Publications by authors named "Enrico Dainese"

43 Publications

Bisphenol A Deranges the Endocannabinoid System of Primary Sertoli Cells with an Impact on Inhibin B Production.

Int J Mol Sci 2020 Nov 26;21(23). Epub 2020 Nov 26.

Department of Applied Clinical and Biotechnological Sciences, University of L'Aquila, 67100 L'Aquila, Italy.

Bisphenol A (BPA) is an endocrine disruptor that negatively affects spermatogenesis, a process where Sertoli cells play a central role. Thus, in the present study we sought to ascertain whether BPA could modulate the endocannabinoid (eCB) system in exposed mouse primary Sertoli cells. Under our experimental conditions, BPA turned out to be cytotoxic to Sertoli cells with an half-maximal inhibitory concentration (IC) of ~6.0 µM. Exposure to a non-cytotoxic dose of BPA (i.e., 0.5 μM for 48 h) increased the expression levels of specific components of the eCB system, namely: type-1 cannabinoid (CB) receptor and diacylglycerol lipase-α (DAGL-α), at mRNA level, type-2 cannabinoid (CB) receptor, transient receptor potential vanilloid 1 (TRPV1) receptors, and DAGL-β, at protein level. Interestingly, BPA also increased the production of inhibin B, but not that of transferrin, and blockade of either CB receptor or TRPV1 receptor further enhanced the BPA effect. Altogether, our study provides unprecedented evidence that BPA deranges the eCB system of Sertoli cells towards CB- and TRPV1-dependent signal transduction, both receptors being engaged in modulating BPA effects on inhibin B production. These findings add CB and TRPV1 receptors, and hence the eCB signaling, to the other molecular targets of BPA already known in mammalian cells.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3390/ijms21238986DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7730056PMC
November 2020

Plasmonic active film integrating gold/silver nanostructures for HO readout.

Talanta 2021 Jan 17;222:121682. Epub 2020 Sep 17.

Faculty of Bioscience and Technology for Food, Agriculture and Environment, University of Teramo, Via Renato Balzarini 1, 64100, Teramo, Italy. Electronic address:

A nanostructured Ag/Au adhesive film for HO reagentless determination is here proposed. The film has been realised onto ELISA polystyrene microplates. Microwells surface has been initially modified with a gold nanoparticles (AuNPs)/polydopamine thin-film. The pristine AuNPs-decorated film was later functionalized with catechin (Au-CT) allowing a uniform formation of a plasmonic active nanostructured silver network in presence of Ag. Changes in localized surface plasmon resonance (LSPR) of the silver network upon addition of HO has been used as analytical signal, taking advantage of the etching phenomenon. The Ag/Au nanocomposite-film is characterized by a well-defined (LSPR = 405 ± 5 nm), reproducible (intraplate RSD ≤ 9.8%, n = 96; inter-plate RSD ≤ 11.4%, n = 480) and stable LSPR signal. The film's analytical features have been tested for HO and glucose (bio)sensing. Satisfactory analytical performances were obtained both for HO (linear range 1-200 μM, R = 0.9992, RSD ≤ 6.3%, LOD = 0.2 μM) and glucose (linear range 2-250 μM, R = 0.9998, RSD ≤ 8.9%, LOD = 0.4 μM). As proof of applicability, the determination of the two analytes in soft drinks has been carried out achieving good and reproducible recoveries (84-111%; RSD ≤ 9%). The developed nanostructured film overcomes analytical drawbacks associated with the use of colloidal dispersions in plasmonic assays carried out in solution; the low cost, robustness, ease of use and possibility of coupling enzymatic reactions appears very promising for (bio)sensors based on the detection of HO.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.talanta.2020.121682DOI Listing
January 2021

Structure of a nucleotide pyrophosphatase/phosphodiesterase (NPP) from Euphorbia characias latex characterized by small-angle X-ray scattering: clues for the general organization of plant NPPs.

Acta Crystallogr D Struct Biol 2020 Sep 17;76(Pt 9):857-867. Epub 2020 Aug 17.

Faculty of Biosciences and Technology for Food Agriculture and Environment, University of Teramo, Via Balzarini 1, 64100 Teramo, Italy.

Little information is available concerning the structural features of nucleotide pyrophosphatase/phosphodiesterases (NPPs) of plant origin and the crystal structures of these proteins have not yet been reported. The aim of this study was to obtain insight into these aspects by carrying out a comparative analysis of the sequences of two different fragments of an NPP from the latex of the Mediterranean shrub Euphorbia characias (ELNPP) and by studying the low-resolution structure of the purified protein in solution by means of small-angle X-ray scattering. This is the first structure of a plant NPP in solution that has been reported to date. It is shown that the ELNPP sequence is highly conserved in many other plant species. Of note, the catalytic domains of these plant NPPs have the same highly conserved PDE-domain organization as mammalian NPPs. Moreover, ELNPP is a dimer in solution and this oligomerization state is likely to be common to other plant enzymes.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1107/S2059798320010207DOI Listing
September 2020

Epigenetic regulation of the cannabinoid receptor CB1 in an activity-based rat model of anorexia nervosa.

Int J Eat Disord 2020 05 10;53(5):432-446. Epub 2020 Apr 10.

Department of Biomedical Sciences, Division of Neuroscience and Clinical Pharmacology, University of Cagliari, Cagliari, Italy.

Objective: Both environmental and genetic factors are known to contribute to the development of anorexia nervosa (AN), but the exact etiology remains poorly understood. Herein, we studied the transcriptional regulation of the endocannabinoid system, an interesting target for body weight maintenance and the control of food intake and energy balance.

Method: We used two well-characterized animal models of AN: (a) the activity-based anorexia (ABA) model in which rats, housed with running wheels and subjected to daily food restriction, show reductions in body weight and increase in physical activity; (b) the genetic anx/anx mouse displaying the core features of AN: low food intake and emaciation.

Results: Among the evaluated endocannabinoid system components, we observed a selective and significant down-regulation of the gene encoding for the type 1 cannabinoid receptor (Cnr1) in ABA rats' hypothalamus and nucleus accumbens and, in the latter area, a consistent, significant and correlated increase in DNA methylation at the gene promoter. No changes were evident in the anx/anx mice except for a down-regulation of Cnr1, in the prefrontal cortex.

Discussion: Our findings support a possible role for Cnr1 in the ABA animal model of AN. In particular, its regulation in the nucleus accumbens appears to be triggered by environmental cues due to the consistent epigenetic modulation of the promoter. These data warrant further studies on Cnr1 regulation as a possible target for treatment of AN.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/eat.23271DOI Listing
May 2020

Author Correction: The endocannabinoid hydrolase FAAH is an allosteric enzyme.

Sci Rep 2020 Mar 31;10(1):5903. Epub 2020 Mar 31.

European Center for Brain Research (CERC)/Santa Lucia Foundation, Rome, Italy.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41598-020-62514-wDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7109033PMC
March 2020

The endocannabinoid hydrolase FAAH is an allosteric enzyme.

Sci Rep 2020 02 10;10(1):2292. Epub 2020 Feb 10.

European Center for Brain Research (CERC)/Santa Lucia Foundation, Rome, Italy.

Fatty acid amide hydrolase (FAAH) is a membrane-bound homodimeric enzyme that in vivo controls content and biological activity of N-arachidonoylethanolamine (AEA) and other relevant bioactive lipids termed endocannabinoids. Parallel orientation of FAAH monomers likely allows both subunits to simultaneously recruit and cleave substrates. Here, we show full inhibition of human and rat FAAH by means of enzyme inhibitors used at a homodimer:inhibitor stoichiometric ratio of 1:1, implying that occupation of only one of the two active sites of FAAH is enough to fully block catalysis. Single W445Y substitution in rat FAAH displayed the same activity as the wild-type, but failed to show full inhibition at the homodimer:inhibitor 1:1 ratio. Instead, F432A mutant exhibited reduced specific activity but was fully inhibited at the homodimer:inhibitor 1:1 ratio. Kinetic analysis of AEA hydrolysis by rat FAAH and its F432A mutant demonstrated a Hill coefficient of ~1.6, that instead was ~1.0 in the W445Y mutant. Of note, also human FAAH catalysed an allosteric hydrolysis of AEA, showing a Hill coefficient of ~1.9. Taken together, this study demonstrates an unprecedented allosterism of FAAH, and represents a case of communication between two enzyme subunits seemingly controlled by a single amino acid (W445) at the dimer interface. In the light of extensive attempts and subsequent failures over the last decade to develop effective drugs for human therapy, these findings pave the way to the rationale design of new molecules that, by acting as positive or negative heterotropic effectors of FAAH, may control more efficiently its activity.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41598-020-59120-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7010751PMC
February 2020

The anti-inflammatory agent bindarit acts as a modulator of fatty acid-binding protein 4 in human monocytic cells.

Sci Rep 2019 10 22;9(1):15155. Epub 2019 Oct 22.

European Center for Brain Research (CERC)/Santa Lucia Foundation IRCCS, Rome, 00143, Italy.

We investigated the cellular and molecular mechanisms by which bindarit, a small indazolic derivative with prominent anti-inflammatory effects, exerts its immunoregulatory activity in lipopolysaccharide (LPS) stimulated human monocytic cells. We found that bindarit differentially regulates the release of interleukin-8 (IL-8) and monocyte chemoattractant protein-1 (MCP-1), enhancing the release of IL-8 and reducing that of MCP-1. These effects specifically required a functional interaction between bindarit and fatty acid binding protein 4 (FABP4), a lipid chaperone that couples intracellular lipid mediators to their biological targets and signaling pathways. We further demonstrated that bindarit can directly interact with FABP4 by increasing its expression and nuclear localization, thus impacting on peroxisome proliferator-activated receptor γ (PPARγ) and LPS-dependent kinase signaling. Taken together, these findings suggest a potential key-role of FABP4 in the immunomodulatory activity of bindarit, and extend the spectrum of its possible therapeutic applications to FABP4 modulation.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41598-019-51691-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6805920PMC
October 2019

On the Role of Adenosine A2A Receptor Gene Transcriptional Regulation in Parkinson's Disease.

Front Neurosci 2019 10;13:683. Epub 2019 Jul 10.

Faculty of Bioscience, University of Teramo, Teramo, Italy.

Adenosine A2A receptors (A2ARs) have attracted considerable attention as an important molecular target for the design of Parkinson's disease (PD) therapeutic compounds. Here, we studied the transcriptional regulation of the A2AR gene in human peripheral blood mononuclear cells (PBMCs) obtained from PD patients and in the striatum of the well-validated, 6-hydroxydopamine (6-OHDA)-induced PD mouse model. We report an increase in A2AR mRNA expression and protein levels in both human cells and mice striata, and in the latter we could also observe a consistent reduction in DNA methylation at gene promoter and an increase in histone H3 acetylation at lysine 9. Of particular relevance in clinical samples, we also observed higher levels in the receptor gene expression in younger subjects, as well as in those with less years from disease onset, and less severe disease according to clinical scores. In conclusion, the present findings provide further evidence of the relevant role of A2AR in PD and, based on the clinical data, highlight its potential role as disease biomarker for PD especially at the initial stages of disease development. Furthermore, our preclinical results also suggest selective epigenetic mechanisms targeting gene promoter as tool for the development of new treatments.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3389/fnins.2019.00683DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6635589PMC
July 2019

Iron-Dependent Trafficking of 5-Lipoxygenase and Impact on Human Macrophage Activation.

Front Immunol 2019 28;10:1347. Epub 2019 Jun 28.

Faculty of Bioscience and Technology for Food Agriculture and Environment, University of Teramo, Teramo, Italy.

5-lipoxygenase (5-LOX) is a non-heme iron-containing dioxygenase expressed in immune cells that catalyzes the two initial steps in the biosynthesis of leukotrienes. It is well known that 5-LOX activation in innate immunity cells is related to different iron-associated pro-inflammatory disorders, including cancer, neurodegenerative diseases, and atherosclerosis. However, the molecular and cellular mechanism(s) underlying the interplay between iron and 5-LOX activation are largely unexplored. In this study, we investigated whether iron (in the form of Fe and hemin) might modulate 5-LOX influencing its membrane binding, subcellular distribution, and functional activity. We proved by fluorescence resonance energy transfer approach that metal removal from the recombinant human 5-LOX, not only altered the catalytic activity of the enzyme, but also impaired its membrane-binding. To ascertain whether iron can modulate the subcellular distribution of 5-LOX in immune cells, we exposed THP-1 macrophages and human primary macrophages to exogenous iron. Cells exposed to increasing amounts of Fe showed a redistribution (ranging from ~45 to 75%) of the cytosolic 5-LOX to the nuclear fraction. Accordingly, confocal microscopy revealed that acute exposure to extracellular Fe, as well as hemin, caused an overt increase in the nuclear fluorescence of 5-LOX, accompanied by a co-localization with the 5-LOX activating protein (FLAP) both in THP-1 macrophages and human macrophages. The functional relevance of iron overloading was demonstrated by a marked induction of the expression of interleukin-6 in iron-treated macrophages. Importantly, pre-treatment of cells with the iron-chelating agent deferoxamine completely abolished the hemin-dependent translocation of 5-LOX to the nuclear fraction, and significantly reverted its effect on interleukin-6 overexpression. These results suggest that exogenous iron modulates the biological activity of 5-LOX in macrophages by increasing its ability to bind to nuclear membranes, further supporting a role for iron in inflammation-based diseases where its homeostasis is altered and suggesting further evidence of risks related to iron overload.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3389/fimmu.2019.01347DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6610208PMC
October 2020

Role of Steroids on the Membrane Binding Ability of Fatty Acid Amide Hydrolase.

Cannabis Cannabinoid Res 2019 13;4(1):42-50. Epub 2019 Mar 13.

European Center for Brain Research (CERC)/Santa Lucia Foundation, Rome, Italy.

Fatty acid amide hydrolase (FAAH) is a membrane-bound homodimeric enzyme that gets in contact with a lipophilic substrate in the lipid bilayer, and then cleaves it into water soluble products. FAAH plays a critical role in modulating content and biological activity of endocannabinoids (eCBs), and its function is affected by membrane lipids. Increasing evidence suggests that also steroids can modulate endocannabinoid signaling, both in the central nervous system and at the periphery. In this study, we interrogated the effect of six steroids with relevant biological activity (testosterone, hydrocortisone, estradiol, pregnenolone, progesterone, and cortisone) on the membrane binding ability of rat FAAH. The experimental data analysis obtained by Fluorescence Resonance Energy Transfer Spectroscopy was paralleled by computational docking analysis. Our data revealed distinct effects of the different steroids on the interaction of rat FAAH with model membranes. Among them, pregnenolone was found to be the most effective in raising rat FAAH affinity for model membranes. A possible binding pocket for steroid molecules was identified by docking analysis in the membrane-embedded region of the enzyme; such a pocket could account for the observed increase of the membrane affinity in the presence of the tested molecules. Overall, the results point to steroids as new regulators of FAAH interaction with membranes, which may impact the biological activity of eCBs.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1089/can.2018.0051DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6446164PMC
March 2019

Amine oxidase from Euphorbia characias: Kinetic and structural characterization.

Biotechnol Appl Biochem 2018 Jan 23;65(1):81-88. Epub 2017 Oct 23.

Department of Sciences of Life and Environment, University of Cagliari, Monserrato, Cagliari, Italy.

This minireview focuses on a plant copper/2,4,5-trihydroxyphenyl alanine quinone amine oxidase isolated from the latex of the shrub Euphorbia characias (ELAO). This enzyme has been investigated in terms of both molecular structure and kinetic mechanism. The characterization of this enzyme allowed us to identify specific amino acids and domains that play a key role in modulating substrate access into the active site not only for ELAO but also for other plant and mammalian amine oxidases. As mammalian amine oxidases are implicated in several physiological and pathological conditions, the deep structural characterization of their active site accession mechanisms could be the starting point for the development of enzyme modulators with high therapeutic potential. Thus, this paper gives structural/functional insights that open new perspectives in the research about the whole amine oxidase family.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/bab.1612DOI Listing
January 2018

In silico mapping of allosteric ligand binding sites in type-1 cannabinoid receptor.

Biotechnol Appl Biochem 2018 Jan 3;65(1):21-28. Epub 2018 Jan 3.

European Center for Brain Research (CERC)/Santa Lucia Foundation IRCCS, Rome, Italy.

The recent resolution of the crystal structure of type-1 cannabinoid receptor (CB ) and the discovery of novel modulators for this target open the way to the possibility of elucidating the structural requirements for CB binding, and thereby facilitate a rational drug design. Compounds that target the orthosteric site of CB in some cases have shown side effects. Allosteric modulators could potentially avoid these side effects by influencing binding and/or efficacy of orthosteric ligands. Here, we summarize and compare previous data on different putative allosteric binding sites observed in CB homology models with an in silico docking study of the recently published crystal structure of the same receptor on endogenous and natural hydrophobic ligands that act as positive allosteric modulators and negative allosteric modulators of CB . In particular, a lipid-exposed pocket targeted by most of the tested molecules is reported and discussed.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/bab.1589DOI Listing
January 2018

Molecular dynamics study on the Apo- and Holo-forms of 5-lipoxygenase.

Biotechnol Appl Biochem 2018 Jan 7;65(1):54-61. Epub 2017 Sep 7.

European Center for Brain Research (CERC)/Santa Lucia Foundation IRCCS, Rome, Italy.

Lipoxygenases (LOXs) are nonheme iron-containing enzymes catalyzing the dioxygenation of polyunsaturated fatty acids. LOX catalytic activity depends on the presence of iron in the active site and the iron removal is also able to affect the membrane binding properties of the enzyme. Leukotrienes biosynthesis is initiated by the action of 5-LOX at the level of nuclear membrane and the mechanism of enzyme-membrane interaction is thought to involve structural flexibility and conformational changes at the level of the protein tertiary structure. In this study, we have analyzed by molecular dynamics simulations the conformational changes induced by iron removal in 5-LOX. The data indicate that the degree of enzyme flexibility is related to the presence of iron into the active site that is able to stabilize the protein increasing its rigidity. These findings provide further evidence that the conformation and the functional activity of LOXs is tuned by the presence of iron at the active site, suggesting new approaches for the design of enzyme inhibitors.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/bab.1583DOI Listing
January 2018

Role of palmitoylation of cysteine 415 in functional coupling CB receptor to Gα protein.

Biotechnol Appl Biochem 2018 Jan 15;65(1):16-20. Epub 2017 Aug 15.

Faculty of Bioscience and Technology for Food Agriculture and Environment, University of Teramo, Teramo, Italy.

In this study, we investigated the role of CB palmitoylation in modulating the functional interaction with G proteins both in the absence and presence of agonist binding. Our data show that the nonpalmitoylated CB receptor significantly reduced its association with Gα . The agonist stimulation induced a partial dissociation of Gα proteins from the wild-type receptor, while on the C415A mutant the agonist binding was not able to induce a significant dissociation of Gα from the receptor. The lack of palmitoyl chain seems to hamper the ability of the receptor to functionally interact with the Gα and indicate that the palmitoyl chain is responsible for the functional transmission of the agonist-induced conformational change in the receptor of the G protein. These data were further corroborated by molecular dynamics simulations. Overall these results suggest that palmitoylation of the CB receptor finely tunes its interaction with G proteins and serves as a targeting signal for its functional regulation. Of note, the possibility to reversibly modulate the palmitoylation of CB receptor may offer a coordinated process of regulation and could open new therapeutic approaches.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/bab.1575DOI Listing
January 2018

A Clinical Update of the Hb Siirt [β27(B9)Ala→Gly; HBB: c.83C>G] Hemoglobin Variant.

Hemoglobin 2017 Jan 10;41(1):53-55. Epub 2017 Apr 10.

a Azzociazione Nazionale per la lotta alla Microcitemia in Italia (ANMI) Onlus, Centro Studi Microcitemie Roma , Roma , Italia.

We report a clinical update of the hemoglobin (Hb) variant [β27(B9)Ala→Gly; HBB: c.83C>G], named Hb Siirt, that was previously described as a silent variant in a 23-year-old Kurdish female. The patient was also a carrier of the codon 5 (-CT) (HBB: c.17_18delCT) frameshift mutation and of the ααα triplication. Her initial moderate β-thalassemia intermedia (β-TI) phenotype worsened with time, causing the patient to become a transfusion-dependent subject at the age of ∼40 years. Subsequent molecular characterization of both parents revealed that the Hb Siirt variant was inherited by the mother, while the other two globin alterations (HBB: c.17_18delCT and ααα triplication) were genetically transmitted by the father. The latter remained a carrier of a mild β-TI phenotype throughout his life, at least until the age of 65 years. We hypothesize that the worsened clinical conditions in the daughter were due to the additional, maternally inherited Hb Siirt variant. However, protein 3D conformational analysis did not seem to reveal substantial overall structural changes. Among the other three described variants [Hb Volga (HBB: c.83C>A), Hb Knossos (HBB: c.82 G>T), Hb Grange-Blanche (HBB: c.83C>T] that are due to nucleotide substitutions at codon 27 of the β-globin gene; only Hb Knossos causes a β-thalassemia (β-thal) phenotype.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1080/03630269.2017.1302469DOI Listing
January 2017

Palmitoylation of cysteine 415 of CB receptor affects ligand-stimulated internalization and selective interaction with membrane cholesterol and caveolin 1.

Biochim Biophys Acta Mol Cell Biol Lipids 2017 May 12;1862(5):523-532. Epub 2017 Feb 12.

European Center for Brain Research (CERC)/Santa Lucia Foundation IRCCS, Rome, Italy; Department of Medicine, Campus Bio-Medico University of Rome, Rome, Italy. Electronic address:

We previously demonstrated that CB receptor is palmitoylated at cysteine 415, and that such a post-translational modification affects its biological activity. To assess the molecular mechanisms responsible for modulation of CB receptor function by S-palmitoylation, in this study biochemical and morphological approaches were paralleled with computational analyses. Molecular dynamics simulations suggested that this acyl chain stabilizes helix 8 as well as the interaction of CB receptor with membrane cholesterol. In keeping with these in silico data, experimental results showed that the non-palmitoylated CB receptor was unable to interact efficaciously with caveolin 1, independently of its activation state. Moreover, in contrast with the wild-type receptor, the lack of S-palmitoylation in the helix 8 made the mutant CB receptor completely irresponsive to agonist-induced effects in terms of both lipid raft partitioning and receptor internalization. Overall, our results support the notion that palmitoylation of cysteine 415 modulates the conformational state of helix 8 and influences the interactions of CB receptor with cholesterol and caveolin 1, suggesting that the palmitoyl chain may serve as a functional interface for CB receptor localization and function.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.bbalip.2017.02.004DOI Listing
May 2017

Measuring ECS Interaction with Biomembranes.

Methods Mol Biol 2016 ;1412:267-76

Faculty of Bioscience and Technology for Food Agriculture and Environment, University of Teramo, Teramo, Italy.

Understanding the correct interaction among the different components of the endocannabinoid system (ECS) is fundamental for a proper assessment of the function of endocannabinoids (eCBs) as signaling molecules. The knowledge of how membrane environment is able to modulate intracellular trafficking of eCBs and their interacting proteins holds a huge potential in unraveling new mechanisms of ECS modulation.Here, fluorescence resonance energy transfer (FRET) technique is applied to measure the binding affinity of ECS proteins to model membranes (i.e., large unilamellar vesicles, LUVs). In particular, we describe in details the paradigmatic example of the interaction of recombinant rat FAAH-ΔTM with LUVs constituted by 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC).
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1007/978-1-4939-3539-0_27DOI Listing
December 2017

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

J Nutr Biochem 2015 Mar 3;26(3):250-8. Epub 2014 Dec 3.

Faculty of Biosciences, University of Teramo, Teramo, Italy; Department of Clinical Neuroscience, Karolinska Institutet, Stockholm, Sweden. Electronic address:

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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.jnutbio.2014.10.013DOI Listing
March 2015

A functional interplay between 5-lipoxygenase and μ-calpain affects survival and cytokine profile of human Jurkat T lymphocyte exposed to simulated microgravity.

Biomed Res Int 2014 16;2014:782390. Epub 2014 Sep 16.

European Center for Brain Research (CERC), IRCCS Santa Lucia Foundation, Via del Fosso di Fiorano 64-65, 00143 Rome, Italy ; Center of Integrated Research, Campus Bio-Medico University of Rome, Via Alvaro del Portillo 21, 00128 Rome, Italy.

A growing body of evidence strongly indicates that both simulated and authentic weightlessness exert a broad range of effects on mammalian tissues and cells, including impairment of immune cell function and increased apoptotic death. We previously reported that microgravity-dependent activation of 5-lipoxygenase (5-LOX) might play a central role in the initiation of apoptosis in human T lymphocytes, suggesting that the upregulation of this enzyme might be (at least in part) responsible for immunodepression observed in astronauts during space flights. Herein, we supplement novel information about the molecular mechanisms underlying microgravity-triggered apoptotic cell death and immune system deregulation, demonstrating that under simulated microgravity human Jurkat T cells increase the content of cytosolic DNA fragments and cytochrome c (typical hallmarks of apoptosis) and have an upregulated expression and activity of µ-calpain. These events were paralleled by the unbalance of interleukin- (IL-) 2 and interferon- (INF-) γ, anti- and proapoptotic cytokines, respectively, that seemed to be dependent on the functional interplay between 5-LOX and µ-calpain. Indeed, we report unprecedented evidence that 5-LOX inhibition reduced apoptotic death, restored the initial IL-2/INF-γ ratio, and more importantly reverted µ-calpain activation induced by simulated microgravity.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1155/2014/782390DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4182306PMC
July 2015

Domain mobility as probed by small-angle X-ray scattering may account for substrate access to the active site of two copper-dependent amine oxidases.

Acta Crystallogr D Biol Crystallogr 2014 Aug 25;70(Pt 8):2101-10. Epub 2014 Jul 25.

European Center for Brain Research (CERC)/Santa Lucia Foundation, Rome, Italy.

Amine oxidases are a family of dimeric enzymes that contain one copper(II) ion and one 2,4,5-trihydroxyphenyalanine quinone per subunit. Here, the low-resolution structures of two Cu/TPQ amine oxidases from lentil (Lens esculenta) seedlings and from Euphorbia characias latex have been determined in solution by small-angle X-ray scattering. The active site of these enzymes is highly buried and requires a conformational change to allow substrate access. The study suggests that the funnel-shaped cavity located between the D3 and D4 domains is narrower within the crystal structure, whereas in solution the D3 domain could undergo movement resulting in a protein conformational change that is likely to lead to easier substrate access.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1107/S1399004714012140DOI Listing
August 2014

Membrane lipids are key modulators of the endocannabinoid-hydrolase FAAH.

Biochem J 2014 Feb;457(3):463-72

¶Departments of Cell Biology and Chemistry, The Skaggs Institute for Chemical Biology, The Scripps Research Institute, La Jolla, CA, U.S.A.

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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1042/BJ20130960DOI Listing
February 2014

Probing conformational changes in lipoxygenases upon membrane binding: fine-tuning by the active site inhibitor ETYA.

Biochim Biophys Acta 2014 Jan 3;1841(1):1-10. Epub 2013 Sep 3.

Department of Experimental Medicine and Surgery, Tor Vergata University of Rome, Via Montpellier 1, 00133 Rome, Italy.

Lipoxygenases (LOXs) are lipid-peroxidizing enzymes that are involved in the metabolism of polyunsaturated fatty acids. Their biological activity includes a membrane binding process whose molecular details are not completely understood. The mechanism of enzyme-membrane interactions is thought to involve conformational changes at the level of the protein tertiary structure, and the extent of such alterations depends on the degree of structural flexibility of the different LOX isoforms. In this study, we have tested the resilience properties of a plant and a mammalian LOX, by using high pressure fluorescence measurements at different temperatures. The binding of LOXs to the lipid bilayer has been characterized using both large and giant unilamellar vesicles and electron transfer particles (inner mitochondrial membranes) as model membranes. The data indicate that the degree of LOXs' flexibility is strictly dependent on the two distinct N- and C-terminal domains that characterize the 3D structure of these enzymes. Furthermore, they demonstrate that increasing the rigidity of protein scaffolding by the presence of an active site ligand impairs the membrane binding ability of LOXs. These findings provide evidence that the amphitropic nature of LOXs is finely tuned by the interaction of the substrate with the residues of the active site, suggesting new strategies for the design of enzyme inhibitors.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.bbalip.2013.08.015DOI Listing
January 2014

Epigenetic control of skin differentiation genes by phytocannabinoids.

Br J Pharmacol 2013 Oct;170(3):581-91

Department of Biomedical Sciences, University of Teramo, Teramo, Italy.

Background And Purpose: Endocannabinoid signalling has been shown to have a role in the control of epidermal physiology, whereby anandamide is able to regulate the expression of skin differentiation genes through DNA methylation. Here, we investigated the possible epigenetic regulation of these genes by several phytocannabinoids, plant-derived cannabinoids that have the potential to be novel therapeutics for various human diseases.

Experimental Approach: The effects of cannabidiol, cannabigerol and cannabidivarin on the expression of skin differentiation genes keratins 1 and 10, involucrin and transglutaminase 5, as well as on DNA methylation of keratin 10 gene, were investigated in human keratinocytes (HaCaT cells). The effects of these phytocannabinoids on global DNA methylation and the activity and expression of four major DNA methyltransferases (DNMT1, 3a, 3b and 3L) were also examined.

Key Results: Cannabidiol and cannabigerol significantly reduced the expression of all the genes tested in differentiated HaCaT cells, by increasing DNA methylation of keratin 10 gene, but cannabidivarin was ineffective. Remarkably, cannabidiol reduced keratin 10 mRNA through a type-1 cannabinoid (CB1 ) receptor-dependent mechanism, whereas cannabigerol did not affect either CB1 or CB2 receptors of HaCaT cells. In addition, cannabidiol, but not cannabigerol, increased global DNA methylation levels by selectively enhancing DNMT1 expression, without affecting DNMT 3a, 3b or 3L.

Conclusions And Implications: These findings show that the phytocannabinoids cannabidiol and cannabigerol are transcriptional repressors that can control cell proliferation and differentiation. This indicates that they (especially cannabidiol) have the potential to be lead compounds for the development of novel therapeutics for skin diseases.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1111/bph.12309DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3791996PMC
October 2013

Involvement of 5-lipoxygenase in Alzheimer's disease: a role for DNA methylation.

J Alzheimers Dis 2013 ;37(1):3-8

Department of Biomedical Sciences, University of Teramo, Teramo, Italy.

Lipoxygenases play a major role in the neuropathology of Alzheimer's disease (AD), even though the underlying mechanisms are as yet poorly understood. Here, we studied the epigenetic regulation of 5-lipoxygenase (5-LOX) in peripheral blood mononuclear cells of subjects with late-onset AD and age-matched controls. We found a significant increase in 5-LOX gene expression in AD subjects compared to healthy controls, paralleled by increased 5-LOX protein and leukotriene B4, the 5-LOX product. In addition, a consistent reduction in DNA methylation at 5-LOX gene promoter was documented in AD versus healthy subjects. Taken together, our findings further support a role for 5-LOX in vulnerability to neurodegeneration.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3233/JAD-130506DOI Listing
April 2014

Rat and human fatty acid amide hydrolases: overt similarities and hidden differences.

Biochim Biophys Acta 2012 Nov 3;1821(11):1425-33. Epub 2012 Aug 3.

Neuromed, Pozzilli, Italy.

Fatty acid amide hydrolase (FAAH) is a membrane protein that plays a relevant role in the metabolism of fatty acid amides and esters. It degrades important neurotransmitters such as oleamide and anandamide, and it has been involved in a number of human pathological conditions, representing therefore a valuable target for biochemical and pharmacological research. In this study, we have investigated in vitro the structure-function relationship of rat and human FAAHs. In particular circular dichroism, fluorescence spectroscopy and light scattering measurements have been performed, in order to characterize the structural features of the two proteins, both in the presence and absence of the irreversible inhibitor methoxyarachidonyl-fluorophosphonate. The results demonstrate that the structural dynamics of the two FAAHs are different, despite their high sequence homology and overall similarity in temperature-dependence. Additionally, membrane binding and kinetic assays of both FAAHs indicate that also the functional properties of the two enzymes are different in their interaction with lipid bilayers and with exogenous inhibitors. These findings suggest that pre-clinical studies of FAAH-dependent human diseases based only on animal models should be interpreted with caution, and that the efficacy of new drugs targeted to FAAH should be tested in vitro, on both rat and human enzymes.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.bbalip.2012.07.021DOI Listing
November 2012

Impact of embedded endocannabinoids and their oxygenation by lipoxygenase on membrane properties.

ACS Chem Neurosci 2012 May 24;3(5):386-92. Epub 2012 Feb 24.

Department of Biomedical Sciences, University of Teramo, Teramo, Italy.

N-Arachidonoylethanolamine (anandamide) and 2-arachidonoylglycerol are the best characterized endocannabinoids. Their biological activity is subjected to metabolic control whereby a dynamic equilibrium among biosynthetic, catabolic, and oxidative pathways drives their intracellular concentrations. In particular, lipoxygenases can generate hydroperoxy derivatives of endocannabinoids, endowed with distinct activities within cells. The in vivo interaction between lipoxygenases and endocannabinoids is likely to occur within cell membranes; thus, we sought to ascertain whether a prototypical enzyme like soybean (Glycine max) 15-lipoxygenase-1 is able to oxygenate endocannabinoids embedded in synthetic vesicles and how these substances could affect the binding ability of the enzyme to different lipid bilayers. We show that (i) embedded endocannabinoids increase membrane fluidity; (ii) 15-lipoxygenase-1 preferentially binds to endocannabinoid-containing bilayers; and that (iii) 15-lipoxygenase-1 oxidizes embedded endocannabinoids and thus reduces fluidity and local hydration of membrane lipids. Together, the present findings reveal further complexity in the regulation of endocannabinoid signaling within the central nervous system, disclosing novel control by oxidative pathways.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1021/cn300016cDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3386857PMC
May 2012

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

FASEB J 2012 May 17;26(5):1791-8. Epub 2012 Jan 17.

Department of Biomedical Sciences, University of Teramo, Piazza A. Moro 45, I-64100 Teramo, Italy.

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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1096/fj.11-199406DOI Listing
May 2012

Effects of palmitoylation of Cys(415) in helix 8 of the CB(1) cannabinoid receptor on membrane localization and signalling.

Br J Pharmacol 2012 Apr;165(8):2635-51

Department of Biomedical Sciences, University of Teramo, Teramo, Italy European Center for Brain Research (CERC)/Santa Lucia Foundation I.R.C.C.S., Rome, Italy.

Background And Purpose: The CB(1) cannabinoid receptor is regulated by its association with membrane microdomains such as lipid rafts. Here, we investigated the role of palmitoylation of the CB(1) receptor by analysing the functional consequences of site-specific mutation of Cys(415) , the likely site of palmitoylation at the end of helix 8, in terms of membrane association, raft targeting and signalling.

Experimental Approach: The palmitoylation state of CB(1) receptors in rat forebrain was assessed by depalmitoylation/repalmitoylation experiments. Cys(415) was replaced with alanine by site-directed mutagenesis. Green fluorescence protein chimeras of both wild-type and mutant receptors were transiently expressed and functionally characterized in SH-SY5Y cells and HEK-293 cells by means of confocal microscopy, cytofluorimetry and competitive binding assays. Confocal fluorescence recovery after photobleaching was used to assess receptor membrane dynamics, whereas signalling activity was assessed by [(35) S]GTPγS, cAMP and co-immunoprecipitation assays.

Key Results: Endogenous CB(1) receptors in rat brain were palmitoylated. Mutation of Cys(415) prevented the palmitoylation of the receptor in transfected cells and reduced its recruitment to plasma membrane and lipid rafts; it also increased protein diffusional mobility. The same mutation markedly reduced the functional coupling of CB(1) receptors with G-proteins and adenylyl cyclase, whereas depalmitoylation abolished receptor association with a specific subset of G-proteins.

Conclusions And Implications: CB(1) receptors were post-translationally modified by palmitoylation. Mutation of Cys(415) provides a receptor that is functionally impaired in terms of membrane targeting and signalling.

Linked Articles: This article is part of a themed section on Cannabinoids in Biology and Medicine. To view the other articles in this section visit http://dx.doi.org/10.1111/bph.2012.165.issue-8. To view Part I of Cannabinoids in Biology and Medicine visit http://dx.doi.org/10.1111/bph.2011.163.issue-7.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1111/j.1476-5381.2011.01658.xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3423250PMC
April 2012

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

J Neurochem 2011 Mar 7;116(5):858-65. Epub 2011 Jan 7.

Department of Biomedical Sciences, University of Teramo, Teramo, Italy.

Endocannabinoid signaling modulates a variety of neuroinflammatory and neurodegenerative diseases, mainly through the activation of type-1 and type-2 (CB(1)R and CB(2)R) cannabinoid receptors. CB(1)R is negatively regulated by membrane cholesterol, while CB(2)R is unaffected. Here, we identified in the transmembrane helix 7 of human CBRs a consensus sequence already known in other proteins as cholesterol recognition/interaction amino acid sequence and consensus pattern. As this motif is different in the two CBR subtypes, we mutated lysine 402 of CB(1)R into glycine, to obtain a cholesterol recognition/interaction amino acid sequence and consensus similar to that of CB(2)R. Both mutated and wild-type receptors were transiently expressed in human neuronal SH-SY5Y cells, and their localization and functioning were investigated using biochemical assays and immunofluorescence labelling. We found a reduced propensity of the mutant CB(1)R to reside in cholesterol-rich microdomains and, by means of fluorescence recovery after photobleaching analysis, we documented its loss of sensitivity to increased membrane cholesterol content. These results seem to uncover the existence of a new structural determinant in cannabinoid receptors, that is likely implicated in directing their interaction with cholesterol-rich microdomains of cell membranes.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1111/j.1471-4159.2010.07041.xDOI Listing
March 2011