In addition to lipidation by isoprenoid or palmitate moieties, proteins may suffer modification by reactive lipid species of varied structure. Cell metabolism under both basal and pathological conditions gives rise to products derived from lipid peroxidation that can form stable adducts with particular protein residues (Bogatcheva et al., 2005). Oxidative and nitrosative stress produce many of these products, which play a role in numerous pathologies such as neurodegenerative diseases (Dianzani, 2003), vascular alterations (Lee and Park, 2013), and cancer (Garzón et al., 2011). Amongst the many electrophilic lipids that are produced in cells, cyclopentenone prostaglandins (cyPG) are a family of reactive lipid species with anti-inflammatory, anti-viral and anti-tumoral effects, which makes them a forthcoming option as exogenous therapeutic compounds (Sánchez-Gómez et al., 2004; Straus and Glass, 2001). cyPG generation is increased in cells undergoing oxidative stress, whereas cyPG production in homeostatic cells is low (Ceaser et al., 2004; Koenitzer and Freeman, 2010). The electrophilic nature of cyPG
structural changes within the whole protein that provide new reactivity and electrophilicity. cyPG of different structural characteristics present specificity towards particular cysteine residues, so that different cyPG enter distinctive intramolecular binding pockets even within the same protein. Moreover, cyPG bind to proteins involved in a multitude of different cellular tasks (Garzón et al., 2011; Renedo et al., 2007). These features account for dual actions: cyPG modification of proteins involved in redox regulation can modulate oxidative stress pathways, whereas binding and subsequent inhibition of NF-κB (nuclear factor kappa-light-chain-enhancer of activated B cells) pathway proteins contributes to cyPG anti-inflammatory effects (Díez-Dacal and Pérez-Sala, 2010; Kim and Surh, 2006). The pleiotropic behavior of these electrophilic lipids is therefore a reflection of the variety of intracellular targets, many of which have been discovered in the past decade through proteomic studies, as reviewed in (Garzón et al., 2011; Oeste and Pérez-Sala, 2014). Interestingly, Ras proteins are included in this expanding list of cyPG targets (Oeste et al., 2011; Stamatakis and Pérez- Sala, 2006), as discussed below in further detail.
As shown in Figure 6, cyPG are derived from peroxidation of membrane fatty acids, in particular arachidonic acid, through the action of cyclooxygenases (COX) or non-enzymatically through the isoprostane pathway (Funk, 2001; Gao et al., 2003). In the case of the COX pathway, prostaglandin synthases act on the precursor PGH2 to give
rise to PGD2 and PGE2 (Funk, 2001). The latter two PG can also be formed by non-
enzymatic epimerization of isoprostanes through the isoprostane pathway (Gao et al., 2003), which gives rise to trans isomers of PG, i.e. isoprostanes such as iso-PGJ2 and iso-
PGA2. J series cyPG arise from dehydration of PGD2 to PGJ2, which can be further
dehydrated to form 15d-PGJ2 (15-deoxy-Δ12,14-Prostaglandin J2) or give rise to Δ12-PGJ2
through an albumin-dependent mechanism (Narumiya and Fukushima, 1985; Shibata et al., 2002). cyPG of the A series are produced upon dehydration of PGE1 or PGE2
(Ohno et al., 1986).
cyPG synthesized in this manner contain an α, β-unsaturated carbonyl group within their cyclopentane ring, which renders highly electrophilic -β carbons. Therefore, they are amenable to forming Michael adducts through nucleophilic attack by thiol moieties from cysteines, as seen at the bottom of Figure 6. The cysteine residues to which cyPG bind present structural characteristics allowing for the entry and
positioning of specific cyPG to form the stable, covalent Michael adducts with consequences on biological functions.
Figure 6. Cyclopentenone prostaglandin formation and covalent binding to proteins through Michael addition.
cyPG (blue shaded boxes) are formed by spontaneous dehydration of their PG precursors (gray shaded boxes). These precursors are generated in turn from unsaturated fatty acids such as arachidonic acid by the action of cyclooxygenases (COX) or through non-enzymatic processes, i.e. the isoprostane pathway (dashed arrows). Arachidonic acid is formed as a product of phospholipase A2 or C cleavage of membrane-bound phospholipids. cyPG contain electrophilic carbons (asterisks) that can undergo attack by nucleophilic groups such as cysteine thiolates and form Michael adducts (green arrows), as shown at the bottom for 15d-PGJ. See text for details.
2.2.1 Cyclopentenone prostaglandin modification of Ras proteins
cyPG containing either one or two electrophilic carbons, i.e. single enones or dienones, can bind to different subsets of proteins depending on the steric attributes of the cysteines in the protein to be modified. Several studies have shown that the dienone cyPG 15d-PGJ2 can establish Michael adducts with cysteine residues of proteins as
structurally and functionally different as actin, vimentin (Stamatakis et al., 2006), NF- κB, PPARγ (Peroxisome proliferator-activated receptor gamma) (Cernuda-Morollón et al., 2001; Shiraki et al., 2005), Keap1 or GSTP1-1 (Glutathione S-transferase P) (Levonen et al., 2004; Sánchez-Gómez et al., 2007). The fact that electrophilic carbon count is particularly important for selectivity is reflected by cyPG binding to Ras proteins. Various cyPG have been found to modify and activate Ras proteins in an isoform- and site-selective manner (Oliva et al., 2003). Specifically, single enone cyPG, e.g. PGA1 or PGJ2 bind preferentially to C118, which is present in the GTP binding site
in all three Ras proteins, namely H-Ras, K-Ras and N-Ras. However, the dienone 15d- PGJ2 can bind two cysteine residues simultaneously (C181 and C184), located at the H-
Ras C-terminus (Renedo et al., 2007). Since other Ras proteins such as N- or K-Ras do not contain this sequence, they are not preferential targets for dienone cyPG binding. Interestingly, modification of the H-Ras C-terminal sequence by cyPG or other small reactive molecules elicits changes in its membrane partitioning and distribution along the endocytic pathway (Oeste et al., 2011). It is thus apparent that cyPG selectivity can fine-tune intracellular pathways by modifying not only specific proteins, but also particular cysteine residues within the same protein, which can have consequences on its subcellular sorting and localization.