Conformational Dynamics of Lipoxygenases and Their Interaction with Biological Membranes.

Lipoxygenases (LOXs) are a family of enzymes that includes different fatty acid oxygenases with a common tridimensional structure. The main functions of LOXs are the production of signaling compounds and the structural modifications of biological membranes. These features of LOXs, their widespread presence in all living organisms, and their involvement in human diseases have attracted the attention of the scientific community over the last decades, leading to several studies mainly focused on understanding their catalytic mechanism and designing effective inhibitors. The aim of this review is to discuss the state-of-the-art of a different, much less explored aspect of LOXs, that is, their interaction with lipid bilayers. To this end, the general architecture of six relevant LOXs (namely human 5-, 12-, and 15-LOX, rabbit 12/15-LOX, coral 8-LOX, and soybean 15-LOX), with different specificity towards the fatty acid substrates, is analyzed through the available crystallographic models. Then, their putative interface with a model membrane is examined in the frame of the conformational flexibility of LOXs, that is due to their peculiar tertiary structure. Finally, the possible future developments that emerge from the available data are discussed.

A possible role for G-quadruplexes formation and DNA methylation at IMOOD gene promoter in Obsessive Compulsive Disorder.

Obsessive Compulsive Disorder (OCD) is a mental health condition still classified and diagnosed with subjective interview-based assessments and which molecular clues have not completely been elucidated. We have recently identified a new regulator of anxiety and OCD-like behavior called Immuno-moodulin (IMOOD) and, here, we report that IMOOD gene promoter is differentially methylated in OCD subjects when compared to genomic material collected from healthy controls and this alteration is significantly correlated with the increased expression of the gene in OCD. We also demonstrated that IMOOD promoter can form G-quadruplexes and we suggest that, in homeostatic conditions, these structures could evoke DNA-methylation silencing the gene, whereas in pathological conditions, like OCD, could induce gene expression making the promoter more accessible to transcriptional factors. We here thus further suggest IMOOD as a new biomarker for OCD and also hypothesize new mechanisms of gene regulation.

Bioinformatics of the Endocannabinoid System: Study of DNA Methylation at Rat Cnr1 Gene Promoter.

In this chapter, we will describe the bioinformatic tools that allow verifying the presence of CpG islands in a gene promoter region. We will also describe the tools needed to identify consensus motifs for specific transcription factors, focusing on the study of rat type-1 cannabinoid receptor gene (R_Cnr1) as a case study.

Measuring Endocannabinoid System Interaction with Biomembranes.

Understanding the correct interaction among the different components of the endocannabinoid (eCB) system is fundamental for a proper assessment of the function of eCBs as signaling molecules. The knowledge of how the membrane environment modulates the intracellular trafficking of the eCB system and its interacting proteins holds a huge potential in unraveling new mechanisms of its modulation. This chapter deals with the application of fluorescence resonance energy transfer technique to measure the binding affinity of eCB proteins to model membranes (i.e., large unilamellar vesicles, LUVs). In particular, we describe in detail the paradigmatic example of the interaction of rat recombinant fatty acid amide hydrolase with LUVs constituted of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine.

Regulation of oxytocin receptor gene expression in obsessive-compulsive disorder: a possible role for the microbiota-host epigenetic axis.

BACKGROUND: Obsessive-compulsive disorder (OCD) is a prevalent and severe clinical condition. Robust evidence suggests a gene-environment interplay in its etiopathogenesis, yet the underlying molecular clues remain only partially understood. In order to further deepen our understanding of OCD, it is essential to ascertain how genes interact with environmental risk factors, a cross-talk that is thought to be mediated by epigenetic mechanisms. The human microbiota may be a key player, because bacterial metabolites can act as epigenetic modulators. We analyzed, in the blood and saliva of OCD subjects and healthy controls, the transcriptional regulation of the oxytocin receptor gene and, in saliva, also the different levels of major phyla. We also investigated the same molecular mechanisms in specific brain regions of socially isolated rats showing stereotyped behaviors reminiscent of OCD as well as short chain fatty acid levels in the feces of rats. RESULTS: Higher levels of oxytocin receptor gene DNA methylation, inversely correlated with gene expression, were observed in the blood as well as saliva of OCD subjects when compared to controls. Moreover, Actinobacteria also resulted higher in OCD and directly correlated with oxytocin receptor gene epigenetic alterations. The same pattern of changes was present in the prefrontal cortex of socially-isolated rats, where also altered levels of fecal butyrate were observed at the beginning of the isolation procedure. CONCLUSIONS: This is the first demonstration of an interplay between microbiota modulation and epigenetic regulation of gene expression in OCD, opening new avenues for the understanding of disease trajectories and for the development of new therapeutic strategies.

Controlled spermatozoa-oocyte interaction improves embryo quality in sheep.

The current protocols of in vitro fertilization and culture in sheep rely on paradigms established more than 25 years ago, where Metaphase II oocytes are co-incubated with capacitated spermatozoa overnight. While this approach maximizes the number of fertilized oocytes, on the other side it exposes them to high concentration of reactive oxygen species (ROS) generated by active and degenerating spermatozoa, and positively correlates with polyspermy. Here we set up to precisely define the time frame during which spermatozoa effectively penetrates and fertilizes the oocyte, in order to drastically reduce spermatozoa-oocyte interaction. To do that, in vitro matured sheep oocytes co-incubated with spermatozoa in IVF medium were sampled every 30 min (start of incubation time 0) to verify the presence of a fertilizing spermatozoon. Having defined the fertilization time frame (4 h, data from 105 oocytes), we next compared the standard IVF procedures overnight (about 16 h spermatozoa/oocyte exposure, group o/nIVF) with a short one (4 h, group shIVF). A lower polyspermic fertilization (> 2PN) was detected in shIVF (6.5%) compared to o/nIVF (17.8%), P < 0.05. The o/nIVF group resulted in a significantly lower 2-cell stage embryos, than shIVF [34.6% (81/234) vs 50.6% (122/241) respectively, P < 0.001]. Likewise, the development to blastocyst stage confirmed a better quality [29% (70/241) vs 23.5% (55/234), shIVF vs o/nIVF respectively] and an increased Total Cell Number (TCN) in shIVF embryos, compared with o/n ones. The data on ROS have confirmed that its generation is IVF time-dependent, with high levels in the o/nIVF group. Overall, the data suggest that a shorter oocyte-spermatozoa incubation results in an improved embryo production and a better embryo quality, very likely as a consequence of a shorter exposure to the free oxygen radicals and the ensuing oxidative stress imposed by overnight culture.

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.

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.

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

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

The endocannabinoid hydrolase FAAH is an allosteric enzyme.

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.

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

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.

Modulation of Endocannabinoid-Binding Receptors in Human Neuroblastoma Cells by Tunicamycin.

Endocannabinoid (eCB)-binding receptors can be modulated by several ligands and membrane environment, yet the effect of glycosylation remains to be assessed. In this study, we used human neuroblastoma SH-SY5Y cells to interrogate whether expression, cellular localization, and activity of eCB-binding receptors may depend on N-linked glycosylation. Following treatment with tunicamycin (a specific inhibitor of N-linked glycosylation) at the non-cytotoxic dose of 1 µg/mL, mRNA, protein levels and localization of eCB-binding receptors, as well as N-acetylglucosamine (GlcNAc) residues, were evaluated in SH-SY5Y cells by means of quantitative real-time reverse transcriptase-polymerase chain reaction (qRT-PCR), fluorescence-activated cell sorting (FACS), and confocal microscopy, respectively. In addition, the activity of type-1 and type-2 cannabinoid receptors (CB1 and CB2) was assessed by means of rapid binding assays. Significant changes in gene and protein expression were found upon tunicamycin treatment for CB1 and CB2, as well as for GPR55 receptors, but not for transient receptor potential vanilloid 1 (TRPV1). Deglycosylation experiments with N-glycosidase-F and immunoblot of cell membranes derived from SH-SY5Y cells confirmed the presence of one glycosylated form in CB1 (70 kDa), that was reduced by tunicamycin. Morphological studies demonstrated the co-localization of CB1 with GlcNAc residues, and showed that tunicamycin reduced CB1 membrane expression with a marked nuclear localization, as confirmed by immunoblotting. Cleavage of the carbohydrate side chain did not modify CB receptor binding affinity. Overall, these results support N-linked glycosylation as an unprecedented post-translational modification that may modulate eCB-binding receptors' expression and localization, in particular for CB1.

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

Background: 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 in vivo 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. Methods: 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. Results: 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. Conclusions: Overall, the results point to steroids as new regulators of FAAH interaction with membranes, which may impact the biological activity of eCBs.

Amine oxidase from Euphorbia characias: Kinetic and structural characterization.

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.

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

The recent resolution of the crystal structure of type-1 cannabinoid receptor (CB1 ) and the discovery of novel modulators for this target open the way to the possibility of elucidating the structural requirements for CB1 binding, and thereby facilitate a rational drug design. Compounds that target the orthosteric site of CB1 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 CB1 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 CB1 . In particular, a lipid-exposed pocket targeted by most of the tested molecules is reported and discussed.

A Clinical Update of the Hb Siirt [ß27(B9)Ala→Gly; HBB: c.83C>G] Hemoglobin Variant.

We report a clinical update of the hemoglobin (Hb) variant [ß27(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 anti 3.7 triplication. Her initial moderate ß-thalassemia intermedia (ß-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 αααanti 3.7 triplication) were genetically transmitted by the father. The latter remained a carrier of a mild ß-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 ß-globin gene; only Hb Knossos causes a ß+-thalassemia (ß+-thal) phenotype.

Measuring ECS Interaction with Biomembranes.

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).

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.

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.

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.

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

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.

A novel role for iron in modulating the activity and membrane-binding ability of a trimmed soybean lipoxygenase-1.

Lipoxygenases (LOXs) are iron-containing enzymes that play critical roles in plants and animals. As yet, metal atom extraction, reconstitution, and substitution have not been successfully applied to soybean LOX-1 [Glycine max (L.) Merrill], a prototype member of the LOX family that is widely used in structural and kinetic studies. Here, tryptic digestion of native LOX-1, used as a control, allowed us to isolate the 60-kDa C-terminal region (termed miniLOX), that retains the catalytically active iron in a more accessible position. Then, iron was removed to obtain an unprecedented apo-miniLOX, which was reconstituted and substituted with different metal ions. These forms of miniLOX were characterized vs. native LOX-1 by kinetic analysis, near UV circular dichroism, steady-state fluorescence, and fluorescence resonance energy transfer. MiniLOX showed a 2-fold increase in the membrane-binding affinity compared with native LOX-1 and a remarkable 4-fold increase compared with apo-miniLOX (K(d)=9.2+/-1.0, 17.9+/-2.0, and 45.4+/-4.3 microM, respectively). Furthermore, miniLOX reconstituted with Fe(II) or Fe(III) partially recovered its membrane-binding ability (K(d)=21.4+/-2.4 and 18.9+/-5.5 microM, respectively), overall supporting a novel noncatalytic role for iron in the LOX family.