutic potential of those antioxidants is strictly limited [48]. There are several studies indicating the pro-oxidant effects of natural antioxidants related to their dose [58], e.g. green tea polyphenols at high concentration reveal pro-oxidant effects in cancer prevention and therapies [59]. On the other hand, the number of careful dose-response studies and the amount of data confirming that these pro-oxidant effects can occur in vivo are limited [60]. HNE: reactivity and metabolism HNE is an α,β-unsaturated hydroxyalkenal and is therefore considered to be one of the most reactive electrophilic end-products of lipid peroxidation. It may represent more than 90% of all generated aldehydes [61]. The HNE molecule is highly reactive due to the following functional groups: a carbonyl group on C1, a double bond (alkene) between carbon C2 and C3, and a hydroxyl group on C4 (Fig. 2). Carbons C1 and C3 are electrophilic sites whereas carbon C1 is also a redox center. These sites make the HNE molecule highly reactive toward nucleophilic groups particularly thiol and amino groups. This reactivity is associated with the Michael addition of thiol or amino compounds to carbon C3 as well as the formation of Schiff bases between a primary amino group and the carbonyl group of carbon C1. Because the Schiff base formation is rather slow, reversible Michael adduct generation prevails. Therefore, HNE can react with macromolecules such as lipids that contain an amino group, nucleic acids (mostly with the guanosine moiety of DNA), and with proteins, particularly those that contain cysteine, histidine and lysine residues [62]. It is very important in this 8

context that, in contrast to most ROS, HNE is a highly diffusible molecule and can spread beyond initial generation sites and can act as stress signaling molecule, e.g., it suppresses both basal and inducible NFκB activity, activates the caspase pathways that lead to cell death, and impairs the functions of glucose and glutamate in brain cells [63]. Therefore, uncontrolled excessive production of HNE could interfere with normal cellular signaling and lead to the development of pathological conditions [64]. However, the effects of HNE reactivity are dependent on its level. The physiological concentrations of this aldehyde appear to be within the range of 0.1–1 μM [65]. HNE at these levels can modify signal transduction by inducing the synthesis of detoxifying enzymes or stimulating the function of other enzymes, e.g., kinases, and thus exerts physiologically beneficial effects that promote cell survival and proliferation [66-67]. At concentrations between 10 and 60 μM, HNE demonstrates genotoxic activity that leads to sister chromatid exchange and DNA fragmentation [68], whereas at concentrations higher than 100 μM, HNE is extremely toxic and inhibits DNA and protein synthesis, glycolytic enzymes, and mitochondrial respiration [69]. HNE can accumulate at concentrations up to 5 mM in response to an oxidative insult. At this concentration, it impacts a wide range of biological activities, including the selective suppression or intensification [70]. HNE metabolism In mammals, HNE is rapidly metabolized as a cell defense against cellular modifications. The half-life of HNE is short: less than 2 min [71]. The key enzymes responsible for HNE metabolism in the cell are glutathione-S-transferases (GSTs), aldehyde dehydrogenases (ALDHs) and aldo-keto reductases (AKRs) [72]. The involvement of these enzymes in HNE detoxification depends on its level [71]. At physiological HNE levels, the main reaction is the adduction to the thiol group of GSH, which results in the formation of glutathionyl-HNE adducts (GS-HNE) that prevent the HNE from reacting with cellular macromolecules [71]. The mechanism of the formation of the GS-HNE adducts involves the reaction of the HNE C2-C3 double bond with the nucleophilic thiol group. This reaction is catalyzed by GSTs, especially the GSTA4–4 and GST5–8 isoforms, which are characterized by high catalytic activities [73]. It was shown that GSTA4-4 plays a major role in protecting the cells from the toxic effects of HNE [74]. GSHNE adducts are not inactive compounds but potential signaling molecules. These adducts are responsible for a mitogenic effect in rat aortic smooth muscle cells and an inflammatory effect in adipocytes [75]. To protect the cells from the HNE toxicity, the GS-HNE adducts are transported out of cells through an active ATP-dependent, primary efflux mechanism in which 9

the Ral-interacting GTPase-activating protein (RLIP76) acts as a transporter [76]. Moreover, the process of GS-HNE adducts transport also involves the multidrug resistance proteins (MRP1 and MRP2), which may interact with the alpha class of GSTs [77]. Because GSH is responsible for maintaining the proper level of reduced thiol groups in cells, the HNE-GSH interactions lead to disturbances in cellular redox homeostasis that cause the activation of redox-sensitive transcription factors [78, 79]. On the other hand, in rapidly proliferating cells, a higher level of GS-HNE adducts arrests the cell cycle before it enters the G2 phase [80], but does not affect their viability [81]. In addition to the consumption of GSH during HNE detoxification, this aldehyde can in turn act as a signaling molecule that induces the expression of the glutamate cysteine ligase (GCL) genes that limit the rate of de novo GSH biosynthesis through the Nrf2/ARE signaling pathway, which is active only during stress [82]. At the stress level of HNE, it may be metabolized by aldose reductases (AKRs) and aldehyde dehydrogenases (ALDHs) [83]. Oxidation of the HNE carbonyl group by ALDHs results in the formation of 4-hydroxynonenoic acid (HNA). Among the various isoenzymes of ALDHs, ALDH1A1 is the major HNE-detoxification enzyme, while ALDH5A and ALDH2 play important roles in HNE-mediated cell death [84]. Moreover, ALDH5A has been demonstrated to be the major HNE-metabolizing enzyme in brain cells [85]. It has been shown that the overexpression of ALDH reduces the HNE toxicity whereas a decreased level of this enzyme increases the toxicity. Furthermore, HNE-protein adduct accumulation has been observed in Parkinson’s disease [86]. Another route for the metabolic detoxification of HNE involves the reduction of the C2-C3 double bond by NAD(P)H-dependent alkenal/one oxidoreductase (AO, an enzyme also known as leukotriene B4 12-hydroxydehydrogenase, 15oxoprostaglandin 13-reductase, and dithiolethione-inducible gene-1), which results in the formation of 1,4-dihydroxy-2-nonene (DHN) [87]. HNA and DHN may be further metabolized in the mitochondria through -oxidation by cytochrome P450 to form 9-hydroxyHNA, and this mechanism predominates at a moderate stress level [71]. Moreover, the aldehyde group of HNE can be oxidized and reduced by separate isoenzymes of cytochrome P450 [88, 89]. The CYP4A family can catalyze the oxidation of HNA and thus participates in the metabolism of HNE [47]. Extracellular HNE may be neutralized by the formation of HNE-cysteine adducts. An increased level of these adducts has been observed in colon cells in which the Adenomatous polyposis coli gene was mutated [90]. Moreover, HNE undergoes catabolic degradation in the liver that involves the phosphorylation and ω-oxidation of the hydroxyl group [91, 92].

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Nevertheless, when the level of HNE is high and the metabolizing systems cannot accomplish its elimination, this aldehyde reacts with the nucleophilic groups of cellular macromolecules including phospholipids, nucleic acids and proteins. Phospholipid modifications One of the main cellular targets for HNE are phospholipids. HNE forms Michael adducts and Schiff bases with phospholipids containing amino groups, such as phosphatidylethanolamines (PE) and phosphatidylserines (PS) with the consequence of altering membrane phospholipid asymmetry and fluidity [93]. However, the formation of HNE adducts with PE predominates over that with PS, the latter of which is a significantly less abundant class of phospholipids [94]. HNE-PE Michael adducts are present in the blood and urine of aged human subjects and diabetics [95]. Moreover, HNE creates covalent adducts with plasmalogens (a subclass of PE), which results in alterations in their antioxidant properties. However, the HNE adducts with PE were shown not to be hydrolyzed by phospholipase A2 and phospholipase D [96]. Therefore, PE adducts with HNE could be considered to be species that can potentially regulate the cell signaling associated with the phospholipases. The biological significance of phospholipid adducts with HNE is still poorly clarified compared to the DNA and protein adducts. Nucleic acids modifications An increased level of HNE may lead to disturbances in protein biosynthesis through direct interactions with their DNA coding sequences and/or modifications of their structure. HNE can react with all four DNA bases to form adducts, but the efficiency for the bases differs: G > C > A > T [97]. The most common adduct is HNE-dG. These are bulky exocyclic adducts that are found in the nuclear and mitochondrial DNA of animal and human tissues [98]. The HNE bond to DNA involves a Michael addition of the N2-amino group of dG to the HNE C2-C3 double bond followed by ring closure of N1 onto the aldehyde, which results in four diastereomeric 1,N2-HNE-dG adducts [99]. The products obtained from these reactions are propano-DNA adducts. However, in the presence of peroxides, HNE is converted to the epoxynonanal, which reacts with the amino groups of guanosine, adenosine and cytidine, and after cyclization, forms the etheno-DNA adducts 1,N2-etheno-2'-deoxyguanosine (εdG) 1,N6etheno-2’-eoxyadenosine (𝜀dA), and 3,N4-etheno-2’-deoxycytidine (𝜀dC). In double-stranded DNA, the reactions with the aldehyde group can be reversed because the etheno-adducts are repaired by the base-excision repair (BER) pathway [100]. Elevated etheno-DNA adduct levels have been found in the injured tissues of subjects with chronic pancreatitis, ulcerative colitis, and Crohn’s disease, which indicates that these species are potential markers of 11

genetic damage that results from the oxidation of the omega-6-PUFAs [101, 102]. These covalent modifications of DNA were therefore postulated to be partially responsible for the biological roles of HNE. However, the HNE-DNA adduct formation inhibits the DNA synthesis on the undamaged template, and the adducts may be fragile sites that are susceptible to the acquisition of mutations due to the easier induction of lesions in these DNA sequences and/or substitution of replicative DNA polymerases with DNA polymerases of lower fidelity, which incorporate non-cognate nucleotides [103]. Moreover, the HNE-DNA adducts induce many irreversible mutations during replication. These mutations are mainly G:C to T:A transversions, with lower levels of G:C to A:T transitions [104]. The HNE-DNA adducts are preferentially formed at -GAGGC/A-sequences in the codon 249 of transcription factor p53 gene, which causes gene mutations and affects diverse biological processes including cell cycle arrest, apoptosis, DNA repair, and differentiation [105]. In mammalian cells, the genotoxicity of HNE depends upon the glutathione levels, which modulate the formation of HNE-DNA adducts. However, the glutathione level is decreased by reaction with HNE [102]. In recent years, increasing amounts of data regarding the HNE-DNA adduct profiling and the importance of these adducts for living organisms have been provided by DNA adductomics studies [106]. Since HNE is not very reactive with DNA, the levels of HNE-DNA adducts are relatively low (only one HNE-dG per 109 bases) [107]. However, HNE may be bound to many proteins that are involved in DNA repair, and this may have detrimental effects on the cellular DNA repair capacity and contribute to the cytotoxicity and carcinogenicity of HNE [108]. The HNE-dG adducts are mainly repaired by the nucleotide excision repair pathway [109]. A study on human colon cell extracts revealed that the HNE-dG adducts are repaired by this mechanism much more quickly than some other adducts, e.g., with acrolein [108]. Irrespective of the formation of adducts, HNE also participates in the remodelling of chromatin that changes the DNA-binding activity of transcription factors by exposing the binding sequence in the promoter regions as well as the interactions of DNA with the histones [110]. All histones contain significant amounts of lysine and arginine residues (30 – 40%), and the physiological acetylation and methylation of the histone lysine residues play major roles in the formation of the chromatin structure and thereby regulate the transcription processes [111]. HNE causes alterations in the histidine, lysine and cysteine residues of histone-H2A and affects its conformation, alters its hydrophobicity, and disrupts its globular tertiary structure. These modifications also make the histones less positively charged, which changes the electrostatic association of histones to DNA [112]. HNE-histone adducts alter the 12

DNA-histone interactions, which may contribute to the vulnerability of DNA to oxidation. Furthermore, the HNE-histones adducts may affect transcription by preventing DNA association with the histone, and the acetylated histones are more susceptible to HNE modifications [113]. In turn, HNE enhances the level of histone acetylation, which uncoils the helical structure of the DNA and exposes the binding sequences, e.g., at the NFκB promoter regions, thus promoting the NFκB–DNA binding activity. However, the mechanism by which HNE influences the histone acetylation is not completely understood [110]. HNE may also affect the interaction between the histones and DNA at the level of posttranscriptional modifications of the histones. HNE forms adduct with the lysine residues of the Nmethyltransferase that is responsible for the addition of methyl groups to either the lysine or arginine residues of the histones. Methylation of histones is generally correlated with transcriptional silencing. Therefore, attenuation of the methyltransferase activity by HNE will simultaneously increase the likelihood of transcription factors attaching to the DNA. Overexpression of cytoprotective or antioxidant proteins is a characteristic of cancer progression, and changes in the histone methylation status were found even in the next generation in the context of leukemia and lymphoma [114]. Protein modifications HNE is much more reactive to proteins than to DNA, and 8% of the HNE generated in the cell is bound to proteins [71]. Modifications of protein amino acids by HNE occur primarily on cysteine and lysine and, to a lesser extent, histidine. These modifications lead to formation of stable covalent Michael adducts, but the HNE carbonyl group may alternatively react with the amino groups to form Schiff bases [70, 115]. However Michael adducts represent more than 99% of the generated protein modifications [115]. Michael addition is due to the C2-C3 double bond in the HNE structure, which is highly susceptible to nucleophilic attack, and the addition of thiol or amino groups at the C3 carbon. HNE creates Michael adducts by reacting with the amino group of lysine or histidine and also with the thiol group of cysteine followed by cyclization and hemiacetal or hemithioacetal formation. The HNE-lysine Michael adducts are reversible [116]. Cysteine is characterized by highest reactivity toward HNE, but these adducts are less stable than the HNE-histidine adducts, which have been reported to be the most stable [116, 117]. The second type of HNE-protein interactions is that Schiff bases are formed by the reaction of the HNE aldehyde group with the free amino group of a lysine residue. Moreover, the Schiff bases are generally regarded as more readily reversible than the Michael adducts [118]. The

13

relative amino acid susceptibility for modifications by HNE is the following: Cys ≫ His > Lys [119]. HNE modifies a large number of proteins including human serum albumin, in which nine nucleophilic sites are modified [120]. In this protein, the most reactive site is Cys34, which is modified by HNE via a Michael addition, whereas Lys199 is primarily modified through the Schiff base formation [121]. Two other good HNE targets are LDL and actin [122, 123]. HNE creates adducts with the Lys residues of the apoB protein in LDL, whereas the active site of actin (Cys374) is modified by HNE via Michael addition. Moreover, HNE affects alcohol dehydrogenase by modifying two cysteine residues [124]. It should be emphasized that the formation of HNE-protein adducts depends on the HNE level. HNE, at low levels, reversibly inhibits ALDH2 via the formation of adducts with cysteine residue in the active site of this enzyme, whereas HNE at a concentration of 10 M irreversibly inhibits the ALDH2 activity [125]. Degradation of modified proteins To maintain the cellular integrity, the removal of oxidized proteins or protein fragments by proteolytic systems and the delivery of the resulting amino acids into cellular metabolism are very important processes. The proteolytic systems are therefore considered to be secondary antioxidant defense systems that can prevent or delay the accumulation of altered proteins [126]. However, modified proteins that lack their functional properties, particularly those that have become irreversibly cross-linked, are resistant to proteolysis, leading to their accumulation and can lead to a pool of useless cellular debris. For these reasons, redox and proteotoxic stresses as well as metabolic imbalance have been suggested to be associated with various diseases and aging [127, 128]. Due its ability to form adducts with proteins, HNE, similarly like ROS, is an important factor that leads to activation of proteolytic pathways that can promote either cell survival or cell death. The choice between these alternatives depends on the severity of the stress incurred [129]. A large body of evidence has demonstrated that damaged proteins may be effectively degraded and recycled by the proteasomal system [130, 131]. It has been shown that the ubiquitin/proteasome system plays a crucial role in the in vivo metabolism of HNE-modified proteins [132]. HNE-modified proteins are significantly ubiquitinated, but the proteasomal degradation of oxidatively modified proteins, which principally involves the 20S proteasome, requires protein unfolding; thus, only mildly oxidized proteins are suitable as proteasome substrates. Moreover, the subunits of the 20S proteasome are also targeted by HNE. The modified proteasome therefore has impaired hydrolytic activities, and proteasomal 14

degradation is not efficient [133]. Additionally, a high level of HNE cross-links the peptide chain which results in protein aggregation that leads to proteasome inhibition [134]. The other mechanism by which defective and aggregated long-lived proteins can be degraded to preserve homeostasis and viability as well as to produce energy is autophagy [135]. ROS, by inducing oxidative stress and HNE generation as well as mitochondrial damage, have been demonstrated to be the main activators of autophagy [136]. In general, a low level of HNE induces autophagy, which promotes cell survival, whereas excessive stress overwhelms the beneficial potential of autophagy and leads to cell death. Autophagy induction may be associated with a mutation in the murine superoxide dismutase (SOD1) gene, which modulates autophagy by inhibiting mTOR and the accumulation of the lipid adducts LC3 [137, 138]. Moreover, the SOD1 mutant interacts directly with p62 (SQSTM1), an LC3 binding partner that is known to target protein aggregates for autophagic degradation [139]. It has been suggested that autophagy is important not only for redox homeostasis but also for mitochondrial quality control, cell proliferation and survival [140]. Because the mitochondria are both a source and a target of oxidative stress, autophagy plays an important role in removing damaged forms of these organelles and their components [135]. HNE was shown to form adducts with respiratory chain subunits and to increase the ROS generation, therefore changes in autophagic activity can modulate the effects of HNE on cellular bioenergetics and consequently have an impact on cell death [136]. HNE changes glucosedependent autophagy [by increased LC3-II formation] and apoptosis [by caspase 3 cleavage] in retinal pigment epithelium cells, vascular smooth muscle cells and neuroblastoma cells in a dose-dependent manner [141].

HNE and cellular signaling Under physiological conditions, the formation of HNE-protein adducts allows HNE to participate in multi-step regulation of metabolic and detoxifying enzymes, signaling molecules or transcription factors that ensure the proper functioning of cellular metabolic pathways. However, the most widely described effect of HNE on the signaling pathways involves the activation of kinases as well as the transcription factors that are responsible for redox homeostasis. MAP kinases One of the most important types of enzymes that participate in signaling processes are mitogen-activated protein kinases (MAPKs). These enzymes are responsible for cellular 15

signal transduction in response to a diverse set of stimulators including oxidative stress and products of ROS action [142]. The activation of MAPKs requires phosphorylation by other kinases, most commonly mitogen-activated protein kinases, which in turn is also dependent on the activity of another kinases, MAPKK kinases. MAPKs cause the phosphorylation of serine, threonine and tyrosine residues of proteins. Kinases from the MAPK family regulate diverse cell functions including proliferation, differentiation, gene expression, cell survival, inflammatory responses and apoptosis. Therefore, activation of the MAPKs including the extracellular signal-regulated kinase (ERK), p38, and Jun N-terminal kinase (JNK) under various conditions can affect cytoprotective or apoptotic signaling. It has been shown that HNE at nontoxic levels forms adducts with ERK, JNK and p38, which activate these MAP kinases. This causes induction of HO-1 in keratinocytes, whereas inhibition of the HNE generation in corneal epithelial cells suppresses the HO-1 expression by inactivating p38 and ERK. This process reduces the natural cellular resistance to oxidative stress [143]. On the other side, pathologically increased HNE levels lead to ERK, JNK, and p38 phosphorylation and strong activation that may initiate the apoptotic processes by increasing the activation of caspases -3, -8, and -9 as was observed in mouse fibroblasts [74]. Moreover, increase in the level of HNE promotes its interaction with JNK. This leads to the nuclear translocation of this kinase, which may stimulate autophagy [144]. ERK activation through phosphorylation, dimerization, and nuclear translocation results in the phosphorylation of transcription factors. However, an enhanced HNE level leads to the formation of adducts with ERK at the Cys-63 and His-230 residues. This prevents the ERK dimerization and consequently, its activation [145]. The inhibition of ERK phosphorylation results in a loss of signal transduction and transcriptional activity, which affects the homeostasis, proliferation and cell survival [146]. HNE at enhanced level activates ERK and p38 in a cultured microglial cell line and leads to upregulation and phosphorylation of cytosolic phospholipase A2 (PLA2), which mainly hydrolyzes phospholipids into fatty acids and signaling molecules, as well as to neuroprotective effects against ethanol-induced oxidative stress [147, 148]. Moreover, the increased formation of HNE-protein adducts is accompanied by an increase in the PLA2 activity in rat brains subjected to ethanol-induced stress [149]. However, in the plasma of patients with tick-borne encephalitis, the enhanced HNE levels are accompanied by a decrease in the PLA2 activity, what further enhances lipid peroxidation in the pathological condition [150]. Moreover, under inflammatory conditions, the increased HNE level in liver epithelial RL34 cells activates p38, which leads to upregulation of COX-2 and the initiation of the prostaglandin-dependent signaling pathway [151]. 16

Protein kinase C (PKC) The various forms of protein kinase C play crucial roles in the transduction of cellular signals that control cell proliferation, survival, and transformation by phosphorylating various targets [152]. Many PKC isoforms are lipid-sensitive and Ca2+-dependent enzymes. They are usually activated by growth factors through the stimulation of phospholipase C (PLC), which generates inositol trisphosphate (IP3) and DAG [152]. HNE stimulates PKC indirectly by inducing the activity of phospholipase C, whereas direct HNE adducts of PKC may affect the activity of its subunits [153, 154]. The selectivity of the HNE interactions for the activity of the various PKC isoforms depends on its level: low levels of HNE enhance the activity of PKC𝛽I, 𝛽II and 𝛿, while the same HNE levels inhibit PKC𝛼 activity. At higher levels, HNE inhibits these enzymes [155]. It has been suggested that HNE-dependent enhancement of the PKC activity that is observed in mouse macrophage cells and in the blood and kidneys of diabetic rats activates NADPH oxidase and inhibits ROS generation [156, 157]. Moreover, the HNE-induced PKC 𝛿 activation prevents triglyceride accumulation in obese mice [157, 158]. Transcription factors Proteins play a major role in maintaining redox homeostasis. For this reason, modifications of their structure as well as the regulation of their gene expression by transcription factors are significant in the cellular responses to oxidative conditions. HNE participates at both levels of protein regulation. Transcription factors control the rate of the transcription of genetic information from DNA to mRNA by binding to highly specific DNA sequences. In eukaryotic organisms, transcription factors are mostly present in the cytoplasm as inactive forms that may be activated through ligand binding, phosphorylation, or interaction with other transcription factors [159]. The active transcription factors translocate to the nucleus where they bind to DNA and initiate gene transcription. The main transcription factors responsible for redox homeostasis are: Ref-1, Nrf2, p53, NFκB, and Hsf1 [142]. There is compelling evidence that the activity of these transcription factors can be directly or indirectly affected by electrophilic aldehydes, particularly HNE (Fig. 3). Ref-1 The particularly important transcription factor Redox effector factor-1 (Ref-1) participates in the maintenance of cellular redox homeostasis. This factor is a dual function protein that regulates the activity of other transcription factors as well as mediating the repair of DNA damages. The transcriptional regulatory function of Ref-1 targets transcription factors such as Nrf2, p53, NFκB, HIF-1 or AP-1 [160]. The redox activity of Ref-1 is 17

associated with the reduction of specific cysteine residues in the DNA-binding domains of p53, NFκB, or AP-1 that enable their binding to their target sequences on DNA [161]. Ref-1 is localized mainly in the cytoplasm but can be translocated to the nucleus under oxidative stress conditions. Nuclear export of Ref-1 is initiated by nitric oxide through S-nitrosylation of the redox-sensitive Cys-93 residue [162]. Ref-1 might also be upregulated to protect cells from oxidative damage to DNA by genotoxic agents such as ROS [163]. Despite the redox regulation of the Ref-1 activity and the nucleophilic character of its active site, there is no clear evidence that electrophilic HNE directly affects the activity of Ref-1. However, the Ref-1-dependent transcription factors described above may be activated by HNE. Nrf2 One of the major mechanisms by which cells are protected from oxidative stress is the transcriptional regulation of cytoprotective genes by Nrf2, whose expression is observed in all types of cells [164]. However, the Nrf2/ARE transcription efficacy may be affected by HNE in several independent ways (Fig. 4). Under physiological conditions, the Nrf2-encoding genes are constitutively expressed such that the Nrf2 molecules are continuously biosynthesized. Moreover, the mRNA that encodes Nrf2 contains IRES sequences (internal ribosomal entry sites). These sequences selectively recruit it into polysomes, which enhance the translation of the Nrf2 protein. Additionally, IRESNrf2 is a redox-sensitive molecule; therefore Nrf2 mRNA translation to protein is activated by an increased level of HNE, thus further increasing the Nrf2 biosynthesis [165]. The Nrf2 level in the cytoplasm is regulated by the formation of the Nrf2-Keap1-Cul3 complex. This permits simultaneous ubiquitination of the Nrf2 molecule by Cul3 and degradation by the 26S proteasome. Nrf2 ubiqitination and degradation are also regulated by PAQR3, a protein that tethers the Nrf2-Keap1 complex [166]. HNE binding to Keap1 modifies its conformation and prevents the formation of the Keap1-Nrf2 complex, which increases the concentration of the free Nrf2 in cytoplasm [167]. Because the free Nrf2 molecule cannot be ubiquitinated and degraded, an enhanced Nrf2 level in the cytoplasm has been suggested to be a factor that promotes cancer cell invasion [168]. A redox imbalance as well as an elevated level of HNE in a cell promotes the oxidation of cysteine residues in the Keap1 molecule [169]. HNE directly reacts with critical cysteine residues in Keap1 (mainly Cys-273 and Cys-288), what causes changes in the Keap1 conformation. Simultaneously, by inducing Keap1 hypermethylation, HNE may affect the 18

conformation of Keap1, which results in the dissociation of Nrf2 from the complex [41]. Moreover, by binding free glutathione, HNE promotes cytoplasmic oxidative conditions that favor oxidative changes in the Keap1 molecule [170]. It has also been suggested that HNEinduced Nrf2 activation may be associated with Hsf-1 activity because proteotoxic stress activates Hsf-1 and causes mitochondrial dysfunction, which results in excessive generation of ROS, and in turn activates Nrf2 [171]. Free Nrf2 is translocated to the nucleus, where it forms a complex with a small Maf protein and then is bound to the DNA at a characteristic sequence, 5'-TGACnnnGCA-3' [ARE], and subsequently initiates the transcription of genes [172]. Moreover, the rate of Nrf2 translocation to the nucleus is dependent on the site of its phosphorylation [173]. Through its interactions with the MAP kinases, HNE leads to Nrf2 phosphorylation at threonine and serine residues, which accelerates the Nrf2 translocation to nucleus [174]. Moreover, by removing from the nuclear protein Bach1, which is a competitor for the binding of Nrf2 to the DNA site, HNE supports the activity of Nrf2 [175]. Nrf2 is also directly activated by Ref-1mediated reduction of the cysteine in its DNA-binding domain as well as indirectly by repression of Ref-1, which potently activates Nrf2 and its downstream select targets such as HMOX-1 [176]. The Nrf2 cytoprotective activity mainly involves antioxidant enzymes such as γglutamylcysteine ligase, glutathione S-transferase, glutathione reductase, thioredoxin reductase, catalase, superoxide dismutase, quinone reductase NAD(P)H, and heme oxygenase-1, as well as some non-enzymatic antioxidant proteins, e.g., thioredoxin and ferritin [164]. The Nrf2 nuclear accumulation under oxidative stress conditions is attenuated in older people. For this reason, Nrf2-dependent cytoprotective gene expression is suppressed in response to aging [177]. Furthermore, HNE, at low level, cause a Nrf2-mediated upregulation of UCP3 expression, which leads to an uncoupling of oxidative phosphorylation. This process may protect against oxidative stress [178]. HNE may also induce Nrf2 binding to the Tfam promoter, what causes an increase in mitochondrial multiplication. This, in turn, results in increased ROS generation and lipid peroxidation [179]. The Nrf2 activity may also be controlled by microRNAs that bind to the Nrf2 mRNA to downregulate its translation [180]. Its target protein e.g. GCL downregulation by microRNA miR-142-5p binding to Nrf2 mRNA leads to reduction of GSH level [181]. Moreover, the binding of microRNAs to the Nrf2-specific mRNA targets decreases the levels of the corresponding protein [182]. HNE can regulate the expression of the miRNAs and their targets although the precise mechanism of these interactions is still under investigation [183]. 19

However, miR-200a targets the Keap1 mRNA in breast cancer, which leads to enhanced Nrf2 activation [184]. High levels of HNE enhance the expression of miR-200a, which leads to an increase in the Nrf2 activity in the kidneys of diabetic mice [185]. MicroRNAs are also involved in regulation of Nrf2 degradation via the 26S proteasome. The expression of some components of the proteasome is dependent on miR-122 expression [186]. On the other hand, microRNAs let-7b, let-7c, and miR-155 decrease the expression of Bach1, which competes with Nrf2 for binding to its target DNA site [187, 188]. It has been shown that high levels of HNE in mousemurine livers under steatohepatitis are also associated with enhanced miR-155 expression and decreased levels of Bach1 [189]. It was also shown that the enhanced miR-144 level is responsible for the post-transcriptional control of the Nrf2 expression and redox homeostasis in neuronal cells [190]. In this context, HNE is also suspected to upregulate the expression of miR-144 during colorectal cancer progression [191]. p53 The cellular response to a variety of stimuli such as oxidative stress, DNA damage, hypoxia, and oncogene expression is regulated by the tumor suppressor protein p53 [192]. HNE affects p53 expression through the formation of HNE-DNA adducts at codon 249 of the p53 gene [105]. This modification results in non-functional p53 biosynthesis. Under physiological conditions, p53 directly binds to DNA. It also interacts with several cellular proteins including Mdm2, SV40 T antigens, TATA-binding proteins, and Sp1, which transport it from the nucleus to the cytosol. The cytosolic form of p53 is ubiquitinated and marked for degradation by the 26S proteasome [193-196]. Through the activation of transcription factor Sp1, HNE induces p53 degradation and consequently its down-regulation [149]. However, during oxidative stress conditions, the activity of p53 is regulated by the reduction of a key cysteine residue by Ref-1 in response to excessive DNA damage, which can act as a 'switch' [192]. The p53 level is largely regulated by changes in the degradation of the protein [197]. Activation of p53 requires phosphorylation of its N-terminal transcriptional activation domain by protein kinases of the MAPK family or kinases that are implicated in the genome integrity checkpoint [198]. p53-induced activation of apoptotic pathway requires increases in the intracellular calcium, ROS and oncogene levels [199]. HNE activates the ERK and JNK members of MAP kinase family in a time-dependent manner. These kinases attenuate the HNE–induced p53 phosphorylation in cardiomyocytes [200]. Moreover, it has been suggested that HNE can induce proapoptotic activity of p53 because HNE–induced

20

apoptosis in cultured cardiomyocytes was inhibited by Pft, which is a specific p53 inhibitor [200]. High levels of HNE favour Michael adducts. In particular, HNE-p53 formation has been observed in the blood cells of Alzheimer's disease patients [201]. This adducts cause changes in the p53 conformation and increased nitration of its tyrosine residues [202]. The consequences of these changes are the loss of its pro-apoptotic activity, which favors the survival of damaged cells [201]. Moreover, the brains from p53 knockdown mice show reduced levels of endogenous HNE and HNE-protein adducts [203]. NFκB The control of DNA transcription, cytokine production and cell survival in cellular responses to stimulators such as ROS, cytokines, oxidized LDL, and bacterial or viral antigens is caused by NFκB, which is present in almost all animal cell types [204]. The transcriptional activity of NFκB leads to elevated expression of antioxidants such as Mn-SOD and ferritin heavy chain, as well as the antiapoptotic Bcl-2 family members, caspase inhibitors, TNFα, and Gadd45, which inhibit JNK-mediated cell death [205]. The NFκB components (RelA/p65, c-Rel, RelB, p50, and p52) are inactivated by IκB in the cytosol [206]. However, stimulators are capable of activating NFκB through the activation of IκB kinase (IKK), which phosphorylates IκB. This leads to the dissociation of NFκB from the inhibitor and phosphorylation of the NFκB subunits [207]. However, by binding to a specific residue of Src, HNE activates this non-receptor tyrosine kinase. As a consequence, Src phosphorylates and activates IKK [208]. HNE can also directly bind to IKK, what leads to a decrease in transcriptional activity of NFκB [209]. HNE, by binding to a specific residue of Src, activates IKK phosphorylation and activates NFκB. HNE-induced inflammation in the aged kidney contributes to age-related nephropathy [208]. NFκB may be directly phosphorylated by redox-sensitive kinases such as MEKK-1, Akt, or PKC [210]. Ref-1, through its redox chaperone activity is required to restore the ability of NFκB to bind DNA in the nucleus [211]. NFκB is a redox-sensitive transcription factor whose activity is changed by moderate levels of ROS [212]. Oxidation of the redox-sensitive site, the Cys-62 residue of the p50 subunit, inhibits its ability to bind DNA [213]. This modification is reversible, so DNA binding can be restored following reduction by thioredoxin [214]. NFκB can also be inactivated by redox modifications of its molecule including glutathionylation and Snitrosylation [215, 216]. However, HNE may inhibit the NFκB activity by interactions with Toll-like receptors (TLRs), which are responsible for the NFκB activation during microbial 21

infection. Under these conditions, even during an infection NFκB remains inactive [217]. Moreover, HNE also reacts with the neuropeptide SP, which-stimulates the translocation of NFκB to the nucleus. This process completely blocks the TNF-α and IL-6 expression in peritoneal mast cells [218] (Fig. 5). Hsf1 Hsf1 is responsible for the transcription of various genes such as the major inducible heat shock proteins including Hsp25. This protein accelerates the reduction of oxidized glutathione to its reduced form by activating glutathione reductase [219]. Moreover, Hsf1 activates Nrf2 through increased expression of p62, which binds to the Nrf2-Keap1 complex and causes the dissociation of Nrf2 [220]. Under physiological conditions, Hsf1 exists in the cytoplasm in a complex with Hsp40/Hsp70 and Hsp90. The nucleophilic character of the cysteine residues of the chaperones Hsp70 and Hsp90 is strongly influenced by surface exposure to electrophilic compounds [221]. Therefore, human Hsp90 and Hsp70 are sensitive to modifications by moderate levels of HNE (~10 μM), which cause the formation of Micheal adducts [222]. HNE was shown to covalently modify the Cys-572 residue of Hsp90. Modification of Hsp70 and Hsp90 is associated with the HNE-induced heat-shock response [223]. This is particularly important because Hsp90 and Hsf1 protect many proteins that control a wide variety of cellular functions. The situation in which the modification of Hsp90 causes liberation of proteins suggests a high potential for HNE and other electrophiles to influence cellular functions. Changes in the structure or conformation of Hsp40/Hsp70 or Hsp90 lead to Hsf1 dissociation from the chaperone complex, followed by the formation of Hsf1 trimers and translocation into the nucleus where hyperphosphorylated binds to DNA fragments that contain heat-shock elements (inverted nGAAn repeats) [224]. HNE activates Hsf1 and promotes Hsp70 expression in colon cancer but not in Hsf1-silenced cells, which are sensitive to HNE-induced apoptosis [225]. HNE also leads to an increased expression of the stressinducible Hsp70 gene in the human retinal pigment epithelial cells [67]. Moreover, HNE, by binding and inhibiting Hsp90, leads to caspase-3-independent apoptosis and reduced cellular viability [226].

HNE: from aging to disease

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The challenge of maintaining redox homeostasis requires cooperation between antioxidants and oxidative stress-induced factors. However, lipid peroxidation results in generation of signalling molecule such as HNE that is also involved in regulation of redox homeostasis. Moreover, HNE-modified macromolecules have been shown to affect various cellular functions and interactions, including the inhibition of DNA, RNA and protein synthesis, as well as causing mitochondrial dysfunction, protein aggregation, cell cycle arrest, and even cell death [227, 228]. Since HNE can cause such debilitating damages to cells and tissues, it plays major roles in aging and several age-related pathologies including neurodegenerative diseases, immune diseases and tumorigenesis [229]. HNE-protein adducts accumulate during aging in the cytosol and mitochondria of animal and human cells [230] as well as in serially passaged cells that are undergoing aging in vitro [230, 231]. HNE-protein adducts are also accumulated in human aortas in an agerelated manner [232]. Increased HNE levels contribute to NFκB activation and the stimulation of the expression of pro-inflammatory mediators such as IL-1, IL-6, or TNFα [208, 233]. The age-related increase in the HNE level also activates the peroxisome proliferator-activated receptor PPARβ/δ, which in turn induces neuronal apoptosis as well as age-related muscle dystrophy [234, 235]. In addition to the processes described above, HNE is also involved in the development of age-related pathologies such as neurodegenerative diseases and cancer transformation, in which the previously described alterations in signalling have been implicated. Increased levels of HNE-protein adducts have been found in Alzheimer’s and Parkinson’s diseases [86, 236]. HNE also plays an important role in cancer promotion by forming of DNA-HNE adducts, inhibiting DNA-repair mechanisms and inducing mutations [105]. On the other hand, by activating Nrf2-mediated gene expression and stimulating the biosynthesis of antioxidants, HNE provides cancer cells with protection against the oxidative burst of neutrophils as well as anticancer therapy [237]. In conclusion, level-dependent dual roles for cellular HNE have been observed. Despite the fact that in stress conditions HNE may create adducts with other molecules and lead to alterations in cellular metabolism, this molecule participates in multi-step regulation of metabolic pathways including effects on signaling molecules or transcription factors. HNE is a key player in the maintenance of redox homeostasis that allows proper functioning of the entire antioxidant system.

Conflicts of interest. 23

The authors declare that they have no potential conflicts of interest.

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Figure 1. An overview of HNE chemistry. HNE generation, metabolism and biological activity.

Figure 2. Reactivity of HNE; HNE interactions with proteins, phospholipids and DNA.

Figure 3. Schematic presentation of the HNE effect on genes transcription: the impact of HNE on the DNA structure and interactions with transcription factors involved in the regulation of redox homeostasis.

Figure 4. Schematic presentation of HNE effects on Nrf2/Keap1 pathway activation. (A) Under redox homeostasis, Nrf2 is constantly ubiquitinated through Keap1 and degraded in the proteasome. (B) However, in the case of redox imbalance, Nrf2 dissociates from the Nrf2Keap1 complex and is phosphorylated and translocated to the nucleus. This pathway may be affected by HNE at several independent levels: HNE decreases the inhibitory ability of Keap1, and accelerates Nrf2 phosphorylation and translocation to nucleus. Simultaneously, HNE removes Bach1, a competitor for Nrf2 binding to the DNA, from the nucleus. Moreover, Nrf2 biosynthesis is stimulated by HNE.

Figure 5. Schematic presentation of HNE effects on NFκB activation. (A) Under redox homeostasis, NFκB is inactivated by IκB in the cytosol. Activation of IκB kinase (IKK), which phosphorylates IκB, leads to dissociation of NFκB from the inhibitor and translocation to the nucleus. (B) In the case of redox imbalance, the enhanced HNE level inhibits the phosphorylation of IKK as well as its activity. Moreover, HNE interacts with the Toll-like receptor (TLR) and prevents NFκB activation. HNE also interacts with neuropeptide SP that stimulates NFκB translocation to nucleus. 43

Highlights 

HNE is a key player in maintaining redox homeostasis.



HNE-protein adducts participate in regulation of cellular metabolic pathways.



HNE activates transcription factors (Ref-1, Nrf2, p53, NFκB, and Hsf1).



Depending on its level, HNE exerts harmful or protective effects.

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