Cellular Prion Protein Expression in the Brain Tissue from Brucella ceti-Infected Striped Dolphins (Stenella coeruleoalba)

Brucella ceti, a zoonotic pathogen of major concern to cetacean health and conservation, is responsible for severe meningo-encephalitic/myelitic lesions in striped dolphins (Stenella coeruleoalba), often leading to their stranding and death. This study investigated, for the first time, the cellular prion protein (PrPc) expression in the brain tissue from B. ceti-infected, neurobrucellosis-affected striped dolphins. Seven B. ceti-infected, neurobrucellosis-affected striped dolphins, found stranded along the Italian coastline (6) and in the Canary Islands (1), were investigated, along with five B. ceti-uninfected striped dolphins from the coast of Italy, carrying no brain lesions, which served as negative controls. Western Blot (WB) and immunohistochemistry (IHC) with an anti-PrP murine monoclonal antibody were carried out on the brain parenchyma of these dolphins. While PrPc IHC yielded inconclusive results, a clear-cut PrPc expression of different intensity was found by means of WB analyses in the brain tissue of all the seven herein investigated, B. ceti-infected and neurobrucellosis-affected cetacean specimens, with two dolphins stranded along the Italian coastline and one dolphin beached in Canary Islands also exhibiting a statistically significant increase in cerebral PrPc expression as compared to the five Brucella spp.-negative control specimens. The significantly increased PrPc expression found in three out of seven B. ceti-infected, neurobrucellosis-affected striped dolphins does not allow us to draw any firm conclusion(s) about the putative role of PrPc as a host cell receptor for B. ceti. Should this be the case, an upregulation of PrPc mRNA in the brain tissue of neurobrucellosis-affected striped dolphins could be hypothesized during the different stages of B. ceti infection, as previously shown in murine bone marrow cells challenged with Escherichia coli. Noteworthy, the inflammatory infiltrates seen in the brain and in the cervico-thoracic spinal cord segments from the herein investigated, B. ceti-infected and neurobrucellosis-affected striped dolphins were densely populated by macrophage/histiocyte cells, often harboring Brucella spp. antigen in their cytoplasm, similarly to what was reported in macrophages from mice experimentally challenged with B. abortus. Notwithstanding the above, much more work is needed in order to properly assess the role of PrPc, if any, as a host cell receptor for B. ceti in striped dolphins

Cellular Prion Protein Expression in the Brain Tissue from Brucella ceti-Infected Striped Dolphins (Stenella coeruleoalba)

Brucella ceti, a zoonotic pathogen of major concern to cetacean health and conservation, is responsible for severe meningo-encephalitic/myelitic lesions in striped dolphins (Stenella coeruleoalba), often leading to their stranding and death. This study investigated, for the first time, the cellular prion protein (PrPc) expression in the brain tissue from B. ceti-infected, neurobrucellosis-affected striped dolphins. Seven B. ceti-infected, neurobrucellosis-affected striped dolphins, found stranded along the Italian coastline (6) and in the Canary Islands (1), were investigated, along with five B. ceti-uninfected striped dolphins from the coast of Italy, carrying no brain lesions, which served as negative controls. Western Blot (WB) and immunohistochemistry (IHC) with an anti-PrP murine monoclonal antibody were carried out on the brain parenchyma of these dolphins. While PrPc IHC yielded inconclusive results, a clear-cut PrPc expression of different intensity was found by means of WB analyses in the brain tissue of all the seven herein investigated, B. ceti-infected and neurobrucellosis-affected cetacean specimens, with two dolphins stranded along the Italian coastline and one dolphin beached in Canary Islands also exhibiting a statistically significant increase in cerebral PrPc expression as compared to the five Brucella spp.-negative control specimens. The significantly increased PrPc expression found in three out of seven B. ceti-infected, neurobrucellosis-affected striped dolphins does not allow us to draw any firm conclusion(s) about the putative role of PrPc as a host cell receptor for B. ceti. Should this be the case, an upregulation of PrPc mRNA in the brain tissue of neurobrucellosis-affected striped dolphins could be hypothesized during the different stages of B. ceti infection, as previously shown in murine bone marrow cells challenged with Escherichia coli. Noteworthy, the inflammatory infiltrates seen in the brain and in the cervico-thoracic spinal cord segments from the herein investigated, B. ceti-infected and neurobrucellosis-affected striped dolphins were densely populated by macrophage/histiocyte cells, often harboring Brucella spp. antigen in their cytoplasm, similarly to what was reported in macrophages from mice experimentally challenged with B. abortus. Notwithstanding the above, much more work is needed in order to properly assess the role of PrPc, if any, as a host cell receptor for B. ceti in striped dolphins

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

Iron modulates the membrane-binding and the intracellular trafficking of 5-lipoxygenase: functional implications in neurodegeneration

Iron modulates the membrane-binding and the intracellular trafficking of 5-lipoxygenase: functional implications in neurodegeneration Beatrice Dufrusine1, Andrea Di Francesco1, Annalaura Sabatucci1, Clotilde Beatrice Angelucci2, Sergio Oddi2,3, Claudio D’Addario1,3, Dieter Steinhilber4, Mauro Maccarrone3,5* and Enrico Dainese1,3* 1Faculty of Biosciences, University of Teramo, Teramo, 64100, Italy; 2 Faculty of Veterinary Medicine, University of Teramo, Teramo, 64100, Italy; 3 European Center for Brain Research (CERC)/Santa Lucia Foundation, Rome, Italy; 4 Institute of Pharmaceutical Chemistry, Goethe University Frankfurt, Germany; 5 Center of Integrated Research, Campus Bio-Medico University of Rome, Italy. *Equally senior authors. Introduction 5-lipoxygenase (5-LOX) is a non-heme iron-containing enzyme catalyzing the initial steps in the biosynthesis of leukotrienes, inflammatory eicosanoids that are capable of promoting neurodegeneration. Iron accumulation has been demonstrated in Alzheimer’s disease (AD). Here we present the structure of 5-LOX in solution and a new molecular mechanism throughout the iron content of the enzyme is able to influence its membrane binding and subcellular distribution in THP-1 macrophages. Methods 3D structure in solution and membrane binding experiments were done using Small angle X-ray scattering (SAXS) and fluorescence resonance energy transfer (FRET) approaches, as reported [1]. Confocal fluorescence microscopy, subcellular fractionation and western blot (WB) analysis were carried out as already described [1]. Late Onset AD (LOAD) patients and non-demented healthy subjects (CT) were recruited as described [2]. Results SAXS analysis revealed that 5-LOX in solution is mostly organized as a homodimer. Iron removal from the recombinant human 5-LOX altered the catalytic activity of the enzyme, and impaired its membrane-binding ability. THP-1 cells exposed to increasing amounts of iron showed a redistribution of the cytosolic 5-LOX to the nuclear compartment. Additionally, a significant correlation between plasma levels of the 5-LOX end-product LTB4 and hemoglobin was observed in a population of healthy subjects and AD patients. Conclusions These results suggest that exogenous iron modulates 5-LOX activity by increasing its ability to bind to nuclear membranes, further supporting a new role for iron in inflammation-based neurological diseases where its homeostasis is altered. Acknowledgements. M.M. and E.D. wish to thank EU for granting the Biostruct-X project within the FP VII programme. [1] E. Dainese et al., FASEB J. 24 (2010) 1725-1736. [2] A. Di Francesco et al., JAD 37 (2013) 3-8

Effects of Rare Phytocannabinoids on the Endocannabinoid System of Human Keratinocytes

The decriminalization and legalization of cannabis has paved the way for investigations into the potential of the use of phytocannabinoids (pCBs) as natural therapeutics for the treatment of human diseases. This growing interest has recently focused on rare (less abundant) pCBs that are non-psychotropic compounds, such as cannabigerol (CBG), cannabichromene (CBC), Δ9-tetrahydrocannabivarin (THCV) and cannabigerolic acid (CBGA). Notably, pCBs can act via the endocannabinoid system (ECS), which is involved in the regulation of key pathophysiological processes, and also in the skin. In this study, we used human keratinocytes (HaCaT cells) as an in vitro model that expresses all major ECS elements in order to systematically investigate the effects of CBG, CBC, THCV and CBGA. To this end, we analyzed the gene and protein expression of ECS components (receptors: CB1, CB2, GPR55, TRPV1 and PPARα/γ/δ; enzymes: NAPE-PLD, FAAH, DAGLα/β and MAGL) using qRT-PCR and Western blotting, along with assessments of their functionality using radioligand binding and activity assays. In addition, we quantified the content of endocannabinoid(-like) compounds (AEA, 2-AG, PEA, etc.) using UHPLC-MS/MS. Our results demonstrated that rare pCBs modulate the gene and protein expression of distinct ECS elements differently, as well as the content of endocannabinoid(-like) compounds. Notably, they all increased CB1/2 binding, TRPV1 channel stimulation and FAAH and MAGL catalytic activity. These unprecedented observations should be considered when exploring the therapeutic potential of cannabis extracts for the treatment of human skin diseases

as manuscript BJ20130960 THIS IS NOT THE VERSION OF RECORD -see Biochemical Journal Immediate Publication

Abstract 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 fatty acid amide hydrolase (FAAH), a critical enzyme in terminating the endocannabinoid signalling and an important therapeutic target. Here, using a combined experimental and computational approach, we show that membrane lipids modulate structure, subcellular localization and activity of FAAH. We report that FAAH dimer is stabilized by the lipid bilayer and shows higher membrane binding affinity and enzymatic activity within membranes containing both cholesterol and the natural FAAH substrate, anandamide (AEA). Additionally, colocalization of cholesterol, AEA, and FAAH in mouse neuroblastoma cells suggests a mechanism through which cholesterol increases the substrate accessibility of FAAH. Keywords: cholesterol/endocannabinoids/FAAH/membrane Summary statement The dimeric structure of FAAH is stabilized by the membrane. Membrane binding affinity of FAAH is relevant for subcellular localization. Free cholesterol within the membrane increases the activity of the enzyme. Molecular dynamic studies suggest that cholesterol increases substrate accessibility. INTRODUCTION Fatty acid amide hydrolase (FAAH) is a membrane-bound enzyme that is responsible for the intracellular hydrolysis of the bioactive lipid anandamide (N-arachidonoylethanolamine, AEA) and other congeners known as endocannabinoids (eCBs) Membrane lipid composition affects eCB uptake and signalling, and accumulated evidence demonstrates that cholesterol is a key determinant of this regulation With the aim of dissecting the requirements for the catalytic activity and those for the enzyme interaction with membranes, we analysed by small angle X-ray scattering (SAXS) and fluorescence resonance energy transfer (FRET) the conformational changes induced by lipid bilayers on FAAH. Both using synthetic or reconstructed lipid vesicles from different cell compartments (i.e., plasma membrane (PM) or endoplasmic reticulum (ER)), we demonstrated a key-role of membrane lipids in stabilizing a dimeric form of FAAH with cholesterol and AEA, that both modulate its enzymatic activity within the membrane. Furthermore, molecular dynamics simulations supported a novel mechanism by which cholesterol may help to open the membrane port of FAA