Protein lipoxidation: Detection strategies and challenges

Abstract

Enzymatic and non-enzymatic lipid metabolism can give rise to reactive species that may covalently modify cellular or plasma proteins through a process known as lipoxidation. Under basal conditions, protein lipoxidation can contribute to normal cell homeostasis and participate in signaling or adaptive mechanisms, as exemplified by lipoxidation of Ras proteins or of the cytoskeletal protein vimentin, both of which behave as sensors of electrophilic species. Nevertheless, increased lipoxidation under pathological conditions may lead to deleterious effects on protein structure or aggregation. This can result in impaired degradation and accumulation of abnormally folded proteins contributing to pathophysiology, as may occur in neurodegenerative diseases. Identification of the protein targets of lipoxidation and its functional consequences under pathophysiological situations can unveil the modification patterns associated with the various outcomes, as well as preventive strategies or potential therapeutic targets. Given the wide structural variability of lipid moieties involved in lipoxidation, highly sensitive and specific methods for its detection are required. Derivatization of reactive carbonyl species is instrumental in the detection of adducts retaining carbonyl groups. In addition, use of tagged derivatives of electrophilic lipids enables enrichment of lipoxidized proteins or peptides. Ultimate confirmation of lipoxidation requires high resolution mass spectrometry approaches to unequivocally identify the adduct and the targeted residue. Moreover, rigorous validation of the targets identified and assessment of the functional consequences of these modifications are essential. Here we present an update on methods to approach the complex field of lipoxidation along with validation strategies and functional assays illustrated with well-studied lipoxidation targets.

Increased hepatocellular protein carbonylation in human end-stage alcoholic cirrhosis

“…Aldehyde modification of vimentin on Cys 328 has previously been demonstrated in THP-1 cells treated with 4-HNE which when combined with our in vivo data validates its susceptibility to reactive species 68 . As important, regulation of vimentin on Cys 328 by reactive aldehydes has recently been described as a mechanism to regulate vimentin self-association and regulation of cellular organelles 69,70 .…”

Albumin Cys34 adducted by acrolein as a marker of oxidative stress in ischemia-reperfusion injury during hepatectomy

“…Several MS-based approaches to identify RCSs in free or adducted forms have been reported [33,34]. We have previously found that free plasma RCSs rapidly disappear (by almost 70% within 5 min) during reperfusion, since they form covalent adducts with plasma proteins [22].…”

Adduct Formation and Context Factors in Drug Hypersensitivity: Insight from Proteomic Studies

“…In some of these studies, endogenous lipid electrophiles, typically generated under inflammatory conditions, have been used as models due to their broad reactivity towards nucleophiles and the availability of detection methods [13,42]. Also, coexistence of inflammatory conditions may potentiate the toxic effects of drugs or reduce the threshold at which they can induce adverse effects.…”

“…We have recently reviewed the methodology and challenges of proteomic analysis of modifications by various inflammatory mediators, which in many cases may be applicable to drug-protein interactions [42,44]. Nonetheless, combinations of approaches will be needed to fully characterize the protein modifications generated in pathophysiological conditions under therapeutic regimes.…”