Modification of Proteins by Isoketal-containing Oxidized Phospholipids

Abstract

Oxidative stress frequently leads to altered function of membrane proteins. Isoketals are highly reactive products of the isoprostane pathway of free radical-induced lipid peroxidation that rapidly form covalent protein adducts and exhibit a remarkable proclivity to form protein cross links in vitro. Examination of isoketal adducts from an animal model of oxidative injury revealed that initial adducts were formed by isoketals esterified in phospholipids, representing a novel oxidative injuryassociated modification of proteins by phospholipids. Maturation of adducts involved cleavage from phospholipids and conversion of adducts to a more stable chemical form that can be detected for extended periods. Because initial adducts were formed by phospholipidesterified isoketals, the functional consequence of isoketal adduction was examined using a model membrane protein (a cardiac K ؉ channel). These studies revealed that isoketal adduction profoundly altered protein function, inhibiting potassium current in a dosedependent manner. These findings indicate that phospholipid-esterified isoketals rapidly adduct membrane proteins and that such modification can alter protein function, suggesting a generalized cellular mechanism for alteration of membrane function as a consequence of oxidative stress.Oxidative stress from the generation of free radicals has been implicated in the pathogenesis of a wide variety of human diseases including atherosclerosis, cancer, and neurodegenerative diseases (1-3). Fatty acids esterified in membrane phospholipids are a major target of attack by free radicals (4 -6). We have described previously the formation of a series of highly electrophilic ␥-ketoaldehyde isomers in vitro, which we term isoketals (IsoKs) 1 (7), by rearrangement of endoperoxide intermediates in the isoprostane pathway of free radical-induced peroxidation of phospholipid-esterified arachidonic acid (8). Incorporation of oxygen at different sites of arachidonyl radical formation results in the formation of 8 structural isomers, each of which is composed of 4 racemic diastereoisomers (see Fig. 1A). Analogous molecules, termed neuroketals, are formed from the free radical-catalyzed oxidation of docosahexanoic acid and form protein adducts analogous to those described below for IsoKs (9).IsoKs adduct covalently with the ⑀-amino group of lysine residues in vitro within seconds, which is orders of magnitude faster than has been reported for other products of lipid peroxidation, including 4-hydroxynonenal (7). This remarkable reactivity has precluded the detection of free IsoKs in vivo because of their sequestration as adducts. Therefore, we elucidated the nature of IsoK protein adducts in vitro as an initial step toward identifying the formation of IsoK adducts in vivo (7, 10). IsoKs initially form reversible Schiff base adducts which irreversibly cyclize to pyrroles. The pyrroles then undergo facile autoxidation to stable lactam and hydroxylactam adducts (see Fig. 1B) (7, 10). Consistent with this process, Schiff base adducts f...

Chemical modification of proteins during peroxidation of phospholipids

“…Oxidation of PUFAs in lipoproteins is known to yield a complex array of products, both protein-bound and free in solution, including isoprostane-derived levuglandin (27,28) and isoketal (29,30) derivatives and numerous keto and hydroxy fatty acids (11). A range of smaller, more polar aldehydes, ketoaldehydes, alkenals, and hydroxyalkenals are also released into solution and react with protein to form Schiff bases, Michael adducts, and pyrrole and pyridinium compounds that contribute to the brown color, fluorescence, and cross-linking of oxidized lipoproteins.…”

Oxidative Mediated Lipid Peroxidation Recapitulates Proarrhythmic Effects on Cardiac Sodium Channels

“…Levels of total IsoK-lysyl-lactam adduct were then measured as previously described. 17,21 In brief, after treatment with 0.2N sodium hydroxide for 2 hours to hydrolyze esterified isoketals, protein was initially subjected to complete proteolytic digestion. After digestion, 500 pg of a ( 13 C 6 )-IsoK-lysyl-lactam internal standard was added to each sample, and IsoK-lysyl-lactam adducts were then quantified after solid phase extraction and an initial preparative HPLC by selective reaction monitoring LC electrospray tandem mass spectrometry for the transition from m/z 479 to 84 and m/z 485 to 89 (internal standard).…”

“…16 The hypothesis that alteration in cardiac ion channel function by free radical-mediated lipid peroxidation is potentially caused by the formation of IsoKs is supported by our recent finding that IsoKs formed in membrane phospholipids adduct to proteins while still esterified, suggesting that membrane proteins are a preferred target for adduction by IsoKs. 17 Moreover, these studies demonstrated that IsoKs dramatically inhibited the function of the delayed rectifier potassium current I Kr in cultured atrial tumor cell line 1 (AT-1) myocytes.…”

Pyridoxamine Analogues Scavenge Lipid-Derived γ-Ketoaldehydes and Protect against H2O2-Mediated Cytotoxicity

“…Levels of γKA adducts significantly increase in pathological conditions including atherosclerosis, end-stage renal disease, and Alzheimer's Disease (5,6). Increased γKA adduct formation has also been characterized in experimental models of oxidative injury and inflammation, including carbon tetrachloride treated rats (7), hyperoxia treated mice (8), septic mice (9), and ex vivo activation of platelets (10). Levels of γKA adducted proteins are expected to be elevated in a wide variety of conditions previously linked to oxidative injury and inflammation (11)(12)(13)(14)(15)(16)(17)(18)(19)(20)(21)(22)(23).…”

“…PM is quite hydrophilic, and we have previously found that IsoK formation initially occurs in situ on membrane phospholipids (7). Therefore, modification of PM in a way that retained its high reactivity but increases its lipophilicity could enhance its effectiveness as a scavenger.…”