2. Lipid Oxidation and Protein Lipoxidation

Lipid oxidation can occur enzymatically, catalysed by cyclooxygenases (COX-1/2/3), lipoxygenases (LOX), and cytochrome P450-dependent enzymes (CYP450), or non-enzymatically, when it is mediated by carbon and oxygen-centred radicals [13,14]. Enzymatic pathways give rise to bioactive mediators, such as prostaglandins (PG), thromboxanes and leukotrienes, among others, which have been broadly studied and can act as physiological signalling molecules, with an important role as immunomodulators [15]. Non-enzymatic mechanisms are adventitious oxidations that commonly occur on phospholipids present in cellular membranes and on lipoproteins [12]. Lipids containing unsaturated fatty acyl chains, and particularly those that are polyunsaturated fatty acids (PUFAs), such as linoleic and arachidonic acids, are more vulnerable to attack by different reactive species of oxygen, nitrogen and halogens [16]. In lipid peroxidation, peroxyl radicals are formed as intermediary products [3]. Hence, in a phospholipid bilayer environment, a single radical attack can entail a chain reaction based on a cascade of radical hydrogen abstractions and oxidations. Peroxyl radicals, and to a lesser extent hydroperoxides, are unstable and undergo subsequent reactions including further oxidation, cleavage and cyclization reactions to form a variety of secondary oxidation products (reviewed by [17,18]).

Many of these secondary products, including full-chain length oxidized phospholipids, phospholipids with a truncated fatty acyl chain, and non-esterified breakdown products, are reactive electrophilic products that undergo further rearrangements. Both full-length and shortened fatty acyl chains can contain epoxides, hydroxides, and carboxylic acids as well as reactive carbonyl moieties and α,β-unsaturated alkenal moieties, which are highly reactive [3,19,20,21]. In general, alkenals, and hydroxy- or oxo-alkenals are the most reactive and versatile in terms of their reactivity [17,22,23,24]. Certain reactive lipid products can be formed by enzymatic and/or non-enzymatic reactions. Dehydration of PG synthetised via COX enzymes or non-enzymatically through the isoprostane pathway leads to the generation of cyclopentenone prostaglandins (cyPG) or of keto-PG, which contain unsaturated carbonyl moieties in the cyclopentenone ring and/or in the lateral chains [25,26,27,28]. Lipids can also be attacked by reactive nitrogen species (RNS) to give rise to nitro-alkenals that also can covalently modify proteins [29].

Protein lipoxidation involves the formation of Schiff’s bases or Michael adducts. Schiff’s bases are formed by the reaction between carbonyls (aldehydes or ketones) and primary amines, and consequently can only form on lysine or amino-terminal residues in proteins. In contrast, Michael adducts are formed by reaction of a nucleophile with the β-carbon of an α,β-alkenal, and reactivity is enhanced by the presence of an electron-withdrawing group on the γ carbon, such as in 4-hydroxynonenal (HNE) or 4-oxononenal (ONE). In proteins, the nucleophilic group is most commonly cysteine (in the thiolate form), lysine (primary amine in deprotonated form) or histidine (secondary amine in deprotonated form). Adducts with asparagine and glutamine side chains have been reported for certain lipid species despite the lower nucleophilicity of the amino group in an amide [30]. Arginine can form Michael adducts only when deprotonated, which is rare in physiological conditions as the guanidino group is highly basic. This different reactivity of protein residues and lipid species influences the selectivity of protein lipoxidation as will be discussed below. A summary of the mechanisms is given in Figure 1, while detailed reaction mechanisms for the formation of these adducts and their subsequent rearrangements can be found in other reviews [19,31]. Some of the most studied and interesting electrophilic lipids involved in protein lipoxidation are considered briefly below and in Table 1.

Figure 1. Formation of Schiff’s base and Michael adducts with protein residues. The structures of the lysine, cysteine and histidine residues are shown at the top, with the moieties involved in nucleophilic attack indicated. The histidine imidazole ring exists in 2 resonance forms where the hydrogen can reside on either nitrogen, so either nitrogen can undertake nucleophilic attack. Schiff’s base formation with an amino group is shown in the centre. The Schiff’s base reactions are reversible, involving a hydrolysis reaction. The bottom panel shows Michael adduct formation by nucleophilic group X, where X represents a primary or secondary amine or a thiol. Michael adducts can also decompose by reversal of these reactions, although they are more stable than Schiff’s bases.

Reactive lipid products can be grouped into chemical families according to their reactive chemical groups, which determine their reactivity in lipoxidation reactions. Owing in part to their availability, as well as their biological actions, some reactive lipid products have been much more extensively studied than others. The small, non-esterified aldehydes malondialdehyde (MDA), acrolein, and HNE fall into this category [23,32]. Of these, HNE is the most toxic, acrolein is the most reactive, and MDA is the most mutagenic [33,34,35], reviewed in [10,22,36]; these effects ultimately relate to their potential to cause lipoxidation. In contrast, there are many fewer publications on other aldehydes such as crotonaldehyde, pentanal, hexenal, 4-hydroxy-hexenal (HHE) and 4-hydroxy-dodecadienal, although some of them may be formed physiologically in sufficient amounts to have biological effects and evidence is emerging that they also modify proteins and affect their functions. Substantial research has also been devoted to long-chain species, especially isoprostanes, isolevuglandins, PG species such as cyPG, and nitrated fatty acids (NO₂-FAs), in part due to their signalling properties [37,38,39,40]. Whereas isoprostanes are important as biomarkers of oxidative stress [41], the behaviour of certain eicosanoids including cyPG, and of NO₂-FAs as transcription factor agonists and mediators of inflammatory resolution has raised high interest in their potential therapeutic applications. Moreover, cyPG have been used as model compounds for the identification of lipoxidation targets in proteomic studies [27]. Interest in oxidized and nitrated phospholipids as potential agents of lipoxidation is more recent but nevertheless of emerging physiological importance. In summary, the propensity of a lipoxidation adduct to be formed depends on the reactivity of the lipid oxidation product, the nucleophilicity of the target amino acid in the protein, and the stability of the product generated [42]. Furthermore, the initial adducts can undergo additional rearrangements, including reactions with other nucleophilic groups to cause inter- or intra-molecular cross-links, resulting in linear or cyclic stable products [19,43]. Thus, protein lipoxidation contributes to the generation of protein diversity through PTMs, with a variety of structural and functional consequences.

Oxidation of lipid components of membranes occurs in all living organisms. Therefore, the process of protein lipoxidation would be expected to be universal. Indeed, although much work has focused on mammalian and clinical samples, protein lipoxidation has also been studied in plants and microorganisms. For example, immunoblot analysis using monoclonal antibodies against reactive aldehyde-derived protein modifications showed that in spinach leaves grown in normal conditions the oxygen-evolving complex protein 33 was modified by MDA, acrolein and crotonaldehyde [44], while salt stress in Arabidopsis thaliana resulted in modification of soluble proteins by HNE, HHE, crotonaldehyde, acrolein and MDA [45]. Excellent reviews that highlight the importance of this PTM in plants are available [46,47]. In contrast, little information on natural lipoxidation and its effects are available for fungi and bacteria, although it has been reported that bactericidal antibiotic treatments lead to the formation of MDA adducts [48]. The chemistry and generic consequences of lipoxidation on protein function are expected to be similar in all of these organisms and will depend on the context and the protein target. However, most of the examples provided in this review will be related to animal models in general and human health in particular, in relation to the pathophysiological consequences of this modification.