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For lipid synthesis, energy production via ␤ -oxidation, or for protein fatty acylation to occur, long-chain fatty acids (LCFAs) must be activated by conversion to their CoA derivatives (LCFA-CoAs) by fatty acyl-CoA synthetase (FAS ). Protein fatty acylation is one of many types of posttranslational modifi cations of proteins by lipids, which also includes isoprenoids, glycosylphosphatidylinositols, and cholesterol. Typically, lipids covalently attached to proteins serve as hydrophobic membrane anchors ( 1-6 ).Protein fatty acylation is mainly divided into two categories: N-myristoylation and S-acylation. The corresponding reactions are catalyzed by N-myristoyl transferases (NMT1 and NMT2) and two families of protein acyltransferases (PATs) referred to as zinc fi nger, Asp-His-His-Cys PATs Abstract Progress in understanding the biology of protein fatty acylation has been impeded by the lack of rapid direct detection and identifi cation methods. We fi rst report that a synthetic -alkynyl-palmitate analog can be readily and specifi cally incorporated into GAPDH or mitochondrial 3-hydroxyl-3-methylglutaryl-CoA synthase in vitro and reacted with an azido-biotin probe or the fl uorogenic probe 3-azido-7-hydroxycoumarin using click chemistry for rapid detection by Western blotting or fl at bed fl uorescence scanning. The acylated cysteine residues were confi rmed by MS. Second, -alkynyl-palmitate is preferentially incorporated into transiently expressed H-or N-Ras proteins (but not nonpalmitoylated K-Ras), compared with -alkynyl-myristate or -alkynyl-stearate, via an alkali sensitive thioester bond. Third, -alkynyl-myristate is specifi cally incorporated into endogenous co-and posttranslationally myristoylated proteins. The competitive inhibitors 2-bromopalmitate and 2-hydroxymyristate prevented incorporation of -alkynylpalmitate and -alkynyl-myristate into palmitoylated and myristoylated proteins, respectively. Labeling cells with -alkynyl-palmitate does not affect membrane association of N-Ras. Furthermore, the palmitoylation of endogenous proteins including H-and N-Ras could be easily detected using -alkynyl-palmitate as label in cultured HeLa, Jurkat, and COS-7 cells, and, promisingly, in mice. The -alkynylmyristate and -palmitate analogs used with click chemistry

oxygen species (ROS) signal vital physiological processes including cell growth, angiogenesis, contraction, and relaxation of vascular smooth muscle. Because cytochrome P-450 family 4 (CYP4)/20-hydroxyeicosatetraenoic acid (20-HETE) has been reported to enhance angiogenesis, pulmonary vascular tone, and endothelial nitric oxide synthase function, we explored the potential of this system to stimulate bovine pulmonary artery endothelial cell (BPAEC) ROS production. Our data are the first to demonstrate that 20-HETE increases ROS in BPAECs in a time-and concentration-dependent manner as detected by enhanced fluorescence of oxidation products of dihydroethidium (DHE) and dichlorofluorescein diacetate. An analog of 20-HETE elicits no increase in ROS and blocks 20-HETE-evoked increments in DHE fluorescence, supporting its function as an antagonist. Endothelial cells derived from bovine aortas exhibit enhanced ROS production to 20-HETE quantitatively similar to that of BPAECs. 20-HETE-induced ROS production in BPAECs is blunted by pretreatment with polyethylene-glycolated SOD, apocynin, inhibition of Rac1, and a peptide-based inhibitor of NADPH oxidase subunit p47 phox association with gp91. These data support 20-HETE-stimulated, NADPH oxidase-derived, and Rac1/2-dependent ROS production in BPAECs. 20-HETE promotes translocation of p47 phox and tyrosine phosphorylation of p47 phox in a time-dependent manner as well as increased activated Rac1/2, providing at least three mechanisms through which 20-HETE activates NADPH oxidase. These observations suggest that 20-HETE stimulates ROS production in BPAECs at least in part through activation of NADPH oxidase within minutes of application of the lipid. superoxide; Rac1/2; hydrogen peroxide; CYP4A; reactive oxygen species PRODUCTS OF CYTOCHROME P-450 (CYP) -hydroxylases (including CYP4 isoforms) mediate key physiological functions including autoregulation of blood flow, tubuloglomerular feedback, Na ϩ reabsorption in the kidney, and relaxation of pulmonary arterioles (29,38). Our studies have focused on the role of CYP4 and its arachidonic acid (AA) metabolite, 20-hydroxyeicosatetraenoic acid (20-HETE), in pulmonary vascular function and biology. 20-HETE is the CYP -hydroxylation product of AA cleaved from membrane phospholipid sources. Enzymes of the CYP4A, -4B, and -4F families catalyze the -hydroxylation of fatty acids, and several isoforms in these families produce 20-HETE when incubated with AA. For example, rat CYP4A1, -4A2, and -4A3 catalyze AA -and -1-hydroxylations with the highest catalytic efficiency accruing to CYP4A1 (35). Although CYP4A2 and CYP4A3 exhibit an additional arachidonate 11,12-epoxidation activity, CYP4A1 operates solely as an -hydroxylase. Most investigators suggest that CYP4 isoforms constitute the major source of 20-HETE synthesis in extrahepatic tissues, including the lung (35, 38). Accordingly, we have investigated the effects of CYP4 product, 20-HETE, in our studies of this system in pulmonary vascular biology.We have identified a unique role f...

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Epoxyeicosatrienoic acids (EETs) contribute importantly to the regulation of vascular tone and blood pressure control. The purpose of this study was to develop stable EET analogs and test their in vivo blood pressure lowering effects in hypertensive rats. Using the pharmacophoric moiety of EETs, ether EET analogs were designed with improved solubility and resistance to auto-oxidation and metabolism by soluble epoxide hydrolase. Ether EET analogs were chosen based on their ability to dilate afferent arterioles and subsequently tested for blood pressure lowering effects in rodent models of hypertension. Initially, 11,12-ether-EET-8-ZE failed to lower blood pressure in angiotensin hypertension or spontaneously hypertensive rats (SHR). Esterification of the carboxylic group of 11,12-ether-EET-8-ZE prevented blood pressure increase in SHR when injected at 2 mg/day for 12 days (MAP Δ change at day 8 of injection was −0.3 ± 2 for treated and 12 ± 1 mmHg for control SHR). Amidation of the carboxylic group with aspartic acid produced another EET analog (NUDSA) with a blood pressure lowering effect when injected at 3 mg/day in SHR for 5 days. Amidation of the carboxylic group with lysine amino acid produced another analog with minimal blood pressure lowering effect. These data suggest that esterification of the carboxylic group of 11,12-ether-EET-8-ZE produced the most effective ether-EET analog in lowering blood pressure in SHR and provide the first evidence to support the use of EET analogs in treatment of cardiovascular diseases.