Oxygenation of Anandamide by Lipoxygenases.

The endocannabinoids anandamide and 2-arachidonoylglycerol are not only metabolized by serine hydrolases, such as fatty acid amide hydrolase, monoacylglycerol lipase, and α,ß-hydrolases 6 and 12, but they also serve as substrates for cyclooxygenases, cytochrome P450s, and lipoxygenases. These enzymes oxygenate the 1Z,4Z-pentadiene system of the arachidonic acid backbone of endocannabinoids, thereby giving rise to an entirely new array of bioactive lipids. Hereby, a protocol is provided for the enzymatic synthesis, purification, and characterization of various oxygenated metabolites of anandamide generated by lipoxygenases, which enables the biological study and detection of these metabolites.

Intramolecular quality control: HIV-1 envelope gp160 signal-peptide cleavage as a functional folding checkpoint.

Removal of the membrane-tethering signal peptides that target secretory proteins to the endoplasmic reticulum is a prerequisite for proper folding. While generally thought to be removed co-translationally, we report two additional post-targeting functions for the HIV-1 gp120 signal peptide, which remains attached until gp120 folding triggers its removal. First, the signal peptide improves folding fidelity by enhancing conformational plasticity of gp120 by driving disulfide isomerization through a redox-active cysteine. Simultaneously, the signal peptide delays folding by tethering the N terminus to the membrane, until assembly with the C terminus. Second, its carefully timed cleavage represents intramolecular quality control and ensures release of (only) natively folded gp120. Postponed cleavage and the redox-active cysteine are both highly conserved and important for viral fitness. Considering the ∼15% proteins with signal peptides and the frequency of N-to-C contacts in protein structures, these regulatory roles of signal peptides are bound to be more common in secretory-protein biogenesis.

Analysis of Protein Folding, Transport, and Degradation in Living Cells by Radioactive Pulse Chase.

Radioactive pulse-chase labeling is a powerful tool for studying the conformational maturation, the transport to their functional cellular location, and the degradation of target proteins in live cells. By using short (pulse) radiolabeling times (<30 min) and tightly controlled chase times, it is possible to label only a small fraction of the total protein pool and follow its folding. When combined with nonreducing/reducing SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoprecipitation with (conformation-specific) antibodies, folding processes can be examined in great detail. This system has been used to analyze the folding of proteins with a huge variation in properties such as soluble proteins, single and multi-pass transmembrane proteins, heavily N- and O-glycosylated proteins, and proteins with and without extensive disulfide bonding. Pulse-chase methods are the basis of kinetic studies into a range of additional features, including co- and posttranslational modifications, oligomerization, and polymerization, essentially allowing the analysis of a protein from birth to death. Pulse-chase studies on protein folding are complementary with other biochemical and biophysical methods for studying proteins in vitro by providing increased temporal resolution and physiological information. The methods as described within this paper are adapted easily to study the folding of almost any protein that can be expressed in mammalian or insect-cell systems.

Analysis of Disulfide Bond Formation.

In this unit, protocols are provided for detection of disulfide bond formation in cultures of intact cells and in an in vitro translation system containing isolated microsomes or semi-permeabilized cells. First, the newly synthesized protein of interest is biosynthetically labeled with radioactive amino acids in a short pulse. The labeled protein then is chased with unlabeled amino acids. At different times during the chase, a sample is collected, membranes are lysed with detergent, and the protein is isolated by immunoprecipitation, as described. A support protocol is provided for analysis of disulfide bonds in the immunoprecipitates by SDS-PAGE with and without prior reduction. The difference in mobility observed between the gels with nonreduced and reduced samples is due to disulfide bonds in the nonreduced protein. An additional support protocol is included that uses PEG-maleimide to modify free thiols and follow disulfide-bond formation by SDS-PAGE. © 2017 by John Wiley & Sons, Inc.

Oxygenation of Anandamide by Lipoxygenases.

The endocannabinoids anandamide and 2-arachidonoylglycerol are not only metabolized by serine hydrolases, such as fatty acid amide hydrolase, monoacylglycerol lipase, and α,ß-hydrolases 6 and 12, but they also serve as substrates for cyclooxygenases and lipoxygenases. These enzymes oxygenate the 1Z,4Z-pentadiene system of the arachidonic acid backbone of endocannabinoids, thereby giving rise to an entirely new array of bioactive lipids. Hereby, a protocol is provided for the enzymatic synthesis, purification, and characterization of various oxygenated metabolites of anandamide generated by lipoxygenases, which enables the biological study and detection of these metabolites.

TORC2 mediates the heat stress response in Drosophila by promoting the formation of stress granules.

The kinase TOR is found in two complexes, TORC1, which is involved in growth control, and TORC2, whose roles are less well defined. Here, we asked whether TORC2 has a role in sustaining cellular stress. We show that TORC2 inhibition in Drosophila melanogaster leads to a reduced tolerance to heat stress, whereas sensitivity to other stresses is not affected. Accordingly, we show that upon heat stress, both in the animal and Drosophila cultured S2 cells, TORC2 is activated and is required for maintaining the level of its known target, Akt1 (also known as PKB). We show that the phosphorylation of the stress-activated protein kinases is not modulated by TORC2 nor is the heat-induced upregulation of heat-shock proteins. Instead, we show, both in vivo and in cultured cells, that TORC2 is required for the assembly of heat-induced cytoprotective ribonucleoprotein particles, the pro-survival stress granules. These granules are formed in response to protein translation inhibition imposed by heat stress that appears to be less efficient in the absence of TORC2 function. We propose that TORC2 mediates heat resistance in Drosophila by promoting the cell autonomous formation of stress granules.

Methylation and acetylation of 15-hydroxyanandamide modulate its interaction with the endocannabinoid system.

The biological activity of endocannabinoids like anandamide (AEA) and 2-arachidonoylglycerol (2-AG) is subjected in vivo to a "metabolic control", exerted mainly by catabolic enzymes. AEA is inactivated by fatty acid amide hydrolase (FAAH), that is inhibited competitively by hydroxyanandamides (HAEAs) generated from AEA by lipoxygenase activity. Among these derivatives, 15-HAEA has been shown to be an effective (K(i) approximately 0.6 muM) FAAH inhibitor, that blocks also type-1 cannabinoid receptor (CB1R) but not other components of the "endocannabinoid system (ECS)", like the AEA transporter (AMT) or CB2R. Here, we extended the study of the effect of 15-HAEA on the AEA synthetase (NAPE-PLD) and the AEA-binding vanilloid receptor (TRPV1), showing that 15-HAEA activates the former (up to approximately 140% of controls) and inhibits the latter protein (down to approximately 70%). We also show that 15-HAEA halves the synthesis of 2-AG and almost doubles the transport of this compound across the membrane. In addition, we synthesized methyl and acetyl derivatives of 15-HAEA (15-MeOAEA and 15-AcOAEA, respectively), in order to check their ability to modulate FAAH and the other ECS elements. In fact, methylation and acetylation are common biochemical reactions in the cellular environment. We show that 15-MeOAEA, unlike 15-AcOAEA, is still a powerful competitive inhibitor of FAAH (K(i) approximately 0.7 muM), and that both derivatives have negligible interactions with the other proteins of ECS. Therefore, 15-MeOAEA is a FAAH inhibitor more selective than 15-HAEA. Further molecular dynamics analysis gave clues to the molecular requirements for the interaction of 15-HAEA and 15-MeOAEA with FAAH.

Calcium as a crucial cofactor for low density lipoprotein receptor folding in the endoplasmic reticulum.

The family of low density lipoprotein (LDL) receptors mediate uptake of a plethora of ligands from the circulation and couple this to signaling, thereby performing a crucial role in physiological processes including embryonic development, cancer development, homeostasis of lipoproteins, viral infection, and neuronal plasticity. Structural integrity of individual ectodomain modules in these receptors depends on calcium, and we showed before that the LDL receptor folds its modules late after synthesis via intermediates with abundant non-native disulfide bonds and structure. Using a radioactive pulse-chase approach, we here show that for proper LDL receptor folding, calcium had to be present from the very early start of folding, which suggests at least some native, essential coordination of calcium ions at the still largely non-native folding phase. As long as the protein was in the endoplasmic reticulum (ER), its folding was reversible, which changed only upon both proper incorporation of calcium and exit from the ER. Coevolution of protein folding with the high calcium concentration in the ER may be the basis for the need for this cation throughout the folding process even though calcium is only stably integrated in native repeats at a later stage.

Structural stability of soybean lipoxygenase-1 in solution as probed by small angle X-ray scattering.

Soybean lipoxygenase-1 (LOX-1) is used widely as a model for studying the structural and functional properties of the homologous family of lipoxygenases. The crystallographic structure revealed that LOX-1 is organized in a beta-sheet N-terminal domain and a larger, mostly helical, C-terminal domain. Here, we describe the overall structural characterization of native unliganded LOX-1 in solution, using small angle X-ray scattering (SAXS). We show that the scattering pattern of the unliganded enzyme in solution does not display any significant difference compared with that calculated from the crystal structure, and that models of the overall shape of the protein calculated ab initio from the SAXS pattern provide a close envelope to the crystal structure. These data, demonstrating that LOX-1 has a compact structure also in solution, rule out any major motional flexibility of the LOX-1 molecule in aqueous solutions. In addition we show that eicosatetraynoic acid, an irreversible inhibitor of lipoxygenase used to mimic the effect of substrate binding, does not alter the overall conformation of LOX-1 nor its ability to bind to membranes. In contrast, the addition of glycerol (to 5%, v/v) causes an increase in the binding of the enzyme to membranes without altering its catalytic efficiency towards linoleic acid nor its SAXS pattern, suggesting that the global conformation of the enzyme is unaffected. Therefore, the compact structure determined in the crystal appears to be essentially preserved in these various solution conditions. During the preparation of this article, a paper by M. Hammel and co-workers showed instead a sharp difference between crystal and solution conformations of rabbit 15-LOX-1. The possible cause of this difference might be the presence of oligomers in the rabbit lipoxygenase preparations.

Conversion of linoleic acid into novel oxylipins by the mushroom Agaricus bisporus.

Oxylipins are associated with important processes of the fungal life cycle, such as spore formation. Here, we report the formation of FA metabolites in Agaricus bisporus. Incubation of a crude extract of lamellae with linoleic acid (18:2) led to the extensive formation of two oxylipins. They were identified as 8(R)-hydroxy-9Z,12Z-octadecadienoic acid (8-HOD) and 8(R),11 (S)-dihydroxy-9Z,12Z-octadecadienoic acid (8,11-diHOD) by using RP-HPLC, GC-MS, IR, GC-MS analysis of diastereomeric derivatives, and 1H NMR and 13C NMR spectroscopy. Neither compound has been reported before in A. bisporus. Oleic (18:1), alpha-linolenic (18:3n-3), and gamma-linolenic (18:3n-6) acids were converted into their 8-hydroxy derivatives as well, and 18:3n-3 was further metabolized to its 8,11-diol derivative. Reactions with [U-13C]18:2 demonstrated that the compounds 8-HOD and 8,11-diHOD were formed from exogenously supplied 18:2. When [U-13C]8-HOD was supplied, it was not converted into 8,11-diHOD, indicating that it was not an intermediate in the formation of 8,11-diHOD. When a crude extract of A. bisporus was incubated under an atmosphere of 16O2/18O2, the two hydroxyl groups of 8,11-diHOD contained either two 180 atoms or two 60 atoms. Species that contained one of each isotope could not be detected. We propose that the formation of the 8,11-dihydroxy compounds occurs through either an 8,11-endoperoxy, an 8-peroxo free radical, or an 8-hydroperoxy intermediate. In the latter case, the reaction should be catalyzed by dioxygenase with novel specificity.

Structure-to-function relationship of mini-lipoxygenase, a 60-kDa fragment of soybean lipoxygenase-1 with lower stability but higher enzymatic activity.

Lipoxygenase-1 (Lox-1) is a member of the lipoxygenase family, a class of dioxygenases that take part in the metabolism of polyunsatured fatty acids in eukaryotes. Tryptic digestion of soybean Lox-1 is known to produce a 60 kDa fragment, termed "mini-Lox," which shows enhanced catalytic efficiency and higher membrane-binding ability than the native enzyme (Maccarrone, M., Salucci, M. L., van Zadelhoff, G., Malatesta, F., Veldink, G. Vliegenthart, J. F. G., and Finazzi-Agrò, A. (2001) Biochemistry 40, 6819-6827). In this study, we have investigated the stability of mini-Lox in guanidinium hydrochloride and under high pressure by fluorescence and circular dichroism spectroscopy. Only a partial unfolding could be obtained at high pressure in the range 1-3000 bar at variance with guanidinium hydrochloride. However, in both cases a reversible denaturation was observed. The denaturation experiments demonstrate that mini-Lox is a rather unstable molecule, which undergoes a two-step unfolding transition at moderately low guanidinium hydrochloride concentration (0-4.5 m). Both chemical- and physical-induced denaturation suggest that mini-Lox is more hydrated than Lox-1, an observation also confirmed by 1-anilino-8-naphthalenesulfonate (ANS) binding studies. We have also investigated the occurrence of substrate-induced changes in the protein tertiary structure by dynamic fluorescence techniques. In particular, eicosatetraynoic acid, an irreversible inhibitor of lipoxygenase, has been used to mimic the effect of substrate binding. We demonstrated that mini-Lox is indeed characterized by much larger conformational changes than those occurring in the native Lox-1 upon binding of eicosatetraynoic acid. Finally, by both activity and fluorescence measurements we have found that 1-anilino-8-naphthalenesulfonate has access to the active site of mini-Lox but not to that of intact Lox-1. These findings strongly support the hypothesis that the larger hydration of mini-Lox renders this molecule more flexible, and therefore less stable.

Oxygenated metabolites of anandamide and 2-arachidonoylglycerol: conformational analysis and interaction with cannabinoid receptors, membrane transporter, and fatty acid amide hydrolase.

This study was aimed at finding structural requirements for the interaction of the acyl chain of endocannabinoids with cannabinoid receptors, membrane transporter protein, and fatty acid amide hydrolase (FAAH). To this end, the flexibility of the acyl chain was restricted by introduction of an 1-hydroxy-2Z,4E-pentadiene system in anandamide (N-arachidonoylethanolamine, AEA) and 2-arachidonoylglycerol (2-AG) at various positions using different lipoxygenases. This brought about selectivity and attenuated the binding potency of AEA and 2-AG. Although the displacement constants were modest, 15(S)-hydroxy-eicosa-5Z,8Z,11Z,13E-tetraenoyl-N-(2-hydroxyethyl)amine was found to bind selectively to the CB(1) receptor, whereas its 1-arachidonoyl-sn-glycerol analogue and 13(S)-hydroxy-octadeca-9Z,11E-dienoyl-N-(2-hydroxyethyl)amine could selectively bind to the CB(2) receptor. 11(S)-Hydroxy-eicosa-5Z,8Z,12E,14Z-tetraenoyl-N-(2-hydroxyethyl)amine did not bind to either receptor, whereas 12(S)-hydroxy-eicosa-5Z,8Z,10E,14Z-tetraenoyl-N-(2-hydroxyethyl)amine did bind to both CB receptors with an affinity similar to that of AEA. All oxygenated anandamide derivatives were good inhibitors of FAAH (low micromolar K(i)) but were ineffective on the AEA transporter. 2-AG rapidly isomerizes into 1(3)-arachidonoyl-sn-glycerol. Both 1- and 3-arachidonoyl-sn-glycerol did not bind to either CB receptor and did not interfere with AEA transport. Thus, after it is isomerized, 2-AG is inactivated, thereby decreasing effective concentrations of 2-AG. Analysis of (1)H NMR spectra revealed that chloroform did not induce notably different conformations in the acyl chain of 15(S)-hydroxy-eicosa-5Z,8Z,11Z,13E-tetraenoic acid as compared with water. Molecular dynamics (MD) simulations of AEA and its analogues in the presence of explicit water molecules revealed that a tightly folded conformation of the acyl chain is not the only requirement for CB(1) binding. Structural details of the C(2)-C(15) loop, such as an sp(2) carbon at position 11, are necessary for receptor binding. The MD simulations may suggest that the average orientations of the pentyl tail of AEA and 12(S)-hydroxy-eicosa-5Z,8Z,10E,14Z-tetraenoyl-N-(2-hydroxyethyl)amine are different from that of the low-affinity, inactive ligands.