How cholesterol interacts with membrane proteins: an exploration of cholesterol-binding sites including CRAC, CARC, and tilted domains

The plasma membrane of eukaryotic cells contains several types of lipids displaying high biochemical variability in both their apolar moiety (e.g., the acyl chain of glycerolipids) and their polar head (e.g., the sugar structure of glycosphingolipids). Among these lipids, cholesterol is unique because its biochemical variability is almost exclusively restricted to the oxidation of its polar −OH group. Although generally considered the most rigid membrane lipid, cholesterol can adopt a broad range of conformations due to the flexibility of its isooctyl chain linked to the polycyclic sterane backbone. Moreover, cholesterol is an asymmetric molecule displaying a planar α face and a rough β face. Overall, these structural features open up a number of possible interactions between cholesterol and membrane lipids and proteins, consistent with the prominent regulatory functions that this unique lipid exerts on membrane components. The aim of this review is to describe how cholesterol interacts with membrane lipids and proteins at the molecular/atomic scale, with special emphasis on transmembrane domains of proteins containing either the consensus cholesterol-binding motifs CRAC and CARC or a tilted peptide. Despite their broad structural diversity, all these domains bind cholesterol through common molecular mechanisms, leading to the identification of a subset of amino acid residues that are overrepresented in both linear and three-dimensional membrane cholesterol-binding sites.Fil: Fantini, J.. Aix-Marseille Université. Interactions Moléculaires et Systèmes Membranaires; FranciaFil: Barrantes, Francisco Jose. Pontificia Universidad Católica Argentina "Santa María de los Buenos Aires". Facultad de Ciencias Médicas. Instituto de Investigaciones Biomédicas; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentin

Nicotinic acetylcholine receptor is internalized via a Rac-dependent, dynamin-independent endocytic pathway

Endocytosis of the nicotinic acetylcholine receptor (AChR) is a proposed major mechanism of neuromodulation at neuromuscular junctions and in the pathology of synapses in the central nervous system. We show that binding of the competitive antagonist α-bungarotoxin (αBTX) or antibody-mediated cross-linking induces the internalization of cell surface AChR to late endosomes when expressed heterologously in Chinese hamster ovary cells or endogenously in C2C12 myocytes. Internalization occurs via sequestration of AChR–αBTX complexes in narrow, tubular, surface-connected compartments, which are indicated by differential surface accessibility of fluorescently tagged αBTX–AChR complexes to small and large molecules and real-time total internal reflection fluorescence imaging. Internalization occurs in the absence of clathrin, caveolin, or dynamin but requires actin polymerization. αBTX binding triggers c-Src phosphorylation and subsequently activates the Rho guanosine triphosphatase Rac1. Consequently, inhibition of c-Src kinase activity, Rac1 activity, or actin polymerization inhibits internalization via this unusual endocytic mechanism. This pathway may regulate AChR levels at ligand-gated synapses and in pathological conditions such as the autoimmune disease myasthenia gravis

Mechanics of Channel Gating of the Nicotinic Acetylcholine Receptor

The nicotinic acetylcholine receptor (nAChR) is a key molecule involved in the propagation of signals in the central nervous system and peripheral synapses. Although numerous computational and experimental studies have been performed on this receptor, the structural dynamics of the receptor underlying the gating mechanism is still unclear. To address the mechanical fundamentals of nAChR gating, both conventional molecular dynamics (CMD) and steered rotation molecular dynamics (SRMD) simulations have been conducted on the cryo-electron microscopy (cryo-EM) structure of nAChR embedded in a dipalmitoylphosphatidylcholine (DPPC) bilayer and water molecules. A 30-ns CMD simulation revealed a collective motion amongst C-loops, M1, and M2 helices. The inward movement of C-loops accompanying the shrinking of acetylcholine (ACh) binding pockets induced an inward and upward motion of the outer β-sheet composed of β9 and β10 strands, which in turn causes M1 and M2 to undergo anticlockwise motions around the pore axis. Rotational motion of the entire receptor around the pore axis and twisting motions among extracellular (EC), transmembrane (TM), and intracellular MA domains were also detected by the CMD simulation. Moreover, M2 helices undergo a local twisting motion synthesized by their bending vibration and rotation. The hinge of either twisting motion or bending vibration is located at the middle of M2, possibly the gate of the receptor. A complementary twisting-to-open motion throughout the receptor was detected by a normal mode analysis (NMA). To mimic the pulsive action of ACh binding, nonequilibrium MD simulations were performed by using the SRMD method developed in one of our laboratories. The result confirmed all the motions derived from the CMD simulation and NMA. In addition, the SRMD simulation indicated that the channel may undergo an open-close (O ↔ C) motion. The present MD simulations explore the structural dynamics of the receptor under its gating process and provide a new insight into the gating mechanism of nAChR at the atomic level

Nicotinic α4 Receptor-Mediated Cholinergic Influences on Food Intake and Activity Patterns in Hypothalamic Circuits.

Nicotinic acetylcholine receptors (nAChRs) play an important role in regulating appetite and have been shown to do so by influencing neural activity in the hypothalamus. To shed light on the hypothalamic circuits governing acetylcholine's (ACh) regulation of appetite this study investigated the influence of hypothalamic nAChRs expressing the α4 subunit. We found that antagonizing the α4β2 nAChR locally in the lateral hypothalamus with di-hydro-ß-erythroidine (DHβE), an α4 nAChR antagonist with moderate affinity, caused an increase in food intake following free access to food after a 12 hour fast, compared to saline-infused animals. Immunocytochemical analysis revealed that orexin/hypocretin (HO), oxytocin, and tyrosine hydroxylase (TH)-containing neurons in the A13 and A12 of the hypothalamus expressed the nAChR α4 subunit in varying amounts (34%, 42%, 50%, and 51%, respectively) whereas melanin concentrating hormone (MCH) neurons did not, suggesting that DHβE-mediated increases in food intake may be due to a direct activation of specific hypothalamic circuits. Systemic DHβE (2 mg/kg) administration similarly increased food intake following a 12 hour fast. In these animals a subpopulation of orexin/hypocretin neurons showed elevated activity compared to control animals and MCH neuronal activity was overall lower as measured by expression of the immediate early gene marker for neuronal activity cFos. However, oxytocin neurons in the paraventricular hypothalamus and TH-containing neurons in the A13 and A12 did not show differential activity patterns. These results indicate that various neurochemically distinct hypothalamic populations are under the influence of α4β2 nAChRs and that cholinergic inputs to the lateral hypothalamus can affect satiety signals through activation of local α4β2 nAChR-mediated transmission.This work was supported by the Royal Society and the European Union (Latin America/European Liason, LAEL).This is the final version of the article. It first appeared from PLOS via http://dx.doi.org/1371/journal.pone.013332

Resolution of complex fluorescence spectra of lipids and nicotinic acetylcholine receptor by multivariate analysis reveals protein-mediated effects on the receptor's immediate lipid microenvironment

Analysis of fluorescent spectra from complex biological systems containing various fluorescent probes with overlapping emission bands is a challenging task. Valuable information can be extracted from the full spectra, however, by using multivariate analysis (MA) of measurements at different wavelengths. We applied MA to spectral data of purified Torpedo nicotinic acetylcholine receptor (AChR) protein reconstituted into liposomes made up of dioleoylphosphatidic acid (DOPA) and dioleoylphosphatidylcholine (DOPC) doped with two extrinsic fluorescent probes (NBD-cholesterol/pyrene-PC). Förster resonance energy transfer (FRET) was observed between the protein and pyrene-PC and between pyrene-PC and NBD-cholesterol, leading to overlapping emission bands. Partial least squares analysis was applied to fluorescence spectra of pyrene-PC in liposomes with different DOPC/DOPA ratios, generating a model that was tested by an internal validation (leave-one-out cross-validation) and was further used to predict the apparent lipid molar ratio in AChR-containing samples. The values predicted for DOPA, the lipid with the highest Tm, indicate that the protein exerts a rigidifying effect on its lipid microenvironment. A similar conclusion was reached from excimer formation of pyrene-PC, a collisional-dependent phenomenon. The excimer/monomer ratio (E/M) at different DOPC/DOPA molar ratios revealed the restricted diffusion of the probe in AChR-containing samples in comparison to pure lipid samples devoid of protein. FRET from the AChR (donor) to pyrene-PC (acceptor) as a function of temperature was found to increase with increasing temperature, suggesting a shorter distance between AChR and pyrene PC. Taken together, the results obtained by MA on complex spectra indicate that the AChR rigidifies its surrounding lipid and prefers DOPA rather than DOPC in its immediate microenvironment

Optimización de una técnica para la detección de patologías virales en <i>Pleoticus muelleri</i> (Bate, 1988) en el estuario de Bahía Blanca, Argentina

La comercialización de animales acuáticos require la implementación de controles sanitarios para la detección de virus patógenos estipulados por la Organización Mundial para la Salud Animal (OIE). De acuerdo con sus normas, y específicamente en lo que se refiere la comercialización de crustáceos, la OIE determina como enfermedades de declaración obligatoria, entre otras, a las denominadas: virus del síndrome de la mancha blanca (WSSV), virus de la cabeza amarilla (YHV) y el virus del síndrome de Taura (TSV), las cuales son altamente peligrosas. El objetivo de este estudio fue determinar la presencia o ausencia de estas virosis en poblaciones de langostinos silvestres {Pleoticus muelleri) en el estuario de Bahía Blanca, aplicando metodologías bioquímicas, de biología molecular y genéticas. Los resultados demuestran que se ha logrado optimizar la metodología para la extracción y purificación de ADN y ARN de tejido y hemolinfa de langostinos y el correcto manejo en la captura, acondicionamiento y transporte de los animales al laboratorio. Los ensayos fueron debidamente convalidados por reacciones de control negativas y positivas. En las muestras estudiadas de Pleoticus muelleri se descarta la presencia de estas tres enfermedades virales. Esta metodología permite no sólo realizar una detección temprana y un diagnóstico de las tres patologías virales sino también establecer y asegurar un status libre de infección con el consiguiente beneficio sanitario para la región.Trade in aquatic animals calls for the implementation of controls for the detection of pathogenic viruses in line with the regulations of the OIE, the World Organization for Animal Heath. In relation specifically to trade in crustaceans, the OIE identifies the following diseases: White Spot syndrome virus disease (WSSV); Yellow Head Virus disease (YHV); and Taura syndrome virus disease (TSV), all of which are highly dangerous. The purpose of this study was to determine whether these viruses occur in wild populations of Pleoticus muelleri in Bahia Blanca estuary, Argentina, using biochemical, molecular biology and genetics methods. The results show that the methods used for DNA and RNA extraction and purification from the tissue and hemolymph of these crustaceans, and the techniques for their capture, packaging and transportation to the laboratory have been optimized. Tests were properly validated by negative and positive controls. None of the named diseases were found in the wild populations of Pleoticus muelleri. The methodology followed in the present research not only enables early detection and diagnosis of the three viral pathologies but also ensures an infection-free status with the concomitant health benefits for the region.Asociación Argentina de Geofísicos y Geodesta

PINO RODRÍGUEZ [Material gráfico]

ÁLBUM FAMILIAR CASA DE COLÓNCopia digital. Madrid : Ministerio de Educación, Cultura y Deporte. Subdirección General de Coordinación Bibliotecaria, 201

Competências do Terapeuta Ocupacional em Cuidados Paliativos

Existência de um Poster com o mesmo título no repositório IC-Online - Poster divulgação Terapia Ocupacional em Cuidados Paliativos (dos autores: Ana Costa; Marlene Monteiro; Rita Salgado; Vanda Varela Pedrosa) que faz referência ao presente documento Original e aos seus Autores: Ana Costa; Elisabete Roldão; Francisco Barrantes; Inês Brito; Thais Cândido - "Competências do Terapeuta Ocupacional em Cuidados Paliativos", http://hdl.handle.net/10400.8/4984Documento produzido no âmbito da APTO - Associação Portuguesa de Terapeutas Ocupacionais, por terapeutas ocupacionais sócios da APTO, envolvidos na pesquisa, recolha documental, seleção e organização da informação que permitiu a elaboração do documento. Este é um documento dinâmico, que pode vir a ser reformulado ao longo do tempo e que se pretende informativo e consultivo.Com este documento o Grupo de Interesse em Cuidados Paliativos e Terapia Ocupacional pretende sistematizar as competências gerais do terapeuta ocupacional a exercer funções na área dos Cuidados Paliativos, tendo como referência de base as competências do terapeuta ocupacional, resultantes do Tunning Project. Este documento não altera a necessidade das competências de base para o terapeuta ocupacional vindo, sim, acrescer a estas, competências específicas para a prática profissional de excelência da Terapia Ocupacional em Cuidados Paliativos, nos diversos níveis da intervenção.N/

Functional nicotinic acetylcholine receptor reconstitution in Au(111)-supported thiolipid monolayers

The insertion and function of the muscle-type nicotinic acetylcholine receptor (nAChR) in Au(111)- supported thiolipid self-assembled monolayers have been studied by atomic force microscopy (AFM), surface plasmon resonance (SPR), and electrochemical techniques. It was possible for the first time to resolve the supramolecular arrangement of the protein spontaneously inserted in a thiolipid monolayer in an aqueous solution. Geometric supramolecular arrays of nAChRs were observed, most commonly in a triangular form compatible with three nAChR dimers of ∼20 nm each. Addition of the full agonist carbamoylcholine activated and opened the nAChR ion channel, as revealed by the increase in capacitance relative to that of the nAChR–thiolipid system under basal conditions. Thus, the self-assembled system appears to be a viable biomimetic model to measure ionic conductance mediated by ion-gated ion channels under different experimental conditions, with potential applications in biotechnology and pharmacology.Instituto de Investigaciones Fisicoquímicas Teóricas y Aplicada