Capturing Proteins that Bind Polyunsaturated Fatty Acids: Demonstration Using Arachidonic Acid and Eicosanoids

Abstract

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.

Glucose-mediated tyrosine nitration in adipocytes: Targets and consequences

“…The association with vesicles might provide a regulated source of GTP for GTPases that control intracellular trafficking, while the potential interaction with integrin cytoplasmic domain-associated protein 1α (ICAP-1α) as a consequence of Rac activation might affect cell morphology and behavior during adhesion [61]. NDK-B further binds polyunsaturated fatty acids [62], interacts with heterotrimeric G protein βγ dimers [63], facilitates coat protein complex II assembly [64], and induces c-myc [65] in various cell types. These functions of NDK-B could, if present in adipocytes, provide additional mechanisms by which NDK-B nitration might alter adipocyte biology.…”

Thermal tolerance of contractile function in oxidative skeletal muscle: no protection by antioxidants and reduced tolerance with eicosanoid enzyme inhibition

“…Blocking global LOX activity, even at normal temperatures, greatly decreases extracellular ROS production and has no effect on intracellular superoxide formation in diaphragm muscle in heat exposure (65). Recent evidence suggests that HETEs, leukotrienes, AA, and other eicosanoid products can directly bind and interact with numerous intracellular proteins, including cytoskeleton components associated with the contractile machinery (10). Such lipid-protein interactions are believed to be important in normal regulatory processes of the cell, such as water balance across the cell membrane (44) and GLUT4 translocation to the muscle cell membrane (56).…”

Effect of Polyunsaturated Fatty Acids and Their Metabolites on Bleomycin-Induced Cytotoxic Action on Human Neuroblastoma Cells In Vitro

“…Previously, we and others showed that several polyunsaturated fatty acids (PUFAs) have selective cytotoxic action on many tumor cells of different types with little or no action on normal cells [1] – [14] . But, PUFAs themselves are not very effective in eliminating cancer cells in an in vivo situation partly, due to the fact that they are tightly bound to albumin and other proteins and hence, are unavailable to bring about their tumoricidal action [15] – [17] . Furthermore, PUFAs may be metabolized into several eicosanoids that may have other unwanted actions.…”