RESUMO
Cancer cells are exposed to major compressive and shearing forces during invasion and metastasis, leading to extensive plasma membrane damage. To survive this mechanical stress, they need to repair membrane injury efficiently. Targeting the membrane repair machinery is thus potentially a new way to prevent invasion and metastasis. We show here that annexin-A2 (ANXA2) is required for membrane repair in invasive breast and pancreatic cancer cells. Mechanistically, we show by fluorescence and electron microscopy that cells fail to reseal shear-stress damaged membrane when ANXA2 is silenced or the protein is inhibited with neutralizing antibody. Silencing of ANXA2 has no effect on proliferation in vitro, and may even accelerate migration in wound healing assays, but reduces tumor cell dissemination in both mice and zebrafish. We expect that inhibiting membrane repair will be particularly effective in aggressive, poor prognosis tumors because they rely on the membrane repair machinery to survive membrane damage during tumor invasion and metastasis. This could be achieved either with anti-ANXA2 antibodies, which have been shown to inhibit metastasis of breast and pancreatic cancer cells, or with small molecule drugs.
Assuntos
Proteínas de Membrana , Neoplasias Pancreáticas , Animais , Camundongos , Linhagem Celular Tumoral , Membrana Celular/metabolismo , Proteínas de Membrana/metabolismo , Neoplasias Pancreáticas/patologia , Peixe-ZebraRESUMO
Adeno-associated virus (AAV) vectors are showing promise in gene therapy trials and have proven to be extremely efficient biological tools in basic neuroscience research. One major limitation to their widespread use in the neuroscience laboratory is the cost, labor, skill and time-intense purification process of AAV. We have recently shown that AAV can associate with exosomes (exo-AAV) when the vector is isolated from conditioned media of producer cells, and the exo-AAV is more resistant to neutralizing anti-AAV antibodies compared with standard AAV. Here, we demonstrate that simple pelleting of exo-AAV from media via ultracentrifugation results in high-titer vector preparations capable of efficient transduction of central nervous system (CNS) cells after systemic injection in mice. We observed that exo-AAV is more efficient at gene delivery to the brain at low vector doses relative to conventional AAV, even when derived from a serotype that does not normally efficiently cross the blood-brain barrier. Similar cell types were transduced by exo-AAV and conventionally purified vector. Importantly, no cellular toxicity was noted in exo-AAV-transduced cells. We demonstrated the utility and robustness of exo-AAV-mediated gene delivery by detecting direct GFP fluorescence after systemic injection, allowing three-dimensional reconstruction of transduced Purkinje cells in the cerebellum using ex vivo serial two-photon tomography. The ease of isolation combined with the high efficiency of transgene expression in the CNS, may enable the widespread use of exo-AAV as a neuroscience research tool. Furthermore, the ability of exo-AAV to evade neutralizing antibodies while still transducing CNS after peripheral delivery is clinically relevant.
Assuntos
Dependovirus/genética , Exossomos , Terapia Genética/métodos , Vetores Genéticos/genética , Animais , Anticorpos Neutralizantes/imunologia , Barreira Hematoencefálica/metabolismo , Encéfalo/metabolismo , Linhagem Celular , Técnicas de Transferência de Genes , Humanos , Camundongos , Transdução Genética , TransgenesRESUMO
Treatments for depression include an adapted lifestyle, physical activity, psychotherapies, antidepressant and mood stabilizing drugs, neuromodulation, chronotherapy, spa treatments. Drug treatments used for major depressive episode are antidepressants and mood stabilizers. For a mild episode, psychotherapy is indicated. It should be combined with an antidepressant (serotonin reuptake inhibitor) for moderate and severe episodes. Suicide risk assessment is essential throughout the depressive episode. It is recommended to monitor at the start of antidepressant treatment for suicidal behavior, a change in mood suggesting an underlying bipolar disorder. The effectiveness of the treatment is evaluated after 4 to 8 weeks. The total duration of antidepressant treatment for an EDC is between 6 months and 1 year after remission, in order to prevent relapses. The use of liaison psychiatry, a real healthcare system within the general hospital, is strongly recommended for better screening and treatment of depression, thus reducing the length of hospital stays, improving the prognosis of depression. The aim of this article is to provide clinicians with a summary of validated data on the efficacy/tolerance of treatment for depression, and to suggest practical action to be taken on the main daily clinical situations: treating comorbid conditions, taking into account interactions drugs, manage the serotonin syndrome, lead to withdrawal from antidepressants, manage treatment in the elderly.
Assuntos
Transtorno Depressivo Maior , Psiquiatria , Antidepressivos/uso terapêutico , Depressão , Transtorno Depressivo Maior/diagnóstico , Transtorno Depressivo Maior/epidemiologia , Transtorno Depressivo Maior/terapia , Humanos , Encaminhamento e ConsultaRESUMO
Well-ordered tubular crystals of acetylcholine receptor were obtained from suspensions of Torpedo marmorata receptor-rich vesicles. They are composed of pairs of oppositely oriented molecules arranged on the surface lattice with the symmetry of the plane group p2 (average unit cell dimensions: a = 90 A, b = 162 A, gamma = 117 degrees). The receptor in this lattice has an asymmetric distribution of mass around its perimeter, yet a regular pentagonal shape; thus its five transmembrane subunits appear to have different lengths, but approximately equal cross sections. The tubes grow by lateral aggregation on the vesicle surface of ribbons of the paired molecules. Both ribbons and tubes were sensitive to dispersal by the disulphide reductant, dithiothreitol. This observation and other evidence suggest that the basic pairing interaction in the tubes may be that of the physiological dimer, involving contact between delta-subunits.
Assuntos
Órgão Elétrico/metabolismo , Receptores Colinérgicos/isolamento & purificação , Animais , Cristalização , Técnica de Congelamento e Réplica , Microscopia Eletrônica , Conformação Proteica , TorpedoRESUMO
We have previously uncovered the impact of oncogenic and differentiation processes on extracellular vesicles (EVs) in cancer. This is of interested in the context of glioma stem cells (GSC) that are responsible for recurrent nature of glioblastoma multiforme (GBM), while retaining the potential to undergo differentiation and self renewal. GSCs reside in vascular niches where they interact with endothelial cells through a number of mediators including bioactive cargo of EVs. GSCs can be classified as proneural (PN) or mesenchymal (MES) subtypes on the basis of their gene expression profiles and distinct biological characteristics. In the present study we investigated how GSC diversity and differentiation programmes influence their EV-mediated communication potentials. Indeed, molecular subtypes of GBMs and GSCs differ with respect to their expression of EV-related genes (vesiculome) and GSCs with PN or MES phenotypes produce EVs with markedly different characteristics, marker profiles, proteomes and endothelial stimulating activities. For example, while EVs of PN GSC are largely devoid of exosomal markers their counterparts from MES GSCs express ample CD9, CD63 and CD81 tetraspanins. In both GSC subtypes serum-induced differentiation results in profound, but distinct changes of cellular phenotypes including the enhanced EV production, reconfiguration of their proteomes and the related functional pathways. Notably, the EV uptake was a function of both subtype and differentiation state of donor cells. Thus, while, EVs produced by differentiated MES GSCs were internalized less efficiently than those from undifferentiated cells they exhibited an increased stimulatory potential for human brain endothelial cells. Such stimulating activity was also observed for EVs derived from differentiated PN GSCs, despite their even weaker uptake by endothelial cells. These findings suggest that the role of EVs as biological mediators and biomarkers in GBM may depend on the molecular subtype and functional state of donor cancer cells, including cancer stem cells. Abbreviations: CryoTEM: cryo-transmission electron microscopy; DIFF: differentiated GSCs; EGF: epidermal growth factor; DUC: differential ultracentrifugation; EV: extracellular vesicle; FGF: fibroblast growth factor; GBM: glioblastoma multiforme; GFAP: glial fibrillary acidic protein; GO: gene ontology; GSC: glioma stem cells; HBEC-5i: human brain endothelial cells; MES: mesenchymal cells; MTS - [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt; PMT1: proneural-to-mesenchyman transition cell line 1; PN: proneural cells; TEM: transmission electron microscopy; WB: western blotting.
RESUMO
We present a new assay for analyzing the specific binding of proteins to lipid ligands contained within vesicles or micelles. This assay, referred to as the electrophoretic migration shift assay, was developed using a model system composed of cholera toxin and of its physiological receptor, monosialoganglioside GM1. Using polyacrylamide gel electrophoresis in non-denaturing conditions, the migration of toxin components known to interact with GM1 was retarded when GM1 was present in either lipid vesicles or micelles. This effect was specific, as the migration of proteins not interacting with GM1 was not modified. The localization of retarded proteins and of lipids on gels was further determined by autoradiography. The stoichiometry of binding between cholera toxin and GM1 was determined, giving a value of five GM1 per one pentameric assembly of cholera toxin B-subunits, in agreement with previous studies. The general applicability of this assay was further established using both streptavidin and annexin V together with specific lipid ligands. This assay is fast, simple, quantitative, and requires only microgram quantities of protein.
Assuntos
Eletroforese em Gel de Poliacrilamida/métodos , Lipídeos de Membrana/química , Proteínas de Membrana/química , Anexina A5/química , Proteínas de Bactérias/química , Toxina da Cólera/química , Gangliosídeo G(M1)/química , Técnicas In Vitro , Ligantes , Lipossomos , Micelas , Fosfatidilcolinas/química , Fosfatidilserinas/química , EstreptavidinaRESUMO
A new method of affinity chromatography purification of the detergent-solubilized nicotinic acetylcholine receptor protein (nAChR) is presented, based on the reversible coupling of a chemically monomodified alpha-toxin from Naja nigricollis to a resin. The alpha-toxin was monothiolated on the epsilon-amino group of its lysine-15 by reaction with N-succinimidly-3-(2-pyridyldithio)propionate and was covalently linked in a reversible manner to a thiopropyl-activated agarose resin by thiol-disulfide exchange. We found that 50% of the immobilized toxin molecules were effective for purifying nAChR, indicating a high accessibility of resin-bound toxins to their binding sites on the receptor protein. Purified alpha-toxin/nAChR complexes were eluted with nearly 100% recovery by reduction of disulfide bridges with dithiothreitol. nAChR solutions of high purity were obtained, as shown by polyacrylamide gel electrophoresis. A comparison was made with two other procedures of affinity chromatography using: (1) alpha-bungarotoxin from Bungarus multicinctus polymodified on several amines and covalently linked to a resin in a reversible manner, and (2) a commercial agarose resin bearing irreversibly immobilized alpha-cobrotoxin from Naja naja kaouthia. We conclude that: (1) the use of a selected regioselective linking of a peptidic ligand to a chromatography resin results in an increased efficiency of protein binding, and (2) a high yield of protein recovery is obtained via reversible covalent linking.
Assuntos
Cromatografia de Afinidade/métodos , Proteínas Neurotóxicas de Elapídeos , Receptores Nicotínicos/isolamento & purificação , Animais , Bungarotoxinas/química , Proteínas Neurotóxicas de Elapídeos/química , Reagentes de Ligações Cruzadas , SuccinimidasRESUMO
The (Anx2)(2)(p11)(2) heterotetramer has been implicated in endo- and exocytosis in vivo and in liposome aggregation in vitro. Here we report on the modelling of the heterotetramer complex using docking algorithms. Two types of models are generated-heterotetramer and heterooctamer. On the basis of the agreement between the calculated (X-ray) electron density and the observed projected density from cryo-electron micrographs on the one hand, and calculated energy criteria on the other hand, the heterotetramer models are proposed as the most probable, and one of them is selected as the best model. Analysis of this model at an atomic level suggests that the interaction between the Anx2 core and p11 has an electrostatic character, being stabilised primarily through charged residues.
Assuntos
Anexinas/química , Proteínas S100/química , Algoritmos , Anexina A2/química , Cristalografia por Raios X , Dimerização , Modelos Químicos , Modelos Moleculares , Fosfoproteínas/química , Eletricidade EstáticaRESUMO
Highly ordered two-dimensional crystals of cholera toxin B-subunit pentamers have been grown by specific interaction with planar lipid films containing monosialoganglioside GM1. Electron diffractograms of frozen-hydrated crystals show diffraction peaks extending to beyond 4 A, while electron images diffract to 8 A. A two-dimensional projected structure of cholera toxin B-subunit-GM1 complex has been calculated at 9 A resolution by combining electron diffraction and image data. Crystals present an approximate pgg projection symmetry, with unit cell dimensions a = 119(+/- 1) A, b = 123(+/- 1) A, gamma = 90 degrees. Each pentameric assembly presents two concentric rings of electron scattering density, separated by an area of lower density. The outer and inner rings are centered at 25 A and and 11 A from the pentamer centre, respectively. The apparent projected density of the outer ring is larger than that of the inner ring. We propose that the outer and inner density rings correspond respectively to the peripheral beta-sheet arrangement and the central alpha-helix barrel, recently identified in the crystal structure of the heat-labile enterotoxin from Escherichia coli.
Assuntos
Toxina da Cólera/metabolismo , Gangliosídeo G(M1)/metabolismo , Toxina da Cólera/química , Cristalização , Gangliosídeo G(M1)/química , Conformação Proteica , Difração de Raios XRESUMO
Annexins are a family of water-soluble proteins that bind to membranes in a calcium-dependent manner. Some members have been shown to exhibit voltage-dependent calcium channel activity, a property characteristic of integral membrane proteins. The structures of human annexin V in crystals obtained from aqueous solution and in two-dimensional crystals when bound to phospholipid layers have been determined by X-ray and electron crystallography, respectively. They are compared here. Both structures show close correspondence, suggesting that annexins attach to phospholipid membranes without substantial structural change. These observations, together with biochemical data, lead to the conclusion that annexin V interacts with phospholipid membranes with its convex face. We propose that binding is mediated by direct interaction between the phosphoryl headgroups and the calcium bound to polypeptide loops protruding from the convex face. The membrane area covered by annexin may thus become disordered and permeable allowing calcium flux through the membrane and the central channel-like structure found in annexin molecules.
Assuntos
Proteínas de Ligação ao Cálcio/química , Proteínas de Membrana/química , Proteínas da Gravidez/química , Sequência de Aminoácidos , Anexina A5 , Proteínas de Ligação ao Cálcio/isolamento & purificação , Proteínas de Ligação ao Cálcio/metabolismo , Humanos , Membranas/metabolismo , Modelos Moleculares , Proteínas da Gravidez/isolamento & purificação , Proteínas da Gravidez/metabolismo , Ligação Proteica , Conformação Proteica , SolubilidadeRESUMO
The structure of the membrane protein from urothelial plasma membrane has been investigated. A three-dimensional map has been obtained at 35 A resolution from negatively stained two-dimensional arrays of membrane proteins. The lattice space group has the symmetry p6. Twelve stain-excluding areas are resolvable on projections perpendicular to the membrane plane. In the three-dimensional reconstruction these areas appear to be restricted to one side of the membrane and form protrusions that extend 50 A out of the membrane. No periodic structure is observed at the cytoplasmic side of the membrane, which suggests that the protein is not a transmembrane protein.
Assuntos
Proteínas de Membrana , Bexiga Urinária/análise , Animais , Cristalografia , Análise de Fourier , Microscopia Eletrônica , Modelos Moleculares , Conformação Proteica , SuínosRESUMO
Numerous protein plaques cover the apical surface of mammalian urinary bladder epithelial cells. These plaques contain four integral membrane proteins, called uroplakins, which form a well-ordered array of hexameric complexes. The 3D structure of these naturally occurring 2D crystals was studied by cryo-electron-crystallographic methods using a slow-scan charged-coupled device (CCD) camera to record the electron micrographs. A 1.2 nm projection map calculated from untilted crystals shows that each hexamer comprises a ring of six inner and six outer domains at a radius of 5.7 nm and 9.2 nm respectively. The 3D structure shows that the mass is distributed strongly asymmetrically with respect to the membrane, with most of the mass protruding from the luminal face. Both domains in the asymmetric unit traverse the membrane and protrude from the membrane on the cytoplasmic side. On the luminal side, the two domains are bridged forming a stretched arc. The total thickness of the complex is about 13.2 nm. A model of the urothelial plaque reveals that contacts between the hexamers are much less extended than within the hexamers.
Assuntos
Microscopia Crioeletrônica , Células Epiteliais/química , Glicoproteínas de Membrana/química , Glicoproteínas de Membrana/ultraestrutura , Bexiga Urinária/química , Amiloidose/metabolismo , Animais , Cristalização , Células Epiteliais/ultraestrutura , Análise de Fourier , Congelamento , Substâncias Macromoleculares , Glicoproteínas de Membrana/metabolismo , Proteínas de Membrana/química , Proteínas de Membrana/metabolismo , Proteínas de Membrana/ultraestrutura , Camundongos , Modelos Moleculares , Peso Molecular , Estrutura Quaternária de Proteína , Tetraspaninas , Bexiga Urinária/citologia , Bexiga Urinária/ultraestrutura , Uroplaquina II , Uroplaquina III , Uroplaquina Ia , Uroplaquina IbRESUMO
Two-dimensional crystals of annexin-V bound to lipid layers containing dioleoylphosphatidylserine have been obtained in the presence of Ca2+. The crystals diffract to 20 A resolution and have the symmetry of the plane group p3 (unit cell dimensions: a = b = 94 A, gamma = 120 degrees). Electron image analysis revealed that the crystals are composed of trimers of annexin-V forming triskelion-like motifs. Each annexin-V molecule has a characteristic elongated shape, about 65 A by 20 A, when observed perpendicularly to the crystal plane. It is composed of two staggered domains of similar size, about 40 A by 20 A. Both domains are made of two sub-domains. The present data suggest that the four resolved sub-domains represent the folding units corresponding to the four 70 amino acid repeating segments characteristic of all annexins.
Assuntos
Proteínas de Ligação ao Cálcio/ultraestrutura , Proteínas de Membrana/ultraestrutura , Anexina A5 , Cálcio , Cristalografia , Análise de Fourier , Humanos , Lipídeos , Substâncias Macromoleculares , Microscopia Eletrônica/métodos , Fosfatidilcolinas , FosfatidilserinasRESUMO
The (annexin II-p11)2 tetramer has been proposed to participate in exocytosis and several other members of the annexin superfamily have been reported to aggregate liposomes in vitro. In this context, the Ca2+-dependent binding of several annexins to chromaffin granules and liposomes was investigated by cryo-electron microscopy. The Ca2+-dependent aggregation of lipid membranes by (annexin II-p11)2 results from the spontaneous self-organization of the protein into two-dimensional plaques, which are visualized in projection as characteristic junctions. The junctions have a constant thickness of 210(+/-10) A and present a symmetrical distribution of electron-dense material arranged into seven stripes. They were observed over a wide range of Ca2+ concentrations, down to 2 microM. The molecular components corresponding to the seven electron-dense stripes were assigned as follows: the two associated membranes give rise to two outer stripes each and the three central stripes correspond to the (annexin II-p11)2 tetramer. Each annexin II molecule interacts with the outer lipid leaflet of one membrane, giving rise to one stripe, while the central stripe is due to the (p11)2 dimer with which both annexin II molecules interact. Both annexin II and annexin I also induced the Ca2+-dependent aggregation of liposomes via junctions that lack the central (p11)2 moiety and present only six high-density stripes. As expected, both annexin V and annexin III bind to liposomes without inducing their aggregation.
Assuntos
Anexinas/metabolismo , Grânulos Cromafim/metabolismo , Lipossomos/metabolismo , Animais , Anexina A1/metabolismo , Anexina A1/ultraestrutura , Anexina A2/metabolismo , Anexina A2/ultraestrutura , Anexina A3/metabolismo , Anexina A3/ultraestrutura , Anexina A5/metabolismo , Anexina A5/ultraestrutura , Anexinas/ultraestrutura , Cálcio , Bovinos , Grânulos Cromafim/ultraestrutura , Fosfatidilcolinas/metabolismo , Fosfatidilgliceróis/metabolismo , Fatores de TempoRESUMO
Annexins are intracellular proteins which bind to membranes in a Ca(2+)-dependent manner and which have been proposed to play regulatory roles in different membrane processes. In the present study, the stoichiometry of the Ca(2+)-dependent binding of annexin V to phosphatidylserine molecules incorporated into liposomes was studied by fluorescence spectroscopy. The Ca(2+)-dependence of the binding was determined using liposomes made of dioleoylphosphatidylserine (PS) and dioleoylphosphatidylcholine (PC), with a PC/PS molar ratio ranging from 1 to 800. These liposomes were shown to be mostly unilamellar by cryoelectron microscopy. [Ca2+]1/2 concentrations required for half-maximal binding of annexin V range from 57 microM at PC/PS = 1 up to 96 mM at PC/PS = 800. Titration of accessible PS molecules showed that annexin V molecules bind equally well to liposomes of PC/PS ratio ranging from 1 to 400. The stoichiometry of the binding between annexin V and PS, determined at low PS content, is eight annexin V molecules per one PS molecule. We propose a novel model of the Ca(2+)-dependent interaction between annexin V and lipid membranes, based on the formation of two-dimensional arrays of annexin V molecules, stabilized by both protein-lipid and protein-protein interactions.
Assuntos
Anexina A5/química , Lipossomos , Fosfatidilserinas , Conformação Proteica , Anexina A5/isolamento & purificação , Anexina A5/metabolismo , Cálcio/farmacologia , Feminino , Humanos , Cinética , Bicamadas Lipídicas , Microscopia Eletrônica , Fosfatidilcolinas , Placenta/metabolismo , GravidezRESUMO
The uptake of mannitol in Escherichia coli is controlled by the phosphoenolpyruvate dependent phosphotransferase system. Enzyme II mannitol (EIIMtl) is part of the phosphotransferase system and consists of three covalently bound domains. IICMtl, the integral membrane domain of EIIMtl, is responsible for mannitol transport across the cytoplasmic membrane. In order to understand this molecular process, two-dimensional crystals of IICMtl were grown by reconstitution into lipid bilayers and their structure was investigated by cryo-electron crystallography. The IICMtl crystals obey p22121 symmetry and have a unit cell of 125 Ax65 A, gamma=90 degrees. A projection structure was determined at 5 A resolution using both electron images and electron diffractograms. The unit cell contains two IICMtl dimers with a size of about 40 Ax90 A, which are oriented up and down in the crystal. Each monomer exhibits six domains of high density which most likely correspond to transmembrane alpha-helices and cytoplasmic loops.
Assuntos
Cristalografia/métodos , Sistema Fosfotransferase de Açúcar do Fosfoenolpiruvato/química , Membrana Celular , Cristalização , Proteínas de Escherichia coli , Congelamento , Bicamadas Lipídicas , Proteínas de Transporte de Monossacarídeos , Conformação ProteicaRESUMO
Annexins constitute a family of phospholipid- and Ca(2+)-binding proteins involved in a variety of membrane-related processes. The property of several annexins, including annexin A5, to self-organize at the surface of lipid membranes into 2D ordered arrays has been proposed to be functionally relevant in cellular contexts. To further address this question, we investigated the high-resolution structure of annexin A5 trimers in membrane-bound 2D crystals by cryo-electron microscopy (Cryo-EM). A new 2D crystal form was discovered, with p32(1) symmetry, which is significantly better ordered than the 2D crystals reported before. A 2D projection map was obtained at 6.5 A resolution, revealing protein densities within each of the four domains characteristic of annexins. A quantitative comparison was performed between this structure and models generated from the structure of the soluble form of annexin A5 in pseudo-R3 3D crystals. This analysis indicated that both structures are essentially identical, except for small local changes attributed to membrane binding. As a consequence, and contrary to the common view, annexin A5 molecules maintain their bent shape and do not flatten upon membrane binding, which implies either that the four putative Ca(2+) and membrane-binding loops present different types of interaction with the membrane surface, or that the membrane surface is locally perturbed. We propose that the trimerization of annexin A5 molecules is the relevant structural change occurring upon membrane binding. The evidence that 2D arrays of annexin A5 trimers are responsible for its in vitro property of blood coagulation inhibition supports this conclusion.
Assuntos
Anexinas/química , Anexinas/metabolismo , Membrana Celular/metabolismo , Microscopia Crioeletrônica , Animais , Cálcio/metabolismo , Cristalografia por Raios X , Proteínas de Membrana/química , Proteínas de Membrana/metabolismo , Modelos Moleculares , Ligação Proteica , Estrutura Quaternária de Proteína , Estrutura Terciária de Proteína , Ratos , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Rotação , SolubilidadeRESUMO
The V-type Na(+)-ATPase of the thermophilic, anaerobic bacterium Caloramator fervidus was purified to homogeneity. The subunit compositions of the catalytic V(1) and membrane-embedded V(0) parts were determined and the structure of the enzyme complex was studied by electron microscopy. The V(1) headpiece consists of seven subunits present in one to three copies, and the V(0) part of two subunits in a ratio of 5:2. An analysis of over 7500 single particle images obtained by electron microscopy of the purified V(1)V(0) enzyme complex revealed that the stalk region, connecting the V(1) and V(0) parts, contains two peripheral stalks in addition to a central stalk. One of the two is connected to the V(0) part, while the other is connected to the first via a bar-like structure that is positioned just above V(0), parallel with the plane of the membrane. In projection, this bar seems to contact the central stalk. The data show that the stator structure that prevents rotation of the static part of V(0) relative to V(1) in the rotary catalysis mechanism of energy coupling in ATPases/ATPsynthases is more complex than previously thought.
Assuntos
Adenosina Trifosfatases/química , Adenosina Trifosfatases/ultraestrutura , Bacillaceae/enzimologia , Proteínas de Transporte de Cátions , Estrutura Quaternária de Proteína , Adenosina Trifosfatases/isolamento & purificação , Adenosina Trifosfatases/metabolismo , Trifosfato de Adenosina/metabolismo , Domínio Catalítico , Estabilidade Enzimática , Proteínas de Membrana/química , Proteínas de Membrana/isolamento & purificação , Proteínas de Membrana/metabolismo , Proteínas de Membrana/ultraestrutura , Microscopia Eletrônica , Modelos Moleculares , Peso Molecular , Desnaturação Proteica , Rotação , Sódio/metabolismo , TemperaturaRESUMO
By a kinetic analysis of 59 variant transcripts of Escherichia coli tRNA(Glu) with glutamyl-tRNA synthetase (GluRS), the U11.A24 base-pair, the U13.G22..A46 base-triple, and the lack of residue 47 (delta47) were found to serve as major determinants for tRNA(Glu) identity. This is the first system for which major identity determinants are reported to be clustered in the "augmented D helix", consisting of the D stem with some neighboring residues and the variable loop. Other identity determinants are U34, U35, C36 and A37 in the anticodon loop, and G1.C72 and U2.A71 in the acceptor stem. Phosphate-group protection by GluRS from ethylnitrosourea was observed most strongly for the minor groove side of D-stem helix, indicating that GluRs tightly binds to the D stem for recognition, on the minor groove side, of the potent identity-determinant groups of the U11.A24 and U13.G22 base-pairs. A46 is not involved in direct recognition by GluRS; the U13.G22..A46 base-triple is required probably for formation of the structural features that are recognized by GluRS. In this context, the essential role of characteristic delta47 in tRNA(Glu) identity may be to maintain the U13.G22..A46 base-triple.