Capturing proteins that bind polyunsaturated fatty acids: demonstration using arachidonic acid and eicosanoids.
Polyunsaturated fatty acids (PUFA) and their biological derivatives, including the eicosanoids, have numerous roles in physiology and pathology. Although some eicosanoids are known to act through receptors, the molecular actions of many PUFA remain obscure. As the three-dimensional structure of eicosanoids allows them to specifically bind and activate their receptors, we hypothesized that the same structure would allow other proteins to associate with PUFA and eicosanoids. Here, we demonstrate that biotinylation of arachidonic acid and its oxygenated derivatives 5-hydroxyeicosatetraenoic acid (5-HETE) and leukotriene (LT) B(4) can be used to pull down associated proteins. Separation of proteins by two-dimensional gel electrophoresis indicated that a large number of proteins bound each lipid and that proteins could distinguish between two enantiomers of 5-HETE. Individual proteins, identified by matrix assisted laser desorption/ionization-time of flight mass spectrometry, included proteins that are known to bind lipids, including albumin and phosphatidylethanolamine-binding protein, as well as several novel proteins. These include cytoskeletal proteins, such as actin, moesin, stathmin and coactosin-like protein, and G protein signaling proteins, such as Rho GDP dissociation inhibitor 1 and nucleoside diphosphate kinase B. This method, then, represents a relatively simple and straightforward way to screen for proteins that directly associate with, and are potentially modulated by, PUFA and their derivatives.
Brock
Thomas G
TG
Department of Internal Medicine, University of Michigan, Ann Arbor, MI 48109, USA. brocko@umich.edu
D000818:Animals; D016718:Arachidonic Acid / Q000737:chemistry / Q000378:metabolism*; D045744:Cell Line, Tumor; D015777:Eicosanoids / Q000737:chemistry / Q000378:metabolism*; D015180:Electrophoresis, Gel, Two-Dimensional; D050556:Fatty Acid-Binding Proteins / Q000737:chemistry* / Q000378:metabolism*; D005231:Fatty Acids, Unsaturated / Q000737:chemistry / Q000378:metabolism*; D006893:Hydroxyeicosatetraenoic Acids / Q000737:chemistry / Q000378:metabolism; D015471:Leukemia, Basophilic, Acute / Q000378:metabolism; D051381:Rats
D015777:Eicosanoids; D050556:Fatty Acid-Binding Proteins; D005231:Fatty Acids, Unsaturated; D006893:Hydroxyeicosatetraenoic Acids; D016718:Arachidonic Acid