Enrico Dainese

The Endocannabinoid System and Its Relevance for Nutrition

Endocannabinoids bind to cannabinoid, vanilloid, and peroxisome proliferator-activated receptors. The biological actions of these polyunsaturated lipids are controlled by key agents responsible for their synthesis, transport and degradation, which together form an endocannabinoid system (ECS). In the past few years, evidence has been accumulated for a role of the ECS in regulating food intake and energy balance, both centrally and peripherally. In addition, up-regulation of the ECS in the gastrointestinal tract has a potential impact on inflammatory bowel diseases. In this review, the main features of the ECS are summarized in order to put in better focus our current knowledge of the nutritional relevance of endocannabinoid signaling and of its role in obesity, cardiovascular pathologies, and gastrointestinal diseases. The central and peripheral pathways that underlie these effects are discussed, as well as the possible exploitation of ECS components as novel drug targets for therapeutic intervention in eating disorders.

Evidence for the intracellular accumulation of anandamide in adiposomes

Abstract.Anandamide is a lipid messenger that carries out a wide variety of biological functions. It has been suggested that anandamide accumulation involves binding to a saturable cellular component. To identify the structure(s) involved in this process, we analyzed the intracellular distribution of both biotinylated and radiolabeled anandamide, providing direct evidence that lipid droplets, also known as adiposomes, constitute a dynamic reservoir for the sequestration of anandamide. In addition, confocal microscopy and biochemical studies revealed that the anandamide-hydrolase is also spatially associated with lipid droplets, and that cells with a larger adiposome compartment have an enhanced catabolism of anandamide. Overall, these findings suggest that adiposomes may have a critical role in accumulating anandamide, possibly by connecting plasma membrane to internal organelles along the metabolic route of this endocannabinoid.

Functional characterization of putative cholesterol binding sequence (CRAC) in human type-1 cannabinoid receptor

J. Neurochem. (2011) 116, 858–865.

The endogenous cannabinoid system in the gut of patients with inflammatory bowel disease

Activation of cannabinoid receptors (CBs) by endocannabinoids impacts on a number of gastrointestinal functions. Recent data indicate that CB1 agonists improve 2,4-dinitrobenzene sulfonic acid-induced colitis in mice, thus suggesting a role for the endocannabinoid agonist anandamide (AEA) in protecting the gut against inflammation. We here examined the gut endocannabinoid system in inflammatory bowel disease (IBD) patients, and investigated the ex vivo and in vitro effects of the non-hydrolysable AEA analog methanandamide (MAEA) on the mucosal proinflammatory response. The content of AEA, but not of 2-arachidonoyl-glycerol and N-palmitoylethanolamine, was significantly lower in inflamed than uninflamed IBD mucosa, and this was paralleled by lower activity of the AEA-synthesizing enzyme N-acyl-phosphatidylethanolamine-specific phospholipase D and higher activity of the AEA-degrading enzyme fatty acid amide hydrolase. MAEA significantly downregulated interferon-γ and tumor necrosis factor-α secretion by both organ culture biopsies and lamina propria mononuclear cells. Although these results are promising, further studies are needed to determine the role of cannabinoid pathways in gut inflammation.

Extravirgin olive oil up-regulates CB1 tumor suppressor gene in human colon cancer cells and in rat colon via epigenetic mechanisms

Extravirgin olive oil (EVOO) represents the typical lipid source of the Mediterranean diet, an eating habit pattern that has been associated with a significant reduction of cancer risk. Diet is the more studied environmental factor in epigenetics, and many evidences suggest dysregulation of epigenetic pathways in cancer. The aim of our study was to investigate the effects of EVOO and its phenolic compounds on endocannabinoid system (ECS) gene expression via epigenetic regulation in both human colon cancer cells (Caco-2) and rats exposed to short- and long-term dietary EVOO. We observed a selective and transient up-regulation of CNR1 gene - encoding for type 1 cannabinoid receptor (CB₁) - that was evoked by exposure of Caco-2 cells to EVOO (100 ppm), its phenolic extracts (OPE, 50 μM) or authentic hydroxytyrosol (HT, 50 μM) for 24 h. None of the other major elements of the ECS (i.e., CB₂; GPR55 and TRPV1 receptors; and NAPE-PLD, DAGL, FAAH and MAGL enzymes) was affected at any time point. The stimulatory effect of OPE and HT on CB₁ expression was inversely correlated to DNA methylation at CNR1 promoter and was associated with reduced proliferation of Caco-2 cells. Interestingly, CNR1 gene was less expressed in Caco-2 cells when compared to normal colon mucosa cells, and again this effect was associated with higher level of DNA methylation at CNR1. Moreover, in agreement with the in vitro studies, we also observed a remarkable (~4-fold) and selective increase in CB₁ expression in the colon of rats receiving dietary EVOO supplementation for 10 days. Consistently, CpG methylation of rat Cnr1 promoter, miR23a and miR-301a, previously shown to be involved in the pathogenesis of colorectal cancer and predicted to target CB₁ mRNA, was reduced after EVOO administration down to ~50% of controls. Taken together, our findings demonstrating CB₁ gene expression modulation by EVOO or its phenolic compounds via epigenetic mechanism, both in vitro and in vivo, may provide a new therapeutic avenue for treatment and/or prevention of colon cancer.

A key structural role for active site type 3 copper ions in human ceruloplasmin.

Human ceruloplasmin is a copper containing serum glycoprotein with multiple functions. The crystal structure shows that its six domains are arranged in three pairs with a pseudo-ternary axis. Both the holo and apo forms of human ceruloplasmin were studied by size exclusion chromatography and small angle x-ray scattering in solution. The experimental curve of the holo form displays conspicuous differences with the scattering pattern calculated from the crystal structure. Once the carbohydrate chains and flexible loops not visible in the crystal are accounted for, remaining discrepancies suggest that the central pair of domains may move as a whole with respect to the rest of the molecule. The quasisymmetrical crystal structure therefore appears to be stabilized by crystal packing forces. Upon copper removal, the scattering pattern of human ceruloplasmin exhibits very large differences with that of the holoprotein, which are interpreted in terms of essentially preserved domains freely moving in solution around flexible linkers and exploring an ensemble of open conformations. This model, which is supported by the analysis of domain interfaces, provides a structural explanation for the differences in copper reincorporation into the apoprotein and activity recovery between human ceruloplasmin and two other multicopper oxidases, ascorbate oxidase and laccase. Our results demonstrate that, beyond catalytic activity, the three-copper cluster at the N-terminal-C-terminal interface plays a crucial role in the structural stability of human ceruloplasmin.

5-Lipoxygenase-dependent apoptosis of human lymphocytes in the International Space Station: data from the ROALD experiment

The functional adaptation of the immune system to the surrounding environment is also a fundamental issue in space. It has been suggested that a decreased number of lymphocytes might be a cause of immunosuppression, possibly due to the induction of apoptosis. Early activation of 5‐lipoxygenase (5‐LOX) might play a central role in the initiation of the apoptotic program. The goal of the role of apoptosis in lymphocyte depression (ROALD) experiment, flown on the International Space Station as part of the BIO‐4 mission of the European Space Agency, was to ascertain the induction of apoptosis in human lymphocytes under authentic microgravity, and to elucidate the possible involvement of 5‐LOX. Our results demonstrate that exposure of human lymphocytes to microgravity for 48 h onboard the ISS remarkably increased apoptotic hallmarks such as DNA fragmentation (~3‐fold compared to ground‐based controls) and cleaved‐poly (ADP‐ribose) polymerase (PARP) protein expression (~3‐fold), as well as mRNA levels of apoptosis‐related markers such as p53 (~3‐fold) and calpain (~4‐fold); these changes were paralleled by an early increase of 5‐LOX activity (~2‐fold). Our findings provide a molecular background for the immune dysfunction observed in astronauts during space missions, and reveal potential new markers to monitor health status of ISS crew members.—Battista, N., Meloni, M. A., Bari, M., Mastrangelo, N., Galleri, G., Rapino, C., Dainese, E., Finazzi Agrò, A., Pippia, P., Maccarrone, M. 5‐Lipoxygenase‐dependent apoptosis of human lymphocytes in the International Space Station: data from the ROALD experiment. FASEB J. 26, 1791‐1798 (2012). www.fasebj.org

Effects of palmitoylation of Cys415 in helix 8 of the CB1 cannabinoid receptor on membrane localization and signalling

BACKGROUND AND PURPOSE The CB1 cannabinoid receptor is regulated by its association with membrane microdomains such as lipid rafts. Here, we investigated the role of palmitoylation of the CB1 receptor by analysing the functional consequences of site‐specific mutation of Cys415, the likely site of palmitoylation at the end of helix 8, in terms of membrane association, raft targeting and signalling.

Conformational Changes of Calpain from Human Erythrocytes in the Presence of Ca2

Small angle x-ray scattering has been used to monitor calpain structural transitions during the activation process triggered by Ca2+ binding. The scattering pattern of the unliganded enzyme in solution does not display any significant difference with that calculated from the crystal structure. The addition of Ca2+ promotes the formation of large aggregates, indicating the exposure of hydrophobic patches on the surface of the protease. In contrast, Ca2+ addition in the presence of the thiol proteinase inhibitor E64 or of the inhibitor leupeptin causes a small conformational change with no dissociation of the heterodimer. The resulting conformation appears to be slightly more extended than the unliganded form. From the comparison between ab initio models derived from our data with the crystal structure, the major observable conformational change appears to be localized at level of the L-subunit and in particular seems to confirm the mutual movement already observed by the crystallographic analysis of the dII (dIIb) and the dI (dIIa) domains creating a functional active site. This work not only provides another piece of supporting evidence for the calpain conformational change in the presence of Ca2+, but actually constitutes the first experimental observation of this change for intact heterodimeric calpain in solution.

Membrane lipids are key modulators of the endocannabinoid-hydrolase FAAH

Lipid composition is expected to play an important role in modulating membrane enzyme activity, in particular if the substrates are themselves lipid molecules. A paradigmatic case is FAAH (fatty acid amide hydrolase), an enzyme critical in terminating endocannabinoid signalling and an important therapeutic target. In the present study, using a combined experimental and computational approach, we show that membrane lipids modulate the structure, subcellular localization and activity of FAAH. We report that the FAAH dimer is stabilized by the lipid bilayer and shows a higher membrane-binding affinity and enzymatic activity within membranes containing both cholesterol and the natural FAAH substrate AEA (anandamide). Additionally, co-localization of cholesterol, AEA and FAAH in mouse neuroblastoma cells suggests a mechanism through which cholesterol increases the substrate accessibility of FAAH.