Exploring the biology of lipid peroxidation-derived protein carbonylation

Abstract

The sustained overproduction of reactive oxygen and nitrogen species results in an imbalance of cellular prooxidant-antioxidant systems and is implicated in numerous disease states, including alcoholic liver disease, cancer, neurological disorders, inflammation, and cardiovascular disease. The accumulation of reactive aldehydes resulting from sustained oxidative stress and lipid peroxidation is an underlying factor in the development of these pathologies. Determining the biochemical factors that elicit cellular responses resulting from protein carbonylation remains a key element to developing therapeutic approaches and ameliorating disease pathologies. This review details our current understanding of the generation of reactive aldehydes via lipid peroxidation resulting in protein carbonylation, focusing on pathophysiologic factors associated with 4-hydroxynonenal-protein modification. Additionally, an overview of in vitro and in vivo model systems used to study the physiologic impact of protein carbonylation is presented. Finally, an update of the methods commonly used in characterizing protein modification by reactive aldehydes provides an overview of isolation techniques, mass spectrometry, and computational biology. It is apparent that research in this area employing state-of-the-art proteomics, mass spectrometry, and computational biology is rapidly evolving, yielding foundational knowledge concerning the molecular mechanisms of protein carbonylation and its relation to a spectrum of diseases associated with oxidative stress.

Figure 1

Lipid peroxidation of PUFAs results in the generation of biogenic aldehydes.

Figure 2. Strategies for isolating oxidative posttranslational protein modifications in vitro and in situ

(A) Biotin hydrazide involves the formation of a reducible hydrazone bond with the carbonyl carbon of Michael-type protein adducts. This method provides specificity for protein carbonyls via avidin-biotin binding, eliminating non-specific binding which may occur with immunoprecipitation techniques. (B) Girard’s P reagent provides an example for modifying existing hydrazide chemistry to enhance specificity and selectivity. The presence of a positively charged quaternary amine in addition to the functional hydrazide allows for additional separation utilizing neutral pH strong cation exchange chromatography. (C) Click Chemistry is a recently developed strategy for isolating protein carbonyls. This method is applied to determine protein targets of reactive aldehyde species. Synthesized alkynyl-4-HNE is utilized to achieve protein carbonylation, which is then reacted with an azido-biotin reagent to form a triazole-linked product. Avidin isolation and subsequent proteomic analysis allows for identification of a broad spectrum of protein targets of 4-HNE.