Methods for imaging and detecting modification of proteins by reactive lipid species

Abstract

Products of lipid peroxidation are generated in a wide range of pathologies characterized by those associated with oxidative stress and inflammation. Many oxidized lipids contain reactive functional groups that can modify proteins, change their structure and function, and affect cell signaling. However, intracellular localization and protein adducts of reactive lipids have been difficult to detect and rely largely on antibodies raised against specific lipid-protein adducts. As an alternative approach for monitoring oxidized lipids in cultured cells, we have tagged the lipid peroxidation substrate arachidonic acid, and an electrophilic lipid, 15-deoxy-Δ 12,14 -prostaglandin-J 2 (15d-PGJ 2 ) with either biotin or the fluorophore BODIPY. Tagged arachidonic acid can be used in combination with conditions of oxidant stress or inflammation to assess the subcellular localization and protein modification by oxidized lipids generated in situ. Furthermore, we show that reactive lipid oxidation products such as 15d-PGJ 2 can also be labeled and used in fluorescent and Western blotting applications. This article describes the synthesis, purification, and selected application of these tagged lipids in vitro.

Hemin causes mitochondrial dysfunction in endothelial cells through promoting lipid peroxidation: the protective role of autophagy

“…We have previously used biotinylated lipids to monitor protein adduct formation with reactive lipid species in cells (14,25,61). To determine whether hemin induces the formation of reactive lipid species we used a biotin-tagged derivative of arachidonic acid (Bt-AA).…”

“…To monitor the induction and location of intracellular lipid peroxidation by hemin we incorporated a fluorescent tag, BODIPY (BD), onto arachidonic acid through conjugation with the carboxyl group on the fatty acid (25). We have shown previously that this does not change the ability of the unsaturated fatty acid to be a substrate for lipid peroxidation (25,39). BAEC were incubated with BD-AA and hemin for 2 h followed by treatment with mitotracker red (500 nM) after which confocal images were taken.…”

“…HEPES, sucrose, potassium chloride, and EGTA were obtained from Fisher Scientific (Fairlawn, NJ). BODIPY-arachidonic acid (BD-AA) was synthesized as described (25) and suspended in an ethanol vehicle for cell treatments. Antibodies against caspase 9, cleaved caspase 3, and ␤-actin were purchased from Cell Signaling Technologies (Boston, MA).…”

Fatty acid binding proteins have the potential to channel dietary fatty acids into enterocyte nuclei

“…Prior to binding assays , the proteins were delipidated in batch using hydroxyalkoxypropyldextran (Sigma-Aldrich) chromatography, equilibrated with 50 mM Tris-HCl, pH 7. ( 55,56 ). A dye-free 4× loading sample buffer containing 200 mM Tris-HCl (pH 6.8), 20% glycerol, and 8% SDS was then added to each sample.…”

Transduction of Redox Signaling by Electrophile-Protein Reactions

“…Once fractionated, peptides and corresponding proteins are identified and sequenced by high-performance liquid chromatography (HPLC)–MS/MS. Addition of biotinylated electrophiles to either intact cells or subcellular fractions (mitochondria, microsomes, nuclei, or cytosol) has provided important new insight into electrophile-sensitive subproteomes (27, 30, 72–74). This strategy has also been extended to the biotin labeling of complex lipids that are then subjected to per-oxyl radical-induced oxidation (75, 76).…”