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Sample records for cell membrane proteins

  1. Cell-free system for synthesizing membrane proteins cell free method for synthesizing membrane proteins

    Science.gov (United States)

    Laible, Philip D; Hanson, Deborah K

    2013-06-04

    The invention provides an in vitro method for producing proteins, membrane proteins, membrane-associated proteins, and soluble proteins that interact with membrane-associated proteins for assembly into an oligomeric complex or that require association with a membrane for proper folding. The method comprises, supplying intracytoplasmic membranes from organisms; modifying protein composition of intracytoplasmic membranes from organism by modifying DNA to delete genes encoding functions of the organism not associated with the formation of the intracytoplasmic membranes; generating appropriate DNA or RNA templates that encode the target protein; and mixing the intracytoplasmic membranes with the template and a transcription/translation-competent cellular extract to cause simultaneous production of the membrane proteins and encapsulation of the membrane proteins within the intracytoplasmic membranes.

  2. Membrane Protein Mobility and Orientation Preserved in Supported Bilayers Created Directly from Cell Plasma Membrane Blebs.

    Science.gov (United States)

    Richards, Mark J; Hsia, Chih-Yun; Singh, Rohit R; Haider, Huma; Kumpf, Julia; Kawate, Toshimitsu; Daniel, Susan

    2016-03-29

    Membrane protein interactions with lipids are crucial for their native biological behavior, yet traditional characterization methods are often carried out on purified protein in the absence of lipids. We present a simple method to transfer membrane proteins expressed in mammalian cells to an assay-friendly, cushioned, supported lipid bilayer platform using cell blebs as an intermediate. Cell blebs, expressing either GPI-linked yellow fluorescent proteins or neon-green fused transmembrane P2X2 receptors, were induced to rupture on glass surfaces using PEGylated lipid vesicles, which resulted in planar supported membranes with over 50% mobility for multipass transmembrane proteins and over 90% for GPI-linked proteins. Fluorescent proteins were tracked, and their diffusion in supported bilayers characterized, using single molecule tracking and moment scaling spectrum (MSS) analysis. Diffusion was characterized for individual proteins as either free or confined, revealing details of the local lipid membrane heterogeneity surrounding the protein. A particularly useful result of our bilayer formation process is the protein orientation in the supported planar bilayer. For both the GPI-linked and transmembrane proteins used here, an enzymatic assay revealed that protein orientation in the planar bilayer results in the extracellular domains facing toward the bulk, and that the dominant mode of bleb rupture is via the "parachute" mechanism. Mobility, orientation, and preservation of the native lipid environment of the proteins using cell blebs offers advantages over proteoliposome reconstitution or disrupted cell membrane preparations, which necessarily result in significant scrambling of protein orientation and typically immobilized membrane proteins in SLBs. The bleb-based bilayer platform presented here is an important step toward integrating membrane proteomic studies on chip, especially for future studies aimed at understanding fundamental effects of lipid interactions

  3. Denaturation of membrane proteins and hyperthermic cell killing

    NARCIS (Netherlands)

    Burgman, Paulus Wilhelmus Johannes Jozef

    1993-01-01

    Summarizing: heat induced denaturation of membrane proteins is probably related to hyperthermic cell killing. Induced resistance of heat sensitive proteins seems to be involved in the development of thermotolerance. Although many questions remain still to be answered, it appears that HSP72, when

  4. Salinity induced changes in cell membrane stability, protein and ...

    African Journals Online (AJOL)

    control), 4.7, 9.4 and 14.1 dS m-1 to determine the effect of salt on vegetative growth, relative water content, cell membrane stability, protein and RNA contents in sand culture experiment. Fresh and dry weights of plants, shoots and roots decreased ...

  5. Polyunsaturation in cell membranes and lipid bilayers and its effects on membrane proteins.

    Science.gov (United States)

    Slater, S J; Kelly, M B; Yeager, M D; Larkin, J; Ho, C; Stubbs, C D

    1996-03-01

    The effect of variation of the degree of cis-unsaturation on cell membrane protein functioning was investigated using a model lipid bilayer system and protein kinase C (PKC). This protein is a key element of signal transduction. Furthermore it is representative of a class of extrinsic membrane proteins that show lipid dependent interactions with cell membranes. To test for dependence of activity on the phospholipid unsaturation, experiments were devised using a vesicle assay system consisting of phosphatidylcholine (PC) and phosphatidylserine (PS) in which the unsaturation was systematically varied. Highly purified PKC alpha and epsilon were obtained using the baculovirus-insect cell expression system. It was shown that increased PC unsaturation elevated the activity of PKC alpha. By contrast, increasing the unsaturation of PS decreased the activity of PKC alpha, and to a lesser extent PKC epsilon. This result immediately rules out any single lipid bilayer physical parameter, such as lipid order, underlying the effect. It is proposed that while PC unsaturation effects are explainable on the basis of a contribution to membrane surface curvature stress, the effects of PS unsaturation may be due to specific protein-lipid interactions. Overall, the results indicate that altered phospholipid unsaturation in cell membranes that occurs in certain disease states such as chronic alcoholism, or by dietary manipulations, are likely to have profound effects on signal transduction pathways involving PKC and similar proteins.

  6. Plasma membrane associated membranes (PAM) from Jurkat cells contain STIM1 protein is PAM involved in the capacitative calcium entry?

    Science.gov (United States)

    Kozieł, Katarzyna; Lebiedzinska, Magdalena; Szabadkai, Gyorgy; Onopiuk, Marta; Brutkowski, Wojciech; Wierzbicka, Katarzyna; Wilczyński, Grzegorz; Pinton, Paolo; Duszyński, Jerzy; Zabłocki, Krzysztof; Wieckowski, Mariusz R

    2009-12-01

    A proper cooperation between the plasma membrane, the endoplasmic reticulum and the mitochondria seems to be essential for numerous cellular processes involved in Ca(2+) signalling and maintenance of Ca(2+) homeostasis. A presence of microsomal and mitochondrial proteins together with those characteristic for the plasma membrane in the fraction of the plasma membrane associated membranes (PAM) indicates a formation of stabile interactions between these three structures. We isolated the plasma membrane associated membranes from Jurkat cells and found its significant enrichment in the plasma membrane markers including plasma membrane Ca(2+)-ATPase, Na(+), K(+)-ATPase and CD3 as well as sarco/endoplasmic reticulum Ca(2+) ATPase as a marker of the endoplasmic reticulum membranes. In addition, two proteins involved in the store-operated Ca(2+) entry, Orai1 located in the plasma membrane and an endoplasmic reticulum protein STIM1 were found in this fraction. Furthermore, we observed a rearrangement of STIM1-containing protein complexes isolated from Jurkat cells undergoing stimulation by thapsigargin. We suggest that the inter-membrane compartment composed of the plasma membrane and the endoplasmic reticulum, and isolated as a stabile plasma membrane associated membranes fraction, might be involved in the store-operated Ca(2+) entry, and their formation and rebuilding have an important regulatory role in cellular Ca(2+) homeostasis.

  7. Identification of glycan structure alterations on cell membrane proteins in desoxyepothilone B resistant leukemia cells.

    Science.gov (United States)

    Nakano, Miyako; Saldanha, Rohit; Göbel, Anja; Kavallaris, Maria; Packer, Nicolle H

    2011-11-01

    Resistance to tubulin-binding agents used in cancer is often multifactorial and can include changes in drug accumulation and modified expression of tubulin isotypes. Glycans on cell membrane proteins play important roles in many cellular processes such as recognition and apoptosis, and this study investigated whether changes to the glycan structures on cell membrane proteins occur when cells become resistant to drugs. Specifically, we investigated the alteration of glycan structures on the cell membrane proteins of human T-cell acute lymphoblastic leukemia (CEM) cells that were selected for resistance to desoxyepothilone B (CEM/dEpoB). The glycan profile of the cell membrane glycoproteins was obtained by sequential release of N- and O-glycans from cell membrane fraction dotted onto polyvinylidene difluoride membrane with PNGase F and β-elimination respectively. The released glycan alditols were analyzed by liquid chromatography (graphitized carbon)-electrospray ionization tandem MS. The major N-glycan on CEM cell was the core fucosylated α2-6 monosialo-biantennary structure. Resistant CEM/dEpoB cells had a significant decrease of α2-6 linked sialic acid on N-glycans. The lower α2-6 sialylation was caused by a decrease in activity of β-galactoside α2-6 sialyltransferase (ST6Gal), and decreased expression of the mRNA. It is clear that the membrane glycosylation of leukemia cells changes during acquired resistance to dEpoB drugs and that this change occurs globally on all cell membrane glycoproteins. This is the first identification of a specific glycan modification on the surface of drug resistant cells and the mechanism of this downstream effect on microtubule targeting drugs may offer a route to new interventions to overcome drug resistance.

  8. Rupturing Giant Plasma Membrane Vesicles to Form Micron-sized Supported Cell Plasma Membranes with Native Transmembrane Proteins.

    Science.gov (United States)

    Chiang, Po-Chieh; Tanady, Kevin; Huang, Ling-Ting; Chao, Ling

    2017-11-09

    Being able to directly obtain micron-sized cell blebs, giant plasma membrane vesicles (GPMVs), with native membrane proteins and deposit them on a planar support to form supported plasma membranes could allow the membrane proteins to be studied by various surface analytical tools in native-like bilayer environments. However, GPMVs do not easily rupture on conventional supports because of their high protein and cholesterol contents. Here, we demonstrate the possibility of using compression generated by the air-water interface to efficiently rupture GPMVs to form micron-sized supported membranes with native plasma membrane proteins. We demonstrated that not only lipid but also a native transmembrane protein in HeLa cells, Aquaporin 3 (AQP3), is mobile in the supported membrane platform. This convenient method for generating micron-sized supported membrane patches with mobile native transmembrane proteins could not only facilitate the study of membrane proteins by surface analytical tools, but could also enable us to use native membrane proteins for bio-sensing applications.

  9. Membrane protein stoichiometry studied in intact mammalian cells using liquid-phase electron microscopy.

    Science.gov (United States)

    DE Jonge, N

    2018-02-01

    Receptor membrane proteins in the plasma membranes of cells respond to extracellular chemical signals by conformational changes, spatial redistribution, and (re-)assembly into protein complexes, for example, into homodimers (pairs of the same protein type). The functional state of the proteins can be determined from information about how subunits are assembled into protein complexes. Stoichiometric information about the protein complex subunits, however, is generally not obtained from intact cells but from pooled material extracted from many cells, resulting in a lack of fundamental knowledge about the functioning of membrane proteins. First, functional states may dramatically differ from cell to cell on account of cell heterogeneity. Second, extracting the membrane proteins from the plasma membrane may lead to many artefacts. Liquid-phase scanning transmission electron microscopy (STEM), in short liquid STEM, is a new technique capable of determining the locations of individual membrane proteins within the intact plasma membranes of cells in liquid. Many tens of whole cells can readily be imaged. It is possible to analyse the stoichiometry of membrane proteins in single cells while accounting for heterogenic cell populations. Liquid STEM was used to image epidermal growth factor receptors in whole COS7 cells. A study of the dimerisation of the HER2 protein in breast cancer cells revealed the presence of rare cancer cells in which HER2 was in a different functional state than in the bulk cells. Stoichiometric information about receptors is essential not only for basic science but also for biomedical application because they present many important pharmaceutical targets. © 2017 The Authors Journal of Microscopy © 2017 Royal Microscopical Society.

  10. High-level cell-free production of membrane proteins with nanodiscs.

    Science.gov (United States)

    Roos, Christian; Kai, Lei; Haberstock, Stefan; Proverbio, Davide; Ghoshdastider, Umesh; Ma, Yi; Filipek, Slawomir; Wang, Xiaoning; Dötsch, Volker; Bernhard, Frank

    2014-01-01

    This chapter addresses two major bottlenecks in cell-free membrane protein production. Firstly, we describe the optimization of expression templates for obtaining membrane proteins in preparative scales. We present details for a newly established tag variation screen providing high success rates in improving expression efficiencies while having only minimal impacts on the target protein structure. Secondly, we present protocols for the efficient co-translational insertion of membrane proteins into defined lipid bilayers. We describe the production of nanodiscs and their implementation into cell-free expression reactions for the co-translational reconstitution of membrane proteins. In addition we give guidelines for the loading of nanodiscs with different lipids in order to systematically analyze effects of lipids on the translocation, functional folding, and stability of cell-free expressed membrane proteins.

  11. Lipid-protein interactions in plasma membranes of fiber cells isolated from the human eye lens.

    Science.gov (United States)

    Raguz, Marija; Mainali, Laxman; O'Brien, William J; Subczynski, Witold K

    2014-03-01

    The protein content in human lens membranes is extremely high, increases with age, and is higher in the nucleus as compared with the cortex, which should strongly affect the organization and properties of the lipid bilayer portion of intact membranes. To assess these effects, the intact cortical and nuclear fiber cell plasma membranes isolated from human lenses from 41- to 60-year-old donors were studied using electron paramagnetic resonance spin-labeling methods. Results were compared with those obtained for lens lipid membranes prepared from total lipid extracts from human eyes of the same age group [Mainali, L., Raguz, M., O'Brien, W. J., and Subczynski, W. K. (2013) Biochim. Biophys. Acta]. Differences were considered to be mainly due to the effect of membrane proteins. The lipid-bilayer portions of intact membranes were significantly less fluid than lipid bilayers of lens lipid membranes, prepared without proteins. The intact membranes were found to contain three distinct lipid environments termed the bulk lipid domain, boundary lipid domain, and trapped lipid domain. However, the cholesterol bilayer domain, which was detected in cortical and nuclear lens lipid membranes, was not detected in intact membranes. The relative amounts of bulk and trapped lipids were evaluated. The amount of lipids in domains uniquely formed due to the presence of membrane proteins was greater in nuclear membranes than in cortical membranes. Thus, it is evident that the rigidity of nuclear membranes is greater than that of cortical membranes. Also the permeability coefficients for oxygen measured in domains of nuclear membranes were significantly lower than appropriate coefficients measured in cortical membranes. Relationships between the organization of lipids into lipid domains in fiber cells plasma membranes and the organization of membrane proteins are discussed. Copyright © 2014 Elsevier Ltd. All rights reserved.

  12. Expression of Trans-Membrane Proteins in vitro Using a Cell Free System

    Science.gov (United States)

    Weisse, Natalie; Noireaux, Vincent; Chalmeau, Jerome

    2010-10-01

    Trans-membrane proteins represent a significant portion of the proteins expressed by cells. The expression of proteins in vitro, however, remains a challenge. Numerous expression approaches have been developed with cell free expression (CFE) being one of the most promising. CFE is based on a transcription-translation system that has been extracted from E. coli bacteria. Adding the desired DNA allows expression of a selected protein, and in the presence of phospholipids the expression of trans-membrane proteins becomes possible. In order to express trans-membrane proteins in a closed native environment, the cell free system (CFS) is encapsulated with a phospholipid bilayer, creating an artificial cell. To verify protein expression, AquaporinZ (AqpZ), a well-known trans-membrane protein tagged with a green fluorescent protein (eGFP), was used so the expressed proteins could be seen under a fluorescent microscope. These artificial cells will serve as an experimental platform for testing the viability of the expressed trans-membrane proteins. Results from the manipulation of these artificial cells by attaching them to the slide surface through streptavidin-biotin bonding will be presented.

  13. In-Situ Observation of Membrane Protein Folding during Cell-Free Expression.

    Directory of Open Access Journals (Sweden)

    Axel Baumann

    Full Text Available Proper insertion, folding and assembly of functional proteins in biological membranes are key processes to warrant activity of a living cell. Here, we present a novel approach to trace folding and insertion of a nascent membrane protein leaving the ribosome and penetrating the bilayer. Surface Enhanced IR Absorption Spectroscopy selectively monitored insertion and folding of membrane proteins during cell-free expression in a label-free and non-invasive manner. Protein synthesis was performed in an optical cell containing a prism covered with a thin gold film with nanodiscs on top, providing an artificial lipid bilayer for folding. In a pilot experiment, the folding pathway of bacteriorhodopsin via various secondary and tertiary structures was visualized. Thus, a methodology is established with which the folding reaction of other more complex membrane proteins can be observed during protein biosynthesis (in situ and in operando at molecular resolution.

  14. Resolving mixed mechanisms of protein subdiffusion at the T cell plasma membrane

    Science.gov (United States)

    Golan, Yonatan; Sherman, Eilon

    2017-06-01

    The plasma membrane is a complex medium where transmembrane proteins diffuse and interact to facilitate cell function. Membrane protein mobility is affected by multiple mechanisms, including crowding, trapping, medium elasticity and structure, thus limiting our ability to distinguish them in intact cells. Here we characterize the mobility and organization of a short transmembrane protein at the plasma membrane of live T cells, using single particle tracking and photoactivated-localization microscopy. Protein mobility is highly heterogeneous, subdiffusive and ergodic-like. Using mobility characteristics, we segment individual trajectories into subpopulations with distinct Gaussian step-size distributions. Particles of low-to-medium mobility consist of clusters, diffusing in a viscoelastic and fractal-like medium and are enriched at the centre of the cell footprint. Particles of high mobility undergo weak confinement and are more evenly distributed. This study presents a methodological approach to resolve simultaneous mixed subdiffusion mechanisms acting on polydispersed samples and complex media such as cell membranes.

  15. Fluorescent in situ folding control for rapid optimization of cell-free membrane protein synthesis.

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    Annika Müller-Lucks

    Full Text Available Cell-free synthesis is an open and powerful tool for high-yield protein production in small reaction volumes predestined for high-throughput structural and functional analysis. Membrane proteins require addition of detergents for solubilization, liposomes, or nanodiscs. Hence, the number of parameters to be tested is significantly higher than with soluble proteins. Optimization is commonly done with respect to protein yield, yet without knowledge of the protein folding status. This approach contains a large inherent risk of ending up with non-functional protein. We show that fluorophore formation in C-terminal fusions with green fluorescent protein (GFP indicates the folding state of a membrane protein in situ, i.e. within the cell-free reaction mixture, as confirmed by circular dichroism (CD, proteoliposome reconstitution and functional assays. Quantification of protein yield and in-gel fluorescence intensity imply suitability of the method for membrane proteins of bacterial, protozoan, plant, and mammalian origin, representing vacuolar and plasma membrane localization, as well as intra- and extracellular positioning of the C-terminus. We conclude that GFP-fusions provide an extension to cell-free protein synthesis systems eliminating the need for experimental folding control and, thus, enabling rapid optimization towards membrane protein quality.

  16. Tritium labelling of a cholesterol amphiphile designed for cell membrane anchoring of proteins.

    Science.gov (United States)

    Schäfer, Balázs; Orbán, Erika; Kele, Zoltán; Tömböly, Csaba

    2015-01-01

    Cell membrane association of proteins can be achieved by the addition of lipid moieties to the polypeptide chain, and such lipid-modified proteins have important biological functions. A class of cell surface proteins contains a complex glycosylphosphatidylinositol (GPI) glycolipid at the C-terminus, and they are accumulated in cholesterol-rich membrane microdomains, that is, lipid rafts. Semisynthetic lipoproteins prepared from recombinant proteins and designed lipids are valuable probes and model systems of the membrane-associated proteins. Because GPI-anchored proteins can be reinserted into the cell membrane with the retention of the biological function, they are appropriate candidates for preparing models via reduction of the structural complexity. A synthetic headgroup was added to the 3β-hydroxyl group of cholesterol, an essential lipid component of rafts, and the resulting cholesterol derivative was used as a simplified GPI mimetic. In order to quantitate the membrane integrated GPI mimetic after the exogenous addition to live cells, a tritium labelled cholesterol anchor was prepared. The radioactive label was introduced into the headgroup, and the radiolabelled GPI mimetic anchor was obtained with a specific activity of 1.37 TBq/mmol. The headgroup labelled cholesterol derivative was applied to demonstrate the sensitive detection of the cell membrane association of the anchor under in vivo conditions. Copyright © 2015 John Wiley & Sons, Ltd.

  17. Repair of Nerve Cell Membrance Damage by Calcium-Dependent, Membrane-Binding Proteins

    Science.gov (United States)

    2013-09-01

    disease amyloid beta protein forms calcium channels in bilayer membranes: blockade by tromethamine and aluminum , Proc Natl Acad Sci U S A 90 (1993) 567... coagulation cascade in blood and the chaperoning of membrane interactions inside cells.4 Common to these diverse functions is the ability to bind...are quickly resolved restoring the permeability barrier. In vivo, such a transient breakdown of the membrane might constitute a significant initial cost

  18. Toward mechanical manipulations of cell membranes and membrane proteins using an atomic force microscope: an invited review.

    Science.gov (United States)

    Ikai, Atsushi; Afrin, Rehana

    2003-01-01

    Recent advances in the use of the atomic force microscope (AFM) for manipulating cell membranes and membrane proteins are reviewed. Early pioneering work on measurements of the magnitude of the force required to create indentations with defined depth on their surfaces and to separate interacting pairs of avidin-biotin, antigen-antibody, and complementary DNA pairs formed the basis of this field. The method has subsequently been applied to map the presence of cell surface receptors and polysaccharides on live cell membranes by force measurement, with promising results. Attempts to extract phospholipids and proteins from lipid bilayers and live cell surfaces have been reported, providing a new tool for the manipulation of cellular activities and biochemical analysis at the single-cell level. An increasing awareness of the effect of the pulling speed (nm/s or microm/s), or more accurately, the force loading rate (pN/s or nN/s) on the magnitude of the rupture force, has led researchers to construct energy diagrams of rupture events based on the parameters available from such studies. Information on such nature of the interplay of force and loading rate is vital for nanomanipulation of living cells and cell membranes. Some relevant work for membrane manipulation using other methods is also reviewed in relation to AFM-based methodology.

  19. Outer Hair Cell Lateral Wall Structure Constrains the Mobility of Plasma Membrane Proteins.

    Science.gov (United States)

    Yamashita, Tetsuji; Hakizimana, Pierre; Wu, Siva; Hassan, Ahmed; Jacob, Stefan; Temirov, Jamshid; Fang, Jie; Mellado-Lagarde, Marcia; Gursky, Richard; Horner, Linda; Leibiger, Barbara; Leijon, Sara; Centonze, Victoria E; Berggren, Per-Olof; Frase, Sharon; Auer, Manfred; Brownell, William E; Fridberger, Anders; Zuo, Jian

    2015-09-01

    Nature's fastest motors are the cochlear outer hair cells (OHCs). These sensory cells use a membrane protein, Slc26a5 (prestin), to generate mechanical force at high frequencies, which is essential for explaining the exquisite hearing sensitivity of mammalian ears. Previous studies suggest that Slc26a5 continuously diffuses within the membrane, but how can a freely moving motor protein effectively convey forces critical for hearing? To provide direct evidence in OHCs for freely moving Slc26a5 molecules, we created a knockin mouse where Slc26a5 is fused with YFP. These mice and four other strains expressing fluorescently labeled membrane proteins were used to examine their lateral diffusion in the OHC lateral wall. All five proteins showed minimal diffusion, but did move after pharmacological disruption of membrane-associated structures with a cholesterol-depleting agent and salicylate. Thus, our results demonstrate that OHC lateral wall structure constrains the mobility of plasma membrane proteins and that the integrity of such membrane-associated structures are critical for Slc26a5's active and structural roles. The structural constraint of membrane proteins may exemplify convergent evolution of cellular motors across species. Our findings also suggest a possible mechanism for disorders of cholesterol metabolism with hearing loss such as Niemann-Pick Type C diseases.

  20. Outer Hair Cell Lateral Wall Structure Constrains the Mobility of Plasma Membrane Proteins.

    Directory of Open Access Journals (Sweden)

    Tetsuji Yamashita

    2015-09-01

    Full Text Available Nature's fastest motors are the cochlear outer hair cells (OHCs. These sensory cells use a membrane protein, Slc26a5 (prestin, to generate mechanical force at high frequencies, which is essential for explaining the exquisite hearing sensitivity of mammalian ears. Previous studies suggest that Slc26a5 continuously diffuses within the membrane, but how can a freely moving motor protein effectively convey forces critical for hearing? To provide direct evidence in OHCs for freely moving Slc26a5 molecules, we created a knockin mouse where Slc26a5 is fused with YFP. These mice and four other strains expressing fluorescently labeled membrane proteins were used to examine their lateral diffusion in the OHC lateral wall. All five proteins showed minimal diffusion, but did move after pharmacological disruption of membrane-associated structures with a cholesterol-depleting agent and salicylate. Thus, our results demonstrate that OHC lateral wall structure constrains the mobility of plasma membrane proteins and that the integrity of such membrane-associated structures are critical for Slc26a5's active and structural roles. The structural constraint of membrane proteins may exemplify convergent evolution of cellular motors across species. Our findings also suggest a possible mechanism for disorders of cholesterol metabolism with hearing loss such as Niemann-Pick Type C diseases.

  1. Bacillus thuringiensis Cyt2Aa2 toxin disrupts cell membranes by forming large protein aggregates.

    Science.gov (United States)

    Tharad, Sudarat; Toca-Herrera, José L; Promdonkoy, Boonhiang; Krittanai, Chartchai

    2016-10-01

    Bacillus thuringiensis (Bt) Cyt2Aa2 showed toxicity against Dipteran insect larvae and in vitro lysis activity on several cells. It has potential applications in the biological control of insect larvae. Although pore-forming and/or detergent-like mechanisms were proposed, the mechanism underlying cytolytic activity remains unclear. Analysis of the haemolytic activity of Cyt2Aa2 with osmotic stabilizers revealed partial toxin inhibition, suggesting a distinctive mechanism from the putative pore formation model. Membrane permeability was studied using fluorescent dye entrapped in large unilamellar vesicles (LUVs) at various protein/lipid molar ratios. Binding of Cyt2Aa2 monomer to the lipid membrane did not disturb membrane integrity until the critical protein/lipid molar ratio was reached, when Cyt2Aa2 complexes and cytolytic activity were detected. The complexes are large aggregates that appeared as a ladder when separated by agarose gel electrophoresis. Interaction of Cyt2Aa2 with Aedes albopictus cells was investigated by confocal microscopy and total internal reflection fluorescent microscopy (TIRF). The results showed that Cyt2Aa2 binds on the cell membrane at an early stage without cell membrane disruption. Protein aggregation on the cell membrane was detected later which coincided with cell swelling. Cyt2Aa2 aggregations on supported lipid bilayers (SLBs) were visualized by AFM. The AFM topographic images revealed Cyt2Aa2 aggregates on the lipid bilayer at low protein concentration and subsequently disrupts the lipid bilayer by forming a lesion as the protein concentration increased. These results supported the mechanism whereby Cyt2Aa2 binds and aggregates on the lipid membrane leading to the formation of non-specific hole and disruption of the cell membrane. © 2016 The Author(s).

  2. Communication Between the Cell Membrane and the Nucleus: Role of Protein Compartmentalization

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    Lelievre, Sophie A; Bissell, Mina J

    1998-10-21

    Understanding how the information is conveyed from outside to inside the cell is a critical challenge for all biologists involved in signal transduction. The flow of information initiated by cell-cell and cell-extracellular matrix contacts is mediated by the formation of adhesion complexes involving multiple proteins. Inside adhesion complexes, connective membrane skeleton (CMS) proteins are signal transducers that bind to adhesion molecules, organize the cytoskeleton, and initiate biochemical cascades. Adhesion complex-mediated signal transduction ultimately directs the formation of supramolecular structures in the cell nucleus, as illustrated by the establishment of multi complexes of DNA-bound transcription factors, and the redistribution of nuclear structural proteins to form nuclear subdomains. Recently, several CMS proteins have been observed to travel to the cell nucleus, suggesting a distinctive role for these proteins in signal transduction. This review focuses on the nuclear translocation of structural signal transducers of the membrane skeleton and also extends our analysis to possible translocation of resident nuclear proteins to the membrane skeleton. This leads us to envision the communication between spatially distant cellular compartments (i.e., membrane skeleton and cell nucleus) as a bidirectional flow of information (a dynamic reciprocity) based on subtle multilevel structural and biochemical equilibria. At one level, it is mediated by the interaction between structural signal transducers and their binding partners, at another level it may be mediated by the balance and integration of signal transducers in different cellular compartments.

  3. A Guide to Transient Expression of Membrane Proteins in HEK-293 Cells for Functional Characterization

    KAUST Repository

    Ooi, Amanda Siok Lee

    2016-07-19

    The human embryonic kidney 293 (HEK-293) cells are commonly used as host for the heterologous expression of membrane proteins not least because they have a high transfection efficiency and faithfully translate and process proteins. In addition, their cell size, morphology and division rate, and low expression of native channels are traits that are particularly attractive for current-voltage measurements. Nevertheless, the heterologous expression of complex membrane proteins such as receptors and ion channels for biological characterization and in particular for single-cell applications such as electrophysiology remains a challenge. Expression of functional proteins depends largely on careful step-by-step optimization that includes the design of expression vectors with suitable identification tags, as well as the selection of transfection methods and detection parameters appropriate for the application. Here, we use the heterologous expression of a plant potassium channel, the Arabidopsis thaliana guard cell outward-rectifying K+ channel, AtGORK (At5G37500) in HEK-293 cells as an example, to evaluate commonly used transfection reagents and fluorescent detection methods, and provide a detailed methodology for optimized transient transfection and expression of membrane proteins for in vivo studies in general and for single-cell applications in particular. This optimized protocol will facilitate the physiological and cellular characterization of complex membrane proteins.

  4. Acinetobacter baumannii invades epithelial cells and outer membrane protein A mediates interactions with epithelial cells

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    Park Tae

    2008-12-01

    Full Text Available Abstract Background Acinetobacter baumannii is a nosocomial pathogen of increasing importance, but the pathogenic mechanism of this microorganism has not been fully explored. This study investigated the potential of A. baumannii to invade epithelial cells and determined the role of A. baumannii outer membrane protein A (AbOmpA in interactions with epithelial cells. Results A. baumannii invaded epithelial cells by a zipper-like mechanism, which is associated with microfilament- and microtubule-dependent uptake mechanisms. Internalized bacteria were located in the membrane-bound vacuoles. Pretreatment of recombinant AbOmpA significantly inhibited the adherence to and invasion of A. baumannii in epithelial cells. Cell invasion of isogenic AbOmpA- mutant significantly decreased as compared with wild-type bacteria. In a murine pneumonia model, wild-type bacteria exhibited a severe lung pathology and induced a high bacterial burden in blood, whereas AbOmpA- mutant was rarely detected in blood. Conclusion A. baumannii adheres to and invades epithelial cells. AbOmpA plays a major role in the interactions with epithelial cells. These findings contribute to the understanding of A. baumannii pathogenesis in the early stage of bacterial infection.

  5. A practical guide for the identification of membrane and plasma membrane proteins in human embryonic stem cells and human embryonal carcinoma cells.

    NARCIS (Netherlands)

    Dormeyer, W.; van Hoof, D.; Mummery, C.L.; Krijgsveld, J.; Heck, A.

    2008-01-01

    The identification of (plasma) membrane proteins in cells can provide valuable insights into the regulation of their biological processes. Pluripotent cells such as human embryonic stem cells and embryonal carcinoma cells are capable of unlimited self-renewal and share many of the biological

  6. Visualization of transfer of a fluorescently-labeled membrane raft protein to T cells using lentivirus

    OpenAIRE

    Byrum, Jennifer; Rodgers, William

    2005-01-01

    Lentivirus vector systems have been developed for the safe delivery of foreign genes to target tissues. However, the use of these systems for delivering specific proteins to target cells has been largely unexplored. To test this concept, the lentivirus expression plasmid pLenti was utilized to overexpress in producer cells a YFP-fusion protein that is specifically targeted to glycolipid-enriched membrane rafts, which is the site of virus assembly. Our data show that virus generated in produce...

  7. [Role of protein kinases of human red cell membrane in deformability and aggregation changes].

    Science.gov (United States)

    Murav'ev, A V; Maĭmistova, A A; Tikhomirova, I A; Bulaeva, S V; Mikhaĭlov, P V; Murav'ev, A A

    2012-01-01

    The proteomic analysis has showed that red cell membrane contains several kinases and phosphatases. Therefore the aim of this study was to investigate the role of protein kinases of human red cell membrane in deformability and aggregation changes. Exposure of red blood cells (RBCs) to some chemical compounds led to change in the RBC microrheological properties. When forskolin (10 microM), an adenylyl cyclase (AC) and a protein kinase A (PKA) stimulator was added to RBC suspension, the RBC deformability (RBCD) was increased by 20% (p RBCA) was significantly decreased under these conditions (p RBCA lowering. The similar effect was found when cells were incubated with cisplatin as a tyrosine protein kinase (TPK) activator. It is important to note that a selective TPK inhibitor--lavendustin eliminated the above mention effects. On the whole the total data clearly show that the red cell aggregation and deformation changes were connected with an activation of the different intracellular signaling pathways.

  8. Selective Labeling of Proteins on Living Cell Membranes Using Fluorescent Nanodiamond Probes

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    Shingo Sotoma

    2016-03-01

    Full Text Available The impeccable photostability of fluorescent nanodiamonds (FNDs is an ideal property for use in fluorescence imaging of proteins in living cells. However, such an application requires highly specific labeling of the target proteins with FNDs. Furthermore, the surface of unmodified FNDs tends to adsorb biomolecules nonspecifically, which hinders the reliable targeting of proteins with FNDs. Here, we combined hyperbranched polyglycerol modification of FNDs with the β-lactamase-tag system to develop a strategy for selective imaging of the protein of interest in cells. The combination of these techniques enabled site-specific labeling of Interleukin-18 receptor alpha chain, a membrane receptor, with FNDs, which eventually enabled tracking of the diffusion trajectory of FND-labeled proteins on the membrane surface.

  9. The cell-based L-glutathione protection assays to study endocytosis and recycling of plasma membrane proteins.

    Science.gov (United States)

    Cihil, Kristine M; Swiatecka-Urban, Agnieszka

    2013-12-13

    Membrane trafficking involves transport of proteins from the plasma membrane to the cell interior (i.e. endocytosis) followed by trafficking to lysosomes for degradation or to the plasma membrane for recycling. The cell based L-glutathione protection assays can be used to study endocytosis and recycling of protein receptors, channels, transporters, and adhesion molecules localized at the cell surface. The endocytic assay requires labeling of cell surface proteins with a cell membrane impermeable biotin containing a disulfide bond and the N-hydroxysuccinimide (NHS) ester at 4 ºC - a temperature at which membrane trafficking does not occur. Endocytosis of biotinylated plasma membrane proteins is induced by incubation at 37 ºC. Next, the temperature is decreased again to 4 ºC to stop endocytic trafficking and the disulfide bond in biotin covalently attached to proteins that have remained at the plasma membrane is reduced with L-glutathione. At this point, only proteins that were endocytosed remain protected from L-glutathione and thus remain biotinylated. After cell lysis, biotinylated proteins are isolated with streptavidin agarose, eluted from agarose, and the biotinylated protein of interest is detected by western blotting. During the recycling assay, after biotinylation cells are incubated at 37 °C to load endocytic vesicles with biotinylated proteins and the disulfide bond in biotin covalently attached to proteins remaining at the plasma membrane is reduced with L-glutathione at 4 ºC as in the endocytic assay. Next, cells are incubated again at 37 °C to allow biotinylated proteins from endocytic vesicles to recycle to the plasma membrane. Cells are then incubated at 4 ºC, and the disulfide bond in biotin attached to proteins that recycled to the plasma membranes is reduced with L-glutathione. The biotinylated proteins protected from L-glutathione are those that did not recycle to the plasma membrane.

  10. Comparative transcriptional analysis of Bacillus subtilis cells overproducing either secreted proteins, lipoproteins or membrane proteins

    Directory of Open Access Journals (Sweden)

    Marciniak Bogumiła C

    2012-05-01

    Full Text Available Abstract Background Bacillus subtilis is a favorable host for the production of industrially relevant proteins because of its capacity of secreting proteins into the medium to high levels, its GRAS (Generally Recognized As Safe status, its genetic accessibility and its capacity to grow in large fermentations. However, production of heterologous proteins still faces limitations. Results This study aimed at the identification of bottlenecks in secretory protein production by analyzing the response of B. subtilis at the transcriptome level to overproduction of eight secretory proteins of endogenous and heterologous origin and with different subcellular or extracellular destination: secreted proteins (NprE and XynA of B. subtilis, Usp45 of Lactococcus lactis, TEM-1 β-lactamase of Escherichia coli, membrane proteins (LmrA of L. lactis and XylP of Lactobacillus pentosus and lipoproteins (MntA and YcdH of B. subtilis. Responses specific for proteins with a common localization as well as more general stress responses were observed. The latter include upregulation of genes encoding intracellular stress proteins (groES/EL, CtsR regulated genes. Specific responses include upregulation of the liaIHGFSR operon under Usp45 and TEM-1 β-lactamase overproduction; cssRS, htrA and htrB under all secreted proteins overproduction; sigW and SigW-regulated genes mainly under membrane proteins overproduction; and ykrL (encoding an HtpX homologue specifically under membrane proteins overproduction. Conclusions The results give better insights into B. subtilis responses to protein overproduction stress and provide potential targets for genetic engineering in order to further improve B. subtilis as a protein production host.

  11. Saccharomyces cerevisiae cells lacking Pex3 contain membrane vesicles that harbor a subset of peroxisomal membrane proteins.

    Science.gov (United States)

    Wróblewska, Justyna P; Cruz-Zaragoza, Luis Daniel; Yuan, Wei; Schummer, Andreas; Chuartzman, Silvia G; de Boer, Rinse; Oeljeklaus, Silke; Schuldiner, Maya; Zalckvar, Einat; Warscheid, Bettina; Erdmann, Ralf; van der Klei, Ida J

    2017-10-01

    Pex3 has been proposed to be important for the exit of peroxisomal membrane proteins (PMPs) from the ER, based on the observation that PMPs accumulate at the ER in Saccharomyces cerevisiae pex3 mutant cells. Using a combination of microscopy and biochemical approaches, we show that a subset of the PMPs, including the receptor docking protein Pex14, localizes to membrane vesicles in S. cerevisiae pex3 cells. These vesicles are morphologically distinct from the ER and do not co-sediment with ER markers in cell fractionation experiments. At the vesicles, Pex14 assembles with other peroxins (Pex13, Pex17, and Pex5) to form a complex with a composition similar to the PTS1 import pore in wild-type cells. Fluorescence microscopy studies revealed that also the PTS2 receptor Pex7, the importomer organizing peroxin Pex8, the ubiquitin conjugating enzyme Pex4 with its recruiting PMP Pex22, as well as Pex15 and Pex25 co-localize with Pex14. Other peroxins (including the RING finger complex and Pex27) did not accumulate at these structures, of which Pex11 localized to mitochondria. In line with these observations, proteomic analysis showed that in addition to the docking proteins and Pex5, also Pex7, Pex4/Pex22 and Pex25 were present in Pex14 complexes isolated from pex3 cells. However, formation of the entire importomer was not observed, most likely because Pex8 and the RING proteins were absent in the Pex14 protein complexes. Our data suggest that peroxisomal membrane vesicles can form in the absence of Pex3 and that several PMPs can insert in these vesicles in a Pex3 independent manner. Copyright © 2017 Elsevier B.V. All rights reserved.

  12. IRES-mediated translation of membrane proteins and glycoproteins in eukaryotic cell-free systems.

    Directory of Open Access Journals (Sweden)

    Andreas K Brödel

    Full Text Available Internal ribosome entry site (IRES elements found in the 5' untranslated region of mRNAs enable translation initiation in a cap-independent manner, thereby representing an alternative to cap-dependent translation in cell-free protein expression systems. However, IRES function is largely species-dependent so their utility in cell-free systems from different species is rather limited. A promising approach to overcome these limitations would be the use of IRESs that are able to recruit components of the translation initiation apparatus from diverse origins. Here, we present a solution to this technical problem and describe the ability of a number of viral IRESs to direct efficient protein expression in different eukaryotic cell-free expression systems. The IRES from the intergenic region (IGR of the Cricket paralysis virus (CrPV genome was shown to function efficiently in four different cell-free systems based on lysates derived from cultured Sf21, CHO and K562 cells as well as wheat germ. Our results suggest that the CrPV IGR IRES-based expression vector is universally applicable for a broad range of eukaryotic cell lysates. Sf21, CHO and K562 cell-free expression systems are particularly promising platforms for the production of glycoproteins and membrane proteins since they contain endogenous microsomes that facilitate the incorporation of membrane-spanning proteins and the formation of post-translational modifications. We demonstrate the use of the CrPV IGR IRES-based expression vector for the enhanced synthesis of various target proteins including the glycoprotein erythropoietin and the membrane proteins heparin-binding EGF-like growth factor receptor as well as epidermal growth factor receptor in the above mentioned eukaryotic cell-free systems. CrPV IGR IRES-mediated translation will facilitate the development of novel eukaryotic cell-free expression platforms as well as the high-yield synthesis of desired proteins in already established

  13. IRES-Mediated Translation of Membrane Proteins and Glycoproteins in Eukaryotic Cell-Free Systems

    Science.gov (United States)

    Brödel, Andreas K.; Sonnabend, Andrei; Roberts, Lisa O.; Stech, Marlitt; Wüstenhagen, Doreen A.; Kubick, Stefan

    2013-01-01

    Internal ribosome entry site (IRES) elements found in the 5′ untranslated region of mRNAs enable translation initiation in a cap-independent manner, thereby representing an alternative to cap-dependent translation in cell-free protein expression systems. However, IRES function is largely species-dependent so their utility in cell-free systems from different species is rather limited. A promising approach to overcome these limitations would be the use of IRESs that are able to recruit components of the translation initiation apparatus from diverse origins. Here, we present a solution to this technical problem and describe the ability of a number of viral IRESs to direct efficient protein expression in different eukaryotic cell-free expression systems. The IRES from the intergenic region (IGR) of the Cricket paralysis virus (CrPV) genome was shown to function efficiently in four different cell-free systems based on lysates derived from cultured Sf21, CHO and K562 cells as well as wheat germ. Our results suggest that the CrPV IGR IRES-based expression vector is universally applicable for a broad range of eukaryotic cell lysates. Sf21, CHO and K562 cell-free expression systems are particularly promising platforms for the production of glycoproteins and membrane proteins since they contain endogenous microsomes that facilitate the incorporation of membrane-spanning proteins and the formation of post-translational modifications. We demonstrate the use of the CrPV IGR IRES-based expression vector for the enhanced synthesis of various target proteins including the glycoprotein erythropoietin and the membrane proteins heparin-binding EGF-like growth factor receptor as well as epidermal growth factor receptor in the above mentioned eukaryotic cell-free systems. CrPV IGR IRES-mediated translation will facilitate the development of novel eukaryotic cell-free expression platforms as well as the high-yield synthesis of desired proteins in already established systems. PMID

  14. IRES-mediated translation of membrane proteins and glycoproteins in eukaryotic cell-free systems.

    Science.gov (United States)

    Brödel, Andreas K; Sonnabend, Andrei; Roberts, Lisa O; Stech, Marlitt; Wüstenhagen, Doreen A; Kubick, Stefan

    2013-01-01

    Internal ribosome entry site (IRES) elements found in the 5' untranslated region of mRNAs enable translation initiation in a cap-independent manner, thereby representing an alternative to cap-dependent translation in cell-free protein expression systems. However, IRES function is largely species-dependent so their utility in cell-free systems from different species is rather limited. A promising approach to overcome these limitations would be the use of IRESs that are able to recruit components of the translation initiation apparatus from diverse origins. Here, we present a solution to this technical problem and describe the ability of a number of viral IRESs to direct efficient protein expression in different eukaryotic cell-free expression systems. The IRES from the intergenic region (IGR) of the Cricket paralysis virus (CrPV) genome was shown to function efficiently in four different cell-free systems based on lysates derived from cultured Sf21, CHO and K562 cells as well as wheat germ. Our results suggest that the CrPV IGR IRES-based expression vector is universally applicable for a broad range of eukaryotic cell lysates. Sf21, CHO and K562 cell-free expression systems are particularly promising platforms for the production of glycoproteins and membrane proteins since they contain endogenous microsomes that facilitate the incorporation of membrane-spanning proteins and the formation of post-translational modifications. We demonstrate the use of the CrPV IGR IRES-based expression vector for the enhanced synthesis of various target proteins including the glycoprotein erythropoietin and the membrane proteins heparin-binding EGF-like growth factor receptor as well as epidermal growth factor receptor in the above mentioned eukaryotic cell-free systems. CrPV IGR IRES-mediated translation will facilitate the development of novel eukaryotic cell-free expression platforms as well as the high-yield synthesis of desired proteins in already established systems.

  15. [Interaction of FABP4 with plasma membrane proteins of endothelial cells].

    Science.gov (United States)

    Saavedra, Paula; Girona, Josefa; Aragonès, Gemma; Cabré, Anna; Guaita, Sandra; Heras, Mercedes; Masana, Lluís

    2015-01-01

    Fatty acid binding protein (FABP4) is an adipose tissue-secreted adipokine implicated in the regulation of the energetic metabolism and inflammation. High levels of circulating FABP4 have been described in people with obesity, atherogenic dyslipidemia, diabetes and metabolic syndrome. Recent studies have demonstrated that FABP4 could have a direct effect on peripheral tissues and, specifically, on vascular function. It is still unknown how the interaction between FABP4 and the endothelial cells is produced to prompt these effects on vascular function. The objective of this work is studying the interaction between FABP4 and the plasma membrane proteins of endothelial cells. HUVEC cells were incubated with and without FABP4 (100 ng/ml) for 5 minutes. Immunolocalization of FABP4 was studied by confocal microscopy. The results showed that FABP4 colocalizates with CD31, a membrane protein marker. A strategy which combines 6XHistidine-tag FABP4 (FABP4-His), incubations with or without FABP4-His (100 ng/ml), formaldehyde cross-linking, cellular membrane protein extraction and western blot, was designed to study the FABP4 interactions with membrane proteins of HUVECs. The results showed different western blot profiles depending of the incubation with or without FABP4-His. The immunoblot revelead three covalent protein complexes of about 108, 77 and 33 kDa containing FAPB4 and its putative receptor. The existence of a specific binding protein complex able to bind FABP4 to endothelial cells is supported by these results. The obtained results will permit us advance in the molecular knowledge of FABP4 effects as well as use this protein and its receptor as therapeutic target to prevent cardiovascular. Copyright © 2014 Sociedad Española de Arteriosclerosis. Published by Elsevier España. All rights reserved.

  16. Investigation of basement membrane proteins in a case of granular cell ameloblastoma

    Science.gov (United States)

    Lapthanasupkul, Puangwan; Poomsawat, Sopee; Chindasombatjaroen, Jira

    2012-01-01

    Granular cell ameloblastoma is a rare, benign neoplasm of the odontogenic epithelium. A case of massive granular cell ameloblastoma in a 44-year-old Thai female is reported. Histopathological features displayed a follicular type of ameloblastoma with an accumulation of granular cells residing within the tumor follicles. After treatment by partial mandibulectomy, the patient showed a good prognosis without recurrence in a 2-year follow-up. To characterize the granular cells in ameloblastoma, we examined the expression of basement membrane (BM) proteins, including collagen type IV, laminins 1 and 5 and fibronectin using immunohistochemistry. Except for the granular cells, the tumor cells demonstrated a similar expression of BM proteins compared to follicular and plexiform ameloblastomas in our previous study, whereas the granular cells showed strong positivity to laminins 1 and 5 and fibronectin. The increased fibronectin expression in granular cells suggests a possibility of age-related transformation of granular cells in ameloblastoma. PMID:22361945

  17. Plasma membrane protein trafficking in plant-microbe interactions: a plant cell point of view

    Directory of Open Access Journals (Sweden)

    Nathalie eLeborgne-Castel

    2014-12-01

    Full Text Available In order to ensure their physiological and cellular functions, plasma membrane (PM proteins must be properly conveyed from their site of synthesis, i.e. the endoplasmic reticulum, to their final destination, the PM, through the secretory pathway. PM protein homeostasis also relies on recycling and/or degradation, two processes that are initiated by endocytosis. Vesicular membrane trafficking events to and from the PM have been shown to be altered when plant cells are exposed to mutualistic or pathogenic microbes. In this review, we will describe the fine-tune regulation of such alterations, and their consequence in PM protein activity. We will consider the formation of intracellular perimicrobial compartments, the PM protein trafficking machinery of the host, and the delivery or retrieval of signaling and transport proteins such as pattern-recognition receptors, producers of reactive oxygen species, and sugar transporters.

  18. Membrane protein nanoclustering as a functional unit of immune cells : from nanoscopy to single molecule dynamics

    OpenAIRE

    Torreño Piña, Juan Andrés

    2015-01-01

    Premi Extraordinari de Doctorat, promoció 2014-2015. Àmbit de Ciències State-of-the-art biophysical techniques featuring high temporal and spatial resolution have allowed for the first time the direct visualization of individual transmembrane proteins on the cell membrane. These techniques have revealed that a large amount of molecular components of the cell membrane do not organize in a random manner but they rather grouped together forming so-called clusters at the nanoscale. Moreover, t...

  19. Multifunctional Transmembrane Protein Ligands for Cell-Specific Targeting of Plasma Membrane-Derived Vesicles.

    Science.gov (United States)

    Zhao, Chi; Busch, David J; Vershel, Connor P; Stachowiak, Jeanne C

    2016-07-01

    Liposomes and nanoparticles that bind selectively to cell-surface receptors can target specific populations of cells. However, chemical conjugation of ligands to these particles is difficult to control, frequently limiting ligand uniformity and complexity. In contrast, the surfaces of living cells are decorated with highly uniform populations of sophisticated transmembrane proteins. Toward harnessing cellular capabilities, here it is demonstrated that plasma membrane vesicles (PMVs) derived from donor cells can display engineered transmembrane protein ligands that precisely target cells on the basis of receptor expression. These multifunctional targeting proteins incorporate (i) a protein ligand, (ii) an intrinsically disordered protein spacer to make the ligand sterically accessible, and (iii) a fluorescent protein domain that enables quantification of the ligand density on the PMV surface. PMVs that display targeting proteins with affinity for the epidermal growth factor receptor (EGFR) bind at increasing concentrations to breast cancer cells that express increasing levels of EGFR. Further, as an example of the generality of this approach, PMVs expressing a single-domain antibody against green fluorescence protein (eGFP) bind to cells expressing eGFP-tagged receptors with a selectivity of ≈50:1. The results demonstrate the versatility of PMVs as cell targeting systems, suggesting diverse applications from drug delivery to tissue engineering. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  20. Identification of chitosan oligosaccharides binding proteins from the plasma membrane of wheat leaf cell.

    Science.gov (United States)

    Liu, Dongdong; Jiao, Siming; Cheng, Gong; Li, Xueming; Pei, Zhichao; Pei, Yuxin; Yin, Heng; Du, Yuguang

    2018-01-26

    Chitosan oligosaccharides (COS) have the ability to improve plant resistance to pests and diseases through activating plant immune system. However, it remains unclear whether stimulating reason of COS was associated with the plasma membrane proteins. Here, the interaction of COS with wheat leaf cell demonstrated that fluorescence-labeled COS were enriched on the cell surface and the interaction of COS with plasma membrane proteins was confirmed by quartz crystal microbalance (QCM) biosensor. What's more, HPLC and SDS-PAGE analysis showed that COS binding proteins exhibited more than three peaks and the molecular weight were 66 kDa to 97 kDa, where the COS binding proteins were fished out from wheat plasma membrane proteins by the COS affinity column. More importantly, LC-MS/MS analysis demonstrated that several candidates, including W5G2U8_WHEAT (a potential wall-associated receptor kinase protein), W5HY42_WHEAT and W5I0R4_WHEAT (potential G-type lectin S-receptor-like serine/threonine-protein kinase), have the potential to be COS receptors. Copyright © 2018. Published by Elsevier B.V.

  1. Protein nanocoatings on synthetic polymeric nanofibrous membranes designed as carriers for skin cells.

    Science.gov (United States)

    Bacakova, Marketa; Pajorova, Julia; Stranska, Denisa; Hadraba, Daniel; Lopot, Frantisek; Riedel, Tomas; Brynda, Eduard; Zaloudkova, Margit; Bacakova, Lucie

    2017-01-01

    Protein-coated resorbable synthetic polymeric nanofibrous membranes are promising for the fabrication of advanced skin substitutes. We fabricated electrospun polylactic acid and poly(lactide- co -glycolic acid) nanofibrous membranes and coated them with fibrin or collagen I. Fibronectin was attached to a fibrin or collagen nanocoating, in order further to enhance the cell adhesion and spreading. Fibrin regularly formed a coating around individual nanofibers in the membranes, and also formed a thin noncontinuous nanofibrous mesh on top of the membranes. Collagen also coated most of the fibers of the membrane and randomly created a soft gel on the membrane surface. Fibronectin predominantly adsorbed onto a thin fibrin mesh or a collagen gel, and formed a thin nanofibrous structure. Fibrin nanocoating greatly improved the attachment, spreading, and proliferation of human dermal fibroblasts, whereas collagen nanocoating had a positive influence on the behavior of human HaCaT keratinocytes. In addition, fibrin stimulated the fibroblasts to synthesize fibronectin and to deposit it as an extracellular matrix. Fibrin coating also showed a tendency to improve the ultimate tensile strength of the nanofibrous membranes. Fibronectin attached to fibrin or to a collagen coating further enhanced the adhesion, spreading, and proliferation of both cell types.

  2. A membrane cell for on-line hydrogen/deuterium exchange to study protein folding and protein-protein interactions by mass spectrometry.

    Science.gov (United States)

    Astorga-Wells, Juan; Landreh, Michael; Johansson, Jan; Bergman, Tomas; Jörnvall, Hans

    2011-09-01

    A membrane cell for hydrogen and deuterium exchange on-line with mass spectrometry has been developed to monitor protein-protein interactions and protein conformations. It consists of two channels separated by a semipermeable membrane, where one channel carries the protein sample and the other deuterium oxide. The membrane allows transfer of deuterium oxide into the sample flow. The labeling time is controlled via the flow rate in the sample channel. This cell was validated against three models commonly used in hydrogen-deuterium exchange mass spectrometry: monitoring of folded and unfolded states in a protein, mapping the protein secondary structure at the peptide level, and detection of protein and antibody interactions. The system avoids the conventionally used sample dilution and handling, allowing for potential automation.

  3. A Membrane Cell for On-line Hydrogen/Deuterium Exchange to Study Protein Folding and Protein-Protein Interactions by Mass Spectrometry*

    Science.gov (United States)

    Astorga-Wells, Juan; Landreh, Michael; Johansson, Jan; Bergman, Tomas; Jörnvall, Hans

    2011-01-01

    A membrane cell for hydrogen and deuterium exchange on-line with mass spectrometry has been developed to monitor protein-protein interactions and protein conformations. It consists of two channels separated by a semipermeable membrane, where one channel carries the protein sample and the other deuterium oxide. The membrane allows transfer of deuterium oxide into the sample flow. The labeling time is controlled via the flow rate in the sample channel. This cell was validated against three models commonly used in hydrogen-deuterium exchange mass spectrometry: monitoring of folded and unfolded states in a protein, mapping the protein secondary structure at the peptide level, and detection of protein and antibody interactions. The system avoids the conventionally used sample dilution and handling, allowing for potential automation. PMID:21610101

  4. Reconstitution of membrane protein complexes involved in pneumococcal septal cell wall assembly.

    Directory of Open Access Journals (Sweden)

    Marjolaine Noirclerc-Savoye

    Full Text Available The synthesis of peptidoglycan, the major component of the bacterial cell wall, is essential to cell survival, yet its mechanism remains poorly understood. In the present work, we have isolated several membrane protein complexes consisting of the late division proteins of Streptococcus pneumoniae: DivIB, DivIC, FtsL, PBP2x and FtsW, or subsets thereof. We have co-expressed membrane proteins from S. pneumoniae in Escherichia coli. By combining two successive affinity chromatography steps, we obtained membrane protein complexes with a very good purity. These complexes are functional, as indicated by the retained activity of PBP2x to bind a fluorescent derivative of penicillin and to hydrolyze the substrate analogue S2d. Moreover, we have evidenced the stabilizing role of protein-protein interactions within each complex. This work paves the way for a complete reconstitution of peptidoglycan synthesis in vitro, which will be critical to the elucidation of its intricate regulation mechanisms.

  5. Efficient Isolation and Quantitative Proteomic Analysis of Cancer Cell Plasma Membrane Proteins for Identification of Metastasis-Associated Cell Surface Markers

    DEFF Research Database (Denmark)

    Lund, Rikke; Leth-Larsen, Rikke; Jensen, Ole N

    2009-01-01

    Cell surface membrane proteins are involved in central processes such as cell signaling, cell-cell interactions, ion and solute transport, and they seem to play a pivotal role in several steps of the metastatic process of cancer cells. The low abundance and hydrophobic nature of cell surface...... membrane proteins complicate their purification and identification by MS. We used two isogenic cell lines with opposite metastatic capabilities in nude mice to optimize cell surface membrane protein purification and to identify potential novel markers of metastatic cancer. The cell surface membrane...... peptides of which 622 (300 at SL80) were membrane proteins. The quantitative proteomic analysis identified 16 cell surface proteins as potential markers of the ability of breast cancer cells to form distant metastases....

  6. Diffusion of Integral Membrane Proteins in Protein-Rich Membranes

    DEFF Research Database (Denmark)

    Javanainen, Matti; Martinez-Seara, Hector; Metzler, Ralf

    2017-01-01

    -like dependence D ∝ 1/R. We propose that this 1/R law mainly arises due to geometrical factors: smaller proteins are able to avoid confinement effects much better than their larger counterparts. The results highlight that the lateral dynamics in the crowded setting found in native membranes is radically different......The lateral diffusion of embedded proteins along lipid membranes in protein-poor conditions has been successfully described in terms of the Saffman-Delbrück (SD) model, which predicts that the protein diffusion coefficient D is weakly dependent on its radius R as D ∝ ln(1/R). However, instead...... of being protein-poor, native cell membranes are extremely crowded with proteins. On the basis of extensive molecular simulations, we here demonstrate that protein crowding of the membrane at physiological levels leads to deviations from the SD relation and to the emergence of a stronger Stokes...

  7. Engineering membrane proteins for nuclear medicine. Applications for gene therapy and cell tracking

    International Nuclear Information System (INIS)

    Bogdanov Jr, A.A.; Simonova, M.; Weissleder, R.

    2000-01-01

    Nuclear imaging techniques such as PET and SPECT imaging are expected to play major roles in evaluating the efficacy of in vivo gene therapy. In particular, the quantification of vector delivery and imaging the efficacy of gene expression are of key interests in testing new treatment paradigms and in designing novel vectors. In this review article it has been illustrated how nuclear imaging can be used to image novel cell-surface expressed fusion proteins and how this strategy can be used to probe for phenotypic changes in genetically manipulated cells. Since the described approach uses new fusion proteins, typically not present on eukaryotic cells, such as artificial receptors can be designed to bind radioisotopes currently in clinical use. The described fusion proteins consists of 1) a binding domain such as a peptide based chelator that binds 99mT c oxotechnetate and 2) a membrane anchoring domain. A variety of fusion proteins have been tested so far and the most promising one to date consists of a metallothionein (MT)-derived C-terminal peptide fused a type II membrane protein markers containing the N-terminal membrane anchoring domain of neutral endopeptidase (PEP). Cell-surface expression of MT in transfected cells has been demonstrated using monoclonal antibodies in vitro. Both in vitro and in vivo transchelation experiments have confirmed expression of 99mT c-binding sites in eukaryotic cells. It was expected the described approach to evolve into a useful strategy to tag transfected cells with 99mT c and thus assessing efficiency of gene delivery and expression

  8. Preferential transfer of certain plasma membrane proteins onto T and B cells by trogocytosis.

    Directory of Open Access Journals (Sweden)

    Sandrine Daubeuf

    Full Text Available T and B cells capture antigens via membrane fragments of antigen presenting cells (APC in a process termed trogocytosis. Whether (and how a preferential transfer of some APC components occurs during trogocytosis is still largely unknown. We analyzed the transfer onto murine T and B cells of a large panel of fluorescent proteins with different intra-cellular localizations in the APC or various types of anchors in the plasma membrane (PM. Only the latter were transferred by trogocytosis, albeit with different efficiencies. Unexpectedly, proteins anchored to the PM's cytoplasmic face, or recruited to it via interaction with phosphinositides, were more efficiently transferred than those facing the outside of the cell. For proteins spanning the PM's whole width, transfer efficiency was found to vary quite substantially, with tetraspanins, CD4 and FcRgamma found among the most efficiently transferred proteins. We exploited our findings to set immunodiagnostic assays based on the capture of preferentially transferred components onto T or B cells. The preferential transfer documented here should prove useful in deciphering the cellular structures involved in trogocytosis.

  9. Biologically Complex Planar Cell Plasma Membranes Supported on Polyelectrolyte Cushions Enhance Transmembrane Protein Mobility and Retain Native Orientation.

    Science.gov (United States)

    Liu, Han-Yuan; Chen, Wei-Liang; Ober, Christopher K; Daniel, Susan

    2018-01-23

    Reconstituted supported lipid bilayers (SLB) are widely used as in vitro cell-surface models because they are compatible with a variety of surface-based analytical techniques. However, one of the challenges of using SLBs as a model of the cell surface is the limited complexity in membrane composition, including the incorporation of transmembrane proteins and lipid diversity that may impact the activity of those proteins. Additionally, it is challenging to preserve the transmembrane protein native orientation, function, and mobility in SLBs. Here, we leverage the interaction between cell plasma membrane vesicles and polyelectrolyte brushes to create planar bilayers from cell plasma membrane vesicles that have budded from the cell surface. This approach promotes the direct incorporation of membrane proteins and other species into the planar bilayer without using detergent or reconstitution and preserves membrane constituents. Furthermore, the structure of the polyelectrolyte brush serves as a cushion between the planar bilayer and rigid supporting surface, limiting the interaction of the cytosolic domains of membrane proteins with this surface. Single particle tracking was used to analyze the motion of GPI-linked yellow fluorescent proteins (GPI-YFP) and neon-green fused transmembrane P2X2 receptors (P2X2-neon) and shows that this platform retains over 75% mobility of multipass transmembrane proteins in its native membrane environment. An enzyme accessibility assay confirmed that the protein orientation is preserved and results in the extracellular domain facing toward the bulk phase and the cytosolic side facing the support. Because the platform presented here retains the complexity of the cell plasma membrane and preserves protein orientation and mobility, it is a better representative mimic of native cell surfaces, which may find many applications in biological assays aimed at understanding cell membrane phenomena.

  10. Membrane toxicity of abnormal prion protein in adrenal chromaffin cells of scrapie infected sheep.

    Directory of Open Access Journals (Sweden)

    Gillian McGovern

    Full Text Available Transmissible spongiform encephalopathies (TSEs or prion diseases are associated with accumulations of disease specific PrP (PrP(d in the central nervous system (CNS and often the lymphoreticular system (LRS. Accumulations have additionally been recorded in other tissues including the peripheral nervous system and adrenal gland. Here we investigate the effect of sheep scrapie on the morphology and the accumulation of PrP(d in the adrenal medulla of scrapie affected sheep using light and electron microscopy. Using immunogold electron microscopy, non-fibrillar forms of PrP(d were shown to accumulate mainly in association with chromaffin cells, occasional nerve endings and macrophages. PrP(d accumulation was associated with distinctive membrane changes of chromaffin cells including increased electron density, abnormal linearity and invaginations. Internalisation of PrP(d from the chromaffin cell plasma membrane occurred in association with granule recycling following hormone exocytosis. PrP(d accumulation and internalisation from membranes is similarly associated with perturbations of membrane structure and trafficking in CNS neurons and tingible body macrophages of the LRS. These data suggest that a major toxic effect of PrP(d is at the level of plasma membranes. However, the precise nature of PrP(d-membrane toxicity is tissue and cell specific suggesting that the normal protein may act as a multi-functional scaffolding molecule. We further suggest that the co-localisation of PrP(d with exocytic granules of the hormone trafficking system may provide an additional source of infectivity in blood.

  11. Palmitoylation of SARS-CoV S protein is necessary for partitioning into detergent-resistant membranes and cell-cell fusion but not interaction with M protein

    International Nuclear Information System (INIS)

    McBride, Corrin E.; Machamer, Carolyn E.

    2010-01-01

    Coronaviruses are enveloped RNA viruses that generally cause mild disease in humans. However, the recently emerged coronavirus that caused severe acute respiratory syndrome (SARS-CoV) is the most pathogenic human coronavirus discovered to date. The SARS-CoV spike (S) protein mediates virus entry by binding cellular receptors and inducing fusion between the viral envelope and the host cell membrane. Coronavirus S proteins are palmitoylated, which may affect function. Here, we created a non-palmitoylated SARS-CoV S protein by mutating all nine cytoplasmic cysteine residues. Palmitoylation of SARS-CoV S was required for partitioning into detergent-resistant membranes and for cell-cell fusion. Surprisingly, however, palmitoylation of S was not required for interaction with SARS-CoV M protein. This contrasts with the requirement for palmitoylation of mouse hepatitis virus S protein for interaction with M protein and may point to important differences in assembly and infectivity of these two coronaviruses.

  12. Proteomics of plasma membranes from poplar trees reveals tissue distribution of transporters, receptors, and proteins in cell wall formation.

    Science.gov (United States)

    Nilsson, Robert; Bernfur, Katja; Gustavsson, Niklas; Bygdell, Joakim; Wingsle, Gunnar; Larsson, Christer

    2010-02-01

    By exploiting the abundant tissues available from Populus trees, 3-4 m high, we have been able to isolate plasma membranes of high purity from leaves, xylem, and cambium/phloem at a time (4 weeks after bud break) when photosynthesis in the leaves and wood formation in the xylem should have reached a steady state. More than 40% of the 956 proteins identified were found in the plasma membranes of all three tissues and may be classified as "housekeeping" proteins, a typical example being P-type H(+)-ATPases. Among the 213 proteins predicted to be integral membrane proteins, transporters constitute the largest class (41%) followed by receptors (14%) and proteins involved in cell wall and carbohydrate metabolism (8%) and membrane trafficking (8%). ATP-binding cassette transporters (all members of subfamilies B, C, and G) and receptor-like kinases (four subfamilies) were two of the largest protein families found, and the members of these two families showed pronounced tissue distribution. Leaf plasma membranes were characterized by a very high proportion of transporters, constituting almost half of the integral proteins. Proteins involved in cell wall synthesis (such as cellulose and sucrose synthases) and membrane trafficking were most abundant in xylem plasma membranes in agreement with the role of the xylem in wood formation. Twenty-five integral proteins and 83 soluble proteins were exclusively found in xylem plasma membranes, which identifies new candidates associated with cell wall synthesis and wood formation. Among the proteins uniquely found in xylem plasma membranes were most of the enzymes involved in lignin biosynthesis, which suggests that they may exist as a complex linked to the plasma membrane.

  13. In silicio identification of glycosyl-phosphatidylinositol-anchored plasma-membrane and cell wall proteins of Saccharomyces cerevisiae.

    Science.gov (United States)

    Caro, L H; Tettelin, H; Vossen, J H; Ram, A F; van den Ende, H; Klis, F M

    1997-12-01

    Use of the Von Heijne algorithm allowed the identification of 686 open reading frames (ORFs) in the genome of Saccharomyces cerevisiae that encode proteins with a potential N-terminal signal sequence for entering the secretory pathway. On further analysis, 51 of these proteins contain a potential glycosyl-phosphatidylinositol (GPI)-attachment signal. Seven additional ORFs were found to belong to this group. Upon examination of the possible GPI-attachment sites, it was found that in yeast the most probable amino acids for GPI-attachment as asparagine and glycine. In yeast, GPI-proteins are found at the cell surface, either attached to the plasma-membrane or as an intrinsic part of the cell wall. It was noted that plasma-membrane GPI-proteins possess a dibasic residue motif just before their predicted GPI-attachment site. Based on this, and on homologies between proteins, families of plasma-membrane and cell wall proteins were assigned, revealing 20 potential plasma-membrane and 38 potential cell wall proteins. For members of three plasma-membrane protein families, a function has been described. On the other hand, most of the cell wall proteins seem to be structural components of the wall, responsive to different growth conditions. The GPI-attachment site of yeast slightly differs from mammalian cells. This might be of use in the development of anti-fungal drugs.

  14. F-BAR family proteins, emerging regulators for cell membrane dynamic changes-from structure to human diseases.

    Science.gov (United States)

    Liu, Suxuan; Xiong, Xinyu; Zhao, Xianxian; Yang, Xiaofeng; Wang, Hong

    2015-05-09

    Eukaryotic cell membrane dynamics change in curvature during physiological and pathological processes. In the past ten years, a novel protein family, Fes/CIP4 homology-Bin/Amphiphysin/Rvs (F-BAR) domain proteins, has been identified to be the most important coordinators in membrane curvature regulation. The F-BAR domain family is a member of the Bin/Amphiphysin/Rvs (BAR) domain superfamily that is associated with dynamic changes in cell membrane. However, the molecular basis in membrane structure regulation and the biological functions of F-BAR protein are unclear. The pathophysiological role of F-BAR protein is unknown. This review summarizes the current understanding of structure and function in the BAR domain superfamily, classifies F-BAR family proteins into nine subfamilies based on domain structure, and characterizes F-BAR protein structure, domain interaction, and functional relevance. In general, F-BAR protein binds to cell membrane via F-BAR domain association with membrane phospholipids and initiates membrane curvature and scission via Src homology-3 (SH3) domain interaction with its partner proteins. This process causes membrane dynamic changes and leads to seven important cellular biological functions, which include endocytosis, phagocytosis, filopodium, lamellipodium, cytokinesis, adhesion, and podosome formation, via distinct signaling pathways determined by specific domain-binding partners. These cellular functions play important roles in many physiological and pathophysiological processes. We further summarize F-BAR protein expression and mutation changes observed in various diseases and developmental disorders. Considering the structure feature and functional implication of F-BAR proteins, we anticipate that F-BAR proteins modulate physiological and pathophysiological processes via transferring extracellular materials, regulating cell trafficking and mobility, presenting antigens, mediating extracellular matrix degradation, and transmitting

  15. Plasma membrane protein polarity and trafficking in RPE cells: Past, present and future

    Science.gov (United States)

    Lehmann, Guillermo L.; Benedicto, Ignacio; Philp, Nancy J.; Rodriguez-Boulan, Enrique

    2015-01-01

    The retinal pigment epithelium (RPE) comprises a monolayer of polarized pigmented epithelial cells that is strategically interposed between the neural retina and the fenestrated choroid capillaries. The RPE performs a variety of vectorial transport functions (water, ions, metabolites, nutrients and waste products) that regulate the composition of the subretinal space and support the functions of photoreceptors (PRs) and other cells in the neural retina. To this end, RPE cells display a polarized distribution of channels, transporters and receptors in their plasma membrane (PM) that is remarkably different from that found in conventional extra-ocular epithelia, e.g. intestine, kidney, and gall bladder. This characteristic PM protein polarity of RPE cells depends on the interplay of sorting signals in the RPE PM proteins and sorting mechanisms and biosynthetic/recycling trafficking routes in the RPE cell. Although considerable progress has been made in our understanding of the RPE trafficking machinery, most available data have been obtained from immortalized RPE cell lines that only partially maintain the RPE phenotype and by extrapolation of data obtained in the prototype Madin–Darby Canine Kidney (MDCK) cell line. The increasing availability of RPE cell cultures that more closely resemble the RPE in vivo together with the advent of advanced live imaging microscopy techniques provides a platform and an opportunity to rapidly expand our understanding of how polarized protein trafficking contributes to RPE PM polarity. PMID:25152359

  16. Modulation of Wound Healing and Scar Formation by MG53 Protein-mediated Cell Membrane Repair*

    Science.gov (United States)

    Li, Haichang; Duann, Pu; Lin, Pei-Hui; Zhao, Li; Fan, Zhaobo; Tan, Tao; Zhou, Xinyu; Sun, Mingzhai; Fu, Minghuan; Orange, Matthew; Sermersheim, Matthew; Ma, Hanley; He, Duofen; Steinberg, Steven M.; Higgins, Robert; Zhu, Hua; John, Elizabeth; Zeng, Chunyu; Guan, Jianjun; Ma, Jianjie

    2015-01-01

    Cell membrane repair is an important aspect of physiology, and disruption of this process can result in pathophysiology in a number of different tissues, including wound healing, chronic ulcer and scarring. We have previously identified a novel tripartite motif family protein, MG53, as an essential component of the cell membrane repair machinery. Here we report the functional role of MG53 in the modulation of wound healing and scarring. Although MG53 is absent from keratinocytes and fibroblasts, remarkable defects in skin architecture and collagen overproduction are observed in mg53−/− mice, and these animals display delayed wound healing and abnormal scarring. Recombinant human MG53 (rhMG53) protein, encapsulated in a hydrogel formulation, facilitates wound healing and prevents scarring in rodent models of dermal injuries. An in vitro study shows that rhMG53 protects against acute injury to keratinocytes and facilitates the migration of fibroblasts in response to scratch wounding. During fibrotic remodeling, rhMG53 interferes with TGF-β-dependent activation of myofibroblast differentiation. The resulting down-regulation of α smooth muscle actin and extracellular matrix proteins contributes to reduced scarring. Overall, these studies establish a trifunctional role for MG53 as a facilitator of rapid injury repair, a mediator of cell migration, and a modulator of myofibroblast differentiation during wound healing. Targeting the functional interaction between MG53 and TGF-β signaling may present a potentially effective means for promoting scarless wound healing. PMID:26306047

  17. Adsorption of vitamin K-dependent proteins to live cell membranes measured under flow conditions.

    Science.gov (United States)

    McGee, M P; Teuschler, H

    1999-07-01

    Mechanisms mediating initial adsorption of coagulation proteins to live cells were investigated. Adsorption kinetics were examined under varying flow conditions using tracer-dilution techniques in perfused spherical monolayers of cells expressing tissue factor. At biologically relevant time and concentration ranges, rates exceeded by 2-12 fold the theoretical maximum calculated for steady-state diffusion. Rates were correlated with aqueous-phase flux of reactants and were found to be largely independent of the density of reactive sites on the membrane. Average adsorption rate of factor VIIa at 4 etaM and flow velocity of 0.8 etam s(-1) was 5 x 10(7) s(-1) cm(-2). Adsorption rates of homologous coagulation factors IX and X under similar conditions were 5 and 9 x 10(7) s(-1)cm(-2). Results indicate that flow can effectively increase the rate of coagulation factor adsorption to the membrane of live cells. They also imply that factors affecting blood flow velocity and vessel permeability influence the rate of membrane-dependent coagulation reactions.

  18. Establishment of insect cell lines expressing green fluorescent protein on cell surface based on AcMNPV GP64 membrane fusion characteristic.

    Science.gov (United States)

    Qi, Ben-Xiang; Chen, Ying-Jian; Su, Rui; Li, Yi-Fei; Zheng, Gui-Ling; Li, Chang-You

    2017-10-01

    Displaying a protein on the surface of cells has been provided a very successful strategy to function research of exogenous proteins. Based on the membrane fusion characteristic of Autographa californica multiple nucleopolyhedrovirus envelope protein GP64, we amplified and cloned N-terminal signal peptide and C-terminal transmembrane domain as well as cytoplasmic tail domain of gp64 gene into vector pIZ/V5-His with multi-cloning sites to construct the cell surface expression vector pIZ/V5-gp64. To verify that the vector can be used to express proteins on the membrane of insect cells, a recombinant plasmid pIZ/V5-gp64-GFP was constructed by introducing the PCR amplified green fluorescent protein (GFP) gene and transfected into insect cell lines Sf9 and H5. The transected cells were screened with zeocin and cell cloning. PCR verification results showed that the GFP gene was successfully integrated into these cells. Green fluorescence in Sf9-GFP and H5-GFP cells was observed by using confocal laser scanning microscopy and immunofluorescence detection indicated that GFP protein was located on the cell membrane. Western blot results showed that a fusion protein GP64-GFP of about 40 kDa was expressed on the membrane of Sf9-GFP and H5-GFP cells. The expression system constructed in this paper can be used for localization and continuous expression of exogenous proteins on insect cell membrane.

  19. Modulation of wound healing and scar formation by MG53 protein-mediated cell membrane repair.

    Science.gov (United States)

    Li, Haichang; Duann, Pu; Lin, Pei-Hui; Zhao, Li; Fan, Zhaobo; Tan, Tao; Zhou, Xinyu; Sun, Mingzhai; Fu, Minghuan; Orange, Matthew; Sermersheim, Matthew; Ma, Hanley; He, Duofen; Steinberg, Steven M; Higgins, Robert; Zhu, Hua; John, Elizabeth; Zeng, Chunyu; Guan, Jianjun; Ma, Jianjie

    2015-10-02

    Cell membrane repair is an important aspect of physiology, and disruption of this process can result in pathophysiology in a number of different tissues, including wound healing, chronic ulcer and scarring. We have previously identified a novel tripartite motif family protein, MG53, as an essential component of the cell membrane repair machinery. Here we report the functional role of MG53 in the modulation of wound healing and scarring. Although MG53 is absent from keratinocytes and fibroblasts, remarkable defects in skin architecture and collagen overproduction are observed in mg53(-/-) mice, and these animals display delayed wound healing and abnormal scarring. Recombinant human MG53 (rhMG53) protein, encapsulated in a hydrogel formulation, facilitates wound healing and prevents scarring in rodent models of dermal injuries. An in vitro study shows that rhMG53 protects against acute injury to keratinocytes and facilitates the migration of fibroblasts in response to scratch wounding. During fibrotic remodeling, rhMG53 interferes with TGF-β-dependent activation of myofibroblast differentiation. The resulting down-regulation of α smooth muscle actin and extracellular matrix proteins contributes to reduced scarring. Overall, these studies establish a trifunctional role for MG53 as a facilitator of rapid injury repair, a mediator of cell migration, and a modulator of myofibroblast differentiation during wound healing. Targeting the functional interaction between MG53 and TGF-β signaling may present a potentially effective means for promoting scarless wound healing. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  20. Outer Membrane Protein 25 of Brucella Activates Mitogen-Activated Protein Kinase Signal Pathway in Human Trophoblast Cells

    Directory of Open Access Journals (Sweden)

    Jing Zhang

    2017-12-01

    Full Text Available Outer membrane protein 25 (OMP25, a virulence factor from Brucella, plays an important role in maintaining the structural stability of Brucella. Mitogen-activated protein kinase (MAPK signal pathway widely exists in eukaryotic cells. In this study, human trophoblast cell line HPT-8 and BALB/c mice were infected with Brucella abortus 2308 strain (S2308 and 2308ΔOmp25 mutant strain. The expression of cytokines and activation of MAPK signal pathway were detected. We found that the expressions of tumor necrosis factor-α, interleukin-1, and interleukin-10 (IL-10 were increased in HPT-8 cells infected with S2308 and 2308ΔOmp25 mutant. S2308 also activated p38 phosphorylation protein, extracellular-regulated protein kinases (ERK, and Jun-N-terminal kinase (JNK from MAPK signal pathway. 2308ΔOmp25 could not activate p38, ERK, and JNK branches. Immunohistochemistry experiments showed that S2308 was able to activate phosphorylation of p38 and ERK in BABL/c mice. However, 2308ΔOmp25 could weakly activate phosphorylation of p38 and ERK. These results suggest that Omp25 played an important role in the process of Brucella activation of the MAPK signal pathway.

  1. Modelling of proteins in membranes

    DEFF Research Database (Denmark)

    Sperotto, Maria Maddalena; May, S.; Baumgaertner, A.

    2006-01-01

    This review describes some recent theories and simulations of mesoscopic and microscopic models of lipid membranes with embedded or attached proteins. We summarize results supporting our understanding of phenomena for which the activities of proteins in membranes are expected to be significantly...... oppositely charged lipid membranes, lipid-induced tilting of proteins embedded in lipid bilayers, protein-induced bilayer deformations, protein insertion and assembly, and lipid-controlled functioning of membrane proteins....

  2. Metastasis-related plasma membrane proteins of human breast cancer cells identified by comparative quantitative mass spectrometry

    DEFF Research Database (Denmark)

    Leth-Larsen, Rikke; Lund, Rikke; Hansen, Helle V

    2009-01-01

    The spread of cancer cells from a primary tumor to form metastasis at distant sites is a complex multi-step process. The cancer cell proteins, and plasma membrane proteins in particular, involved in this process are poorly defined and a study of the very early events of the metastatic process using...... clinical samples or in vitro assays is not feasible. We have used a unique model system consisting of two isogenic human breast cancer cell lines that are equally tumorigenic in mice, but while one gives rise to metastasis, the other disseminates single cells that remain dormant at distant organs. Membrane......'-nucleotidase (ecto-5'-NT, CD73), Ndrg1, integrin beta1, CD44, CD74 and MHC class II proteins. The altered expression levels of proteins identified by LC-MS/MS were validated using flow cytometry, Western blotting, immunocyto- and immunohisto-chemistry. Analysis of clinical breast cancer biopsies demonstrated...

  3. Surface and protein analyses of normal human cell attachment on PIII-modified chitosan membranes

    Energy Technology Data Exchange (ETDEWEB)

    Saranwong, N. [Plasma and Beam Physics Research Facility, Department of Physics and Materials Science, Faculty of Science, Chiang Mai University, Chiang Mai 50200 (Thailand); Inthanon, K. [Human and Animal Cell Technology Research Laboratory, Department of Biology, Faculty of Science, Chiang Mai University, Chiang Mai 50200 (Thailand); Wongkham, W., E-mail: weerah@chiangmai.ac.th [Human and Animal Cell Technology Research Laboratory, Department of Biology, Faculty of Science, Chiang Mai University, Chiang Mai 50200 (Thailand); Wanichapichart, P. [Nanotechnology Center of Excellence and Membrane Science and Technology Research Center, Department of Physics, Faculty of Science, Prince of Songkla University, Hat Yai, Songkla 90110 (Thailand); Suwannakachorn, D. [Plasma and Beam Physics Research Facility, Department of Physics and Materials Science, Faculty of Science, Chiang Mai University, Chiang Mai 50200 (Thailand); Yu, L.D., E-mail: yuld@fnrf.science.cmu.ac.th [Plasma and Beam Physics Research Facility, Department of Physics and Materials Science, Faculty of Science, Chiang Mai University, Chiang Mai 50200 (Thailand); Thailand Center of Excellence in Physics, Commission on Higher Education, 328 Si Ayutthaya Road, Bangkok 10400 (Thailand)

    2012-02-01

    Surface of chitosan membrane was modified with argon (Ar) and nitrogen (N) plasma immersion ion implantation (PIII) for human skin fibroblasts F1544 cell attachment. The modified surfaces were characterized by Fourier transform infrared spectroscopy (FTIR) and atomic force microscopy (AFM). Cell attachment patterns were evaluated by scanning electron microscopy (SEM). The enzyme-linked immunosorbent assay (ELISA) was used to quantify levels of focal adhesion kinase (FAK). The results showed that Ar PIII had an enhancement effect on the cell attachment while N-PIII had an inhibition effect. Filopodial analysis revealed more microfilament cytoplasmic spreading on the edge of cells attached on the Ar-treated membranes than N-treated membranes. Higher level FAK was found in Ar-treated membranes than that in N-treated membranes.

  4. Mechanisms of multidrug resistance in HL60 cells. Analysis of resistance associated membrane proteins and levels of mdr gene expression.

    Science.gov (United States)

    McGrath, T; Latoud, C; Arnold, S T; Safa, A R; Felsted, R L; Center, M S

    1989-10-15

    HL60 cells isolated for resistance to Adriamycin do not contain P-glycoprotein, as determined with immunological probes. These cells, however, are multidrug resistant and defective in the cellular accumulation of drug. In view of these findings, we have examined in greater detail certain properties of the HL60/Adr cells and have compared these properties to an HL60 drug-resistant isolate (HL60/Vinc) which contains high levels of P-glycoprotein. The results of these studies demonstrated that verapamil induces a major increase in cellular drug accumulation in both HL60/Adr and HL60/Vinc isolates. An 125I-labeled photoaffinity analog of verapamil labeled P-glycoprotein contained in membranes of HL60/Vinc cells. In contrast, this agent did not label any protein selectively associated with drug resistance in membranes of the HL60/Adr isolate. The photoactive dihydropyridine calcium channel blocker [3H]azidopine and [125I]NASV, a photoaffinity analog of vinblastine, labelled P-glycoprotein in membranes from HL60/Vinc cells, whereas in experiments with the HL60/Adr isolate there was no detectable labeling of a drug resistance associated membrane protein. Additional studies have been carried out to analyze membrane proteins of HL60/Adr cells labeled with the photoaffinity agent 8-azido-alpha-[32P]ATP (AzATP32). The results demonstrate that this agent labeled a resistance associated membrane protein of 190 kilodaltons (P190). P190 is essentially absent in membranes of drug-sensitive cells. Labeling of P190 with AzATP32 in membranes of resistant cells was blocked completely when incubations were carried out in the presence of excess unlabeled ATP. Additional studies were carried out to analyze mdr gene amplification and expression in sensitive and resistant cells. Experiments carried out with human 5',mdr1 (1.1 kb) and mdr3 (1.0 kb) cDNAs demonstrate that both of these sequences were highly amplified in the HL60/Vinc isolate. Only the mrd1 gene sequence however, was

  5. Membrane bending by protein-protein crowding.

    Science.gov (United States)

    Stachowiak, Jeanne C; Schmid, Eva M; Ryan, Christopher J; Ann, Hyoung Sook; Sasaki, Darryl Y; Sherman, Michael B; Geissler, Phillip L; Fletcher, Daniel A; Hayden, Carl C

    2012-09-01

    Curved membranes are an essential feature of dynamic cellular structures, including endocytic pits, filopodia protrusions and most organelles. It has been proposed that specialized proteins induce curvature by binding to membranes through two primary mechanisms: membrane scaffolding by curved proteins or complexes; and insertion of wedge-like amphipathic helices into the membrane. Recent computational studies have raised questions about the efficiency of the helix-insertion mechanism, predicting that proteins must cover nearly 100% of the membrane surface to generate high curvature, an improbable physiological situation. Thus, at present, we lack a sufficient physical explanation of how protein attachment bends membranes efficiently. On the basis of studies of epsin1 and AP180, proteins involved in clathrin-mediated endocytosis, we propose a third general mechanism for bending fluid cellular membranes: protein-protein crowding. By correlating membrane tubulation with measurements of protein densities on membrane surfaces, we demonstrate that lateral pressure generated by collisions between bound proteins drives bending. Whether proteins attach by inserting a helix or by binding lipid heads with an engineered tag, protein coverage above ~20% is sufficient to bend membranes. Consistent with this crowding mechanism, we find that even proteins unrelated to membrane curvature, such as green fluorescent protein (GFP), can bend membranes when sufficiently concentrated. These findings demonstrate a highly efficient mechanism by which the crowded protein environment on the surface of cellular membranes can contribute to membrane shape change.

  6. Eukaryotic membrane protein overproduction in Lactococcus lactis

    NARCIS (Netherlands)

    Kunji, Edmund R.S.; Chan, Ka Wai; Slotboom, Dirk Jan; Floyd, Suzanne; O’Connor, Rosemary; Monné, Magnus

    2005-01-01

    Eukaryotic membrane proteins play many vital roles in the cell and are important drug targets. Approximately 25% of all genes identified in the genome are known to encode membrane proteins, but the vast majority have no assigned function. Although the generation of structures of soluble proteins has

  7. Biopores/membrane proteins in synthetic polymer membranes.

    Science.gov (United States)

    Garni, Martina; Thamboo, Sagana; Schoenenberger, Cora-Ann; Palivan, Cornelia G

    2017-04-01

    Mimicking cell membranes by simple models based on the reconstitution of membrane proteins in lipid bilayers represents a straightforward approach to understand biological function of these proteins. This biomimetic strategy has been extended to synthetic membranes that have advantages in terms of chemical and mechanical stability, thus providing more robust hybrid membranes. We present here how membrane proteins and biopores have been inserted both in the membrane of nanosized and microsized compartments, and in planar membranes under various conditions. Such bio-hybrid membranes have new properties (as for example, permeability to ions/molecules), and functionality depending on the specificity of the inserted biomolecules. Interestingly, membrane proteins can be functionally inserted in synthetic membranes provided these have appropriate properties to overcome the high hydrophobic mismatch between the size of the biomolecule and the membrane thickness. Functional insertion of membrane proteins and biopores in synthetic membranes of compartments or in planar membranes is possible by an appropriate selection of the amphiphilic copolymers, and conditions of the self-assembly process. These hybrid membranes have new properties and functionality based on the specificity of the biomolecules and the nature of the synthetic membranes. Bio-hybrid membranes represent new solutions for the development of nanoreactors, artificial organelles or active surfaces/membranes that, by further gaining in complexity and functionality, will promote translational applications. This article is part of a Special Issue entitled: Lipid order/lipid defects and lipid-control of protein activity edited by Dirk Schneider. Copyright © 2016. Published by Elsevier B.V.

  8. Bee venom phospholipase A2 as a membrane-binding vector for cell surface display or internalization of soluble proteins.

    Science.gov (United States)

    Babon, Aurélie; Wurceldorf, Thibault; Almunia, Christine; Pichard, Sylvain; Chenal, Alexandre; Buhot, Cécile; Beaumelle, Bruno; Gillet, Daniel

    2016-06-15

    We showed that bee venom phospholipase A2 can be used as a membrane-binding vector to anchor to the surface of cells a soluble protein fused to its C-terminus. ZZ, a two-domain derivative of staphylococcal protein A capable of binding constant regions of antibodies was fused to the C-terminus of the phospholipase or to a mutant devoid of enzymatic activity. The fusion proteins bound to the surface of cells and could themselves bind IgGs. Their fate depended on the cell type to which they bound. On the A431 carcinoma cell line the proteins remained exposed on the cell surface. In contrast, on human dendritic cells the proteins were internalized into early endosomes. Copyright © 2015 Elsevier Ltd. All rights reserved.

  9. Towards the development of Bacillus subtilis as a cell factory for membrane proteins and protein complexes

    NARCIS (Netherlands)

    Zweers, Jessica C.; Barak, Imrich; Becher, Doerte; Driessen, Arnold J. M.; Hecker, Michael; Kontinen, Vesa P.; Saller, Manfred J.; Vavrova, L'udmila; van Dijl, Jan Maarten

    2008-01-01

    Background: The Gram-positive bacterium Bacillus subtilis is an important producer of high quality industrial enzymes and a few eukaryotic proteins. Most of these proteins are secreted into the growth medium, but successful examples of cytoplasmic protein production are also known. Therefore, one

  10. SecA is required for membrane targeting of the cell division protein DivIVA in vivo

    Directory of Open Access Journals (Sweden)

    Sven eHalbedel

    2014-02-01

    Full Text Available The conserved protein DivIVA is involved in different morphogenetic processes in Gram-positive bacteria. In Bacillus subtilis, the protein localises to the cell division site and cell poles, and functions as a scaffold for proteins that regulate division site selection, and for proteins that are required for sporulation. To identify other proteins that bind to DivIVA, we performed an in vivo cross-linking experiment. A possible candidate that emerged was the secretion motor ATPase SecA. SecA mutants have been described that inhibit sporulation, and since DivIVA is necessary for sporulation, we examined the localisation of DivIVA in these mutants. Surprisingly, DivIVA was delocalised, suggesting that SecA is required for DivIVA targeting. To further corroborate this, we performed SecA depletion and inhibition experiments, which provided further indications that DivIVA localisation depends on SecA. Cell fractionation experiments showed that SecA is important for binding of DivIVA to the cell membrane. This was unexpected since DivIVA does not contain a signal sequence, and is able to bind to artificial lipid membranes in vitro without support of other proteins. SecA is required for protein secretion and membrane insertion, and therefore its role in DivIVA localisation is likely indirect. Possible alternative roles of SecA in DivIVA folding and/or targeting are discussed.

  11. Vesicle-associated membrane protein 7 (VAMP-7) is essential for target cell killing in a natural killer cell line

    International Nuclear Information System (INIS)

    Marcet-Palacios, Marcelo; Odemuyiwa, Solomon O.; Coughlin, Jason J.; Garofoli, Daniella; Ewen, Catherine; Davidson, Courtney E.; Ghaffari, Mazyar; Kane, Kevin P.; Lacy, Paige; Logan, Michael R.; Befus, A. Dean; Bleackley, R. Chris; Moqbel, Redwan

    2008-01-01

    Natural killer cells recognize and induce apoptosis in foreign, transformed or virus-infected cells through the release of perforin and granzymes from secretory lysosomes. Clinically, NK-cell mediated killing is a major limitation to successful allo- and xenotransplantation. The molecular mechanisms that regulate the fusion of granzyme B-containing secretory lysosomes to the plasma membrane in activated NK cells, prior to target cell killing, are not fully understood. Using the NK cell line YT-Indy as a model, we have investigated the expression of SNAP REceptors (SNAREs), both target (t-) and vesicular (v-) SNAREs, and their function in granzyme B-mediated target cell killing. Our data showed that YT-Indy cells express VAMP-7 and SNAP-23, but not VAMP-2. VAMP-7 was associated with granzyme B-containing lysosomal granules. Using VAMP-7 small interfering RNA (siRNA), we successfully knocked down the expression of VAMP-7 protein in YT-Indy to less than 10% of untreated cells in 24 h. VAMP7-deficient YT-Indy cells activated via co-culture with Jurkat cells released <1 ng/mL of granzyme B, compared to 1.5-2.5 μg/mL from controls. Using Jurkat cells as targets, we showed a 7-fold reduction in NK cell-mediated killing by VAMP-7 deficient YT-Indy cells. Our results show that VAMP-7 is a crucial component of granzyme B release and target cell killing in the NK cell line YT-Indy. Thus, targeting VAMP-7 expression specifically with siRNA, following transplantation, may be a viable strategy for preventing NK cell-mediated transplant rejection, in vivo

  12. Comparative transcriptional analysis of Bacillus subtilis cells overproducing either secreted proteins, lipoproteins or membrane proteins

    NARCIS (Netherlands)

    Marciniak, Bogumila C.; Trip, Hein; van-der Veek, Patricia J.; Kuipers, Oscar P.; Marciniak, Bogumiła C.

    2012-01-01

    Background: Bacillus subtilis is a favorable host for the production of industrially relevant proteins because of its capacity of secreting proteins into the medium to high levels, its GRAS (Generally Recognized As Safe) status, its genetic accessibility and its capacity to grow in large

  13. Membrane labeling of coral gastrodermal cells by biotinylation: the proteomic identification of surface proteins involving cnidaria-dinoflagellate endosymbiosis.

    Directory of Open Access Journals (Sweden)

    Hsing-Hui Li

    Full Text Available The cellular and molecular-scale processes underlying the stability of coral-Symbiodinium endosymbioses remain unclear despite decades of investigation. As the coral gastroderm is the only tissue layer characterized by this unique symbiotic association, the membranes of these symbiotic gastrodermal cells (SGCs may play important roles in the initiation and maintenance of the endosymbiosis. In order to elucidate the interactions between the endosymbiotic dinoflagellates and their coral hosts, a thorough characterization of SGC membranes is therefore required. Cell surface proteins of isolated SGCs were biotinylated herein by a cell impermeant agent, biotin-XX sulfosuccinimidyl ester. The in situ distribution of these biotinylated proteins was uncovered by both fluorescence and transmission electron microscopic imaging of proteins bound to Alexa Fluor® 488-conjugated streptavidin. The identity of these proteins was then determined by two-dimensional gel electrophoresis followed by liquid chromatography-tandem mass spectrometry. Nineteen (19 proteins were identified, and they are known to be involved in the molecular chaperone/stress response, cytoskeletal remodeling, and energy metabolism. These results not only reveal the molecular characters of the host SGC membrane, but also provide critical insight into understanding the possible role of host membranes in this ecologically important endosymbiotic association.

  14. Differential expression profiling of membrane proteins by quantitative proteomics in a human mesenchymal stem cell line undergoing osteoblast differentiation

    DEFF Research Database (Denmark)

    Foster, Leonard J; Zeemann, Patricia A; Li, Chen

    2005-01-01

    in a cell model of hMSCs established by overexpression of human telomerase reverse-transcriptase gene. We identified 463 unique proteins with extremely high confidence, including all known markers of hMSCs (e.g., SH3 [CD71], SH2 [CD105], CD166, CD44, Thy1, CD29, and HOP26 [CD63]) among 148 integral membrane...

  15. Cationic amphipathic peptides accumulate sialylated proteins and lipids in the plasma membrane of eukaryotic host cells.

    Science.gov (United States)

    Weghuber, Julian; Aichinger, Michael C; Brameshuber, Mario; Wieser, Stefan; Ruprecht, Verena; Plochberger, Birgit; Madl, Josef; Horner, Andreas; Reipert, Siegfried; Lohner, Karl; Henics, Tamás; Schütz, Gerhard J

    2011-10-01

    Cationic antimicrobial peptides (CAMPs) selectively target bacterial membranes by electrostatic interactions with negatively charged lipids. It turned out that for inhibition of microbial growth a high CAMP membrane concentration is required, which can be realized by the incorporation of hydrophobic groups within the peptide. Increasing hydrophobicity, however, reduces the CAMP selectivity for bacterial over eukaryotic host membranes, thereby causing the risk of detrimental side-effects. In this study we addressed how cationic amphipathic peptides-in particular a CAMP with Lysine-Leucine-Lysine repeats (termed KLK)-affect the localization and dynamics of molecules in eukaryotic membranes. We found KLK to selectively inhibit the endocytosis of a subgroup of membrane proteins and lipids by electrostatically interacting with negatively charged sialic acid moieties. Ultrastructural characterization revealed the formation of membrane invaginations representing fission or fusion intermediates, in which the sialylated proteins and lipids were immobilized. Experiments on structurally different cationic amphipathic peptides (KLK, 6-MO-LF11-322 and NK14-2) indicated a cooperation of electrostatic and hydrophobic forces that selectively arrest sialylated membrane constituents. Copyright © 2011 Elsevier B.V. All rights reserved.

  16. Protein 90 Recognized as an Iron-Binding Protein Associated with the Plasma Membrane of HeLa Cells

    Czech Academy of Sciences Publication Activity Database

    Kovář, Jan; Štýbrová, Hana; Novák, J.; Ehrlichová, Marie; Truksa, Jaroslav; Koc, Michal; Kriegerbecková, Karin; Scheiber-Mojdehkar, B.; Goldenberg, H.

    1-2, č. 14 (2004), s. 41-46 ISSN 1015-8987 R&D Projects: GA AV ČR IAA5052702; GA ČR GA301/01/0041 Keywords : heat shock protein 90 * iron -binding protein * plasma membrane Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 3.093, year: 2004

  17. Fluorescence Recovery After Photobleaching Analysis of the Diffusional Mobility of Plasma Membrane Proteins: HER3 Mobility in Breast Cancer Cell Membranes.

    Science.gov (United States)

    Sarkar, Mitul; Koland, John G

    2016-01-01

    The fluorescence recovery after photobleaching (FRAP) method is a straightforward means of assessing the diffusional mobility of membrane-associated proteins that is readily performed with current confocal microscopy instrumentation. We describe here the specific application of the FRAP method in characterizing the lateral diffusion of genetically encoded green fluorescence protein (GFP)-tagged plasma membrane receptor proteins. The method is exemplified in an examination of whether the previously observed segregation of the mammalian HER3 receptor protein in discrete plasma membrane microdomains results from its physical interaction with cellular entities that restrict its mobility. Our FRAP measurements of the diffusional mobility of GFP-tagged HER3 reporters expressed in MCF7 cultured breast cancer cells showed that despite the observed segregation of HER3 receptors within plasma membrane microdomains their diffusion on the macroscopic scale is not spatially restricted. Thus, in FRAP analyses of various HER3 reporters a near-complete recovery of fluorescence after photobleaching was observed, indicating that HER3 receptors are not immobilized by long-lived physical interactions with intracellular species. An examination of HER3 proteins with varying intracellular domain sequence truncations also indicated that a proposed formation of oligomeric HER3 networks, mediated by physical interactions involving specific HER3 intracellular domain sequences, either does not occur or does not significantly reduce HER3 mobility on the macroscopic scale.

  18. Efficient Overproduction of Membrane Proteins in Lactococcus lactis Requires the Cell Envelope Stress Sensor/Regulator Couple CesSR

    Science.gov (United States)

    Pinto, Joao P. C.; Kuipers, Oscar P.; Marreddy, Ravi K. R.; Poolman, Bert; Kok, Jan

    2011-01-01

    Background Membrane proteins comprise an important class of molecules whose study is largely frustrated by several intrinsic constraints, such as their hydrophobicity and added requirements for correct folding. Additionally, the complexity of the cellular mechanisms that are required to insert membrane proteins functionally in the membrane and to monitor their folding state makes it difficult to foresee the yields at which one can obtain them or to predict which would be the optimal production host for a given protein. Methods and Findings We describe a rational design approach to improve the lactic acid bacterium Lactococcus lactis as a producer of membrane proteins. Our transcriptome data shows that the two-component system CesSR, which senses cell envelope stresses of different origins, is one of the major players when L. lactis is forced to overproduce the endogenous membrane protein BcaP, a branched-chain amino acid permease. Growth of the BcaP-producing L. lactis strain and its capability to produce membrane proteins are severely hampered when the CesSR system itself or particular members of the CesSR regulon are knocked out, notably the genes ftsH, oxaA2, llmg_2163 and rmaB. Overexpressing cesSR reduced the growth defect, thus directly improving the production yield of BcaP. Applying this rationale to eukaryotic proteins, some of which are notoriously more difficult to produce, such as the medically-important presenilin complex, we were able to significantly diminish the growth defect seen in the wild-type strain and improve the production yield of the presenilin variant PS1Δ9-H6 more than 4-fold. Conclusions The results shed light into a key, and perhaps central, membrane protein quality control mechanism in L. lactis. Modulating the expression of CesSR benefited the production yields of membrane proteins from different origins. These findings reinforce L. lactis as a legitimate alternative host for the production of membrane proteins. PMID:21818275

  19. Regulation of B cell differentiation by intracellular membrane associated proteins and microRNAs: role in the antibody response

    Directory of Open Access Journals (Sweden)

    Zheng eLou

    2015-10-01

    Full Text Available B cells are central to adaptive immunity and their functions in antibody responses are exquisitely regulated. As suggested by recent findings, B cell differentiation is mediated by intracellular membrane structures (including endosomes, lysosomes and autophagosomes and protein factors specifically associated with these membranes, including Rab7, Atg5 and Atg7. These factors participate in vesicle formation/trafficking, signal transduction and induction of gene expression to promote antigen presentation, CSR/SHM, and generation/maintenance of plasma cells and memory B cells. Their expression is induced in B cells activated to differentiate and further fine-tuned by immune-modulating microRNAs, which coordinates CSR/SHM, plasma cell differentiation and memory B cell differentiation. These short non-coding RNAs would individually target multiple factors associated with the same intracellular membrane compartments and collaboratively target a single factor in addition to regulate AID and Blimp-1. These, together with regulation of microRNA biogenesis and activities by endosomes and autophagosomes, show that intracellular membranes and microRNAs, two broadly relevant cell constituents, play important roles in balancing gene expression to specify B cell differentiation processes for optimal antibody responses.

  20. Membrane fission by protein crowding.

    Science.gov (United States)

    Snead, Wilton T; Hayden, Carl C; Gadok, Avinash K; Zhao, Chi; Lafer, Eileen M; Rangamani, Padmini; Stachowiak, Jeanne C

    2017-04-18

    Membrane fission, which facilitates compartmentalization of biological processes into discrete, membrane-bound volumes, is essential for cellular life. Proteins with specific structural features including constricting rings, helical scaffolds, and hydrophobic membrane insertions are thought to be the primary drivers of fission. In contrast, here we report a mechanism of fission that is independent of protein structure-steric pressure among membrane-bound proteins. In particular, random collisions among crowded proteins generate substantial pressure, which if unbalanced on the opposite membrane surface can dramatically increase membrane curvature, leading to fission. Using the endocytic protein epsin1 N-terminal homology domain (ENTH), previously thought to drive fission by hydrophobic insertion, our results show that membrane coverage correlates equally with fission regardless of the hydrophobicity of insertions. Specifically, combining FRET-based measurements of membrane coverage with multiple, independent measurements of membrane vesiculation revealed that fission became spontaneous as steric pressure increased. Further, fission efficiency remained equally potent when helices were replaced by synthetic membrane-binding motifs. These data challenge the view that hydrophobic insertions drive membrane fission, suggesting instead that the role of insertions is to anchor proteins strongly to membrane surfaces, amplifying steric pressure. In line with these conclusions, even green fluorescent protein (GFP) was able to drive fission efficiently when bound to the membrane at high coverage. Our conclusions are further strengthened by the finding that intrinsically disordered proteins, which have large hydrodynamic radii yet lack a defined structure, drove fission with substantially greater potency than smaller, structured proteins.

  1. Phytochemicals prevent mitochondrial membrane permeabilization and protect SH-SY5Y cells against apoptosis induced by PK11195, a ligand for outer membrane translocator protein.

    Science.gov (United States)

    Wu, Yuqiu; Shamoto-Nagai, Masayo; Maruyama, Wakako; Osawa, Toshihiko; Naoi, Makoto

    2017-01-01

    Epidemiological studies present the beneficial effects of dietary habits on prevention of aging-associated decline of brain function. Phytochemicals, the second metabolites of food, protect neuronal cells from cell death in cellular models of neurodegenerative disorders, and the neuroprotective activity has been ascribed to the anti-oxidant and anti-inflammatory functions. In this paper, the cellular mechanism of neuroprotection by phytochemicals was investigated, using the cellular model of mitochondrial apoptosis induced by PK11195, a ligand of outer membrane translocator protein, in SH-SY5Y cells. PK11195 induced mitochondrial membrane permeabilization with rapid transit production of superoxide (superoxide flashes) and calcium release from mitochondria, and activated apoptosis signal pathway. Study on the structure-activity relationship of astaxanthin, ferulic acid derivatives, and sesame lignans revealed that these phytochemicals inhibited mitochondrial membrane permeabilization and protected cells from apoptosis. Ferulic acid derivatives and sesame lignans inhibited or enhanced the mitochondrial pore formation and cell death by PK11195 according to their amphiphilic properties, not directly depending on the antioxidant activity. Regulation of pore formation at mitochondrial membrane is discussed as a novel mechanism behind neuroprotective activity of phytochemicals in aging and age-associated neurodegenerative disorders, and also behind dual functions of phytochemicals in neuronal and cancer cells.

  2. Latent Membrane Protein LMP2A Impairs Recognition of EBV-Infected Cells by CD8+ T Cells.

    Directory of Open Access Journals (Sweden)

    Chiara Rancan

    2015-06-01

    Full Text Available The common pathogen Epstein-Barr virus (EBV transforms normal human B cells and can cause cancer. Latent membrane protein 2A (LMP2A of EBV supports activation and proliferation of infected B cells and is expressed in many types of EBV-associated cancer. It is not clear how latent EBV infection and cancer escape elimination by host immunity, and it is unknown whether LMP2A can influence the interaction of EBV-infected cells with the immune system. We infected primary B cells with EBV deleted for LMP2A, and established lymphoblastoid cell lines (LCLs. We found that CD8+ T cell clones showed higher reactivity against LMP2A-deficient LCLs compared to LCLs infected with complete EBV. We identified several potential mediators of this immunomodulatory effect. In the absence of LMP2A, expression of some EBV latent antigens was elevated, and cell surface expression of MHC class I was marginally increased. LMP2A-deficient LCLs produced lower amounts of IL-10, although this did not directly affect CD8+ T cell recognition. Deletion of LMP2A led to several changes in the cell surface immunophenotype of LCLs. Specifically, the agonistic NKG2D ligands MICA and ULBP4 were increased. Blocking experiments showed that NKG2D activation contributed to LCL recognition by CD8+ T cell clones. Our results demonstrate that LMP2A reduces the reactivity of CD8+ T cells against EBV-infected cells, and we identify several relevant mechanisms.

  3. A study of membrane protein defects and alpha hemoglobin chains of red blood cells in human beta thalassemia

    International Nuclear Information System (INIS)

    Rouyer-Fessard, P.; Garel, M.C.; Domenget, C.; Guetarni, D.; Bachir, D.; Colonna, P.; Beuzard, Y.

    1989-01-01

    The soluble pool of alpha hemoglobin chains present in blood or bone marrow cells was measured with a new affinity method using a specific probe, beta A hemoglobin chain labeled with [ 3 H]N-ethylmaleimide. This pool of soluble alpha chains was 0.067 ± 0.017% of hemoglobin in blood of normal adult, 0.11 ± 0.03% in heterozygous beta thalassemia and ranged from 0.26 to 1.30% in homozygous beta thalassemia intermedia. This elevated pool of soluble alpha chains observed in human beta thalassemia intermedia decreased 33-fold from a value of 10% of total hemoglobin in bone marrow cells to 0.3% in the most dense red blood cells. The amount of insoluble alpha chains was measured by using the polyacrylamide gel electrophoresis in urea and Triton X-100. In beta thalassemia intermedia the amount of insoluble alpha chains was correlated with the decreased spectrin content of red cell membrane and was associated with a decrease in ankyrin and with other abnormalities of the electrophoretic pattern of membrane proteins. The loss and topology of the reactive thiol groups of membrane proteins was determined by using [ 3 H]N-ethylmaleimide added to membrane ghosts prior to urea and Triton X-100 electrophoresis. Spectrin and ankyrin were the major proteins with the most important decrease of thiol groups

  4. Podoplanin, novel 43-kd membrane protein of glomerular epithelial cells, is down-regulated in puromycin nephrosis.

    Science.gov (United States)

    Breiteneder-Geleff, S.; Matsui, K.; Soleiman, A.; Meraner, P.; Poczewski, H.; Kalt, R.; Schaffner, G.; Kerjaschki, D.

    1997-01-01

    Puromycin aminonucleoside nephrosis (PAN), a rat model of human minimal change nephropathy, is characterized by extensive flattening of glomerular epithelial cell (podocyte) foot processes and by severe proteinuria. For comparison of expression of glomerular membrane proteins of normal and PAN rats, a membrane protein fraction of isolated rat glomeruli was prepared and monoclonal antibodies were raised against it. An IgG-secreting clone designated LF3 was selected that specifically immunolabeled podocytes of normal but not of PAN rats. The antigen of LF3 IgG was identified as a 43-kd glycoprotein. Molecular cloning of its cDNA was performed in a delta gt11 expression library prepared from mRNA of isolated rat glomeruli. The predicted amino acid sequence indicated a 166-amino-acid integral membrane protein with a single membrane-spanning domain, two potential phosphorylation sites in its short cytoplasmic tail, and six potential O-glycosylation sites in the large ectodomain. High amino acid sequence identities were found to membrane glycoproteins of rat lung and bone and mouse thymus epithelial cells as well as to a phorbol-ester-induced protein in a mouse osteoblast cell line and to a canine influenza C virus receptor. In PAN, expression of this 43-kd protein was selectively reduced to < 30%, as determined by quantitative immunogold electron microscopy, immunoblotting, and Northern blotting. These data provide evidence that transcription of the 43-kd transmembrane podocyte glycoprotein is specifically down-regulated in PAN. To indicate that this protein could be associated with transformation of arborized foot processes to flat feet (Latin, pes planus) we have called it podoplanin. Images Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 10 Figure 12 Figure 13 Figure 14 Figure 15 PMID:9327748

  5. Quantifying protein densities on cell membranes using super-resolution optical fluctuation imaging

    Czech Academy of Sciences Publication Activity Database

    Lukeš, T.; Glatzová, Daniela; Kvíčalová, Zuzana; Levet, F.; Benda, Aleš; Letschert, S.; Sauer, M.; Brdička, Tomáš; Lasser, T.; Cebecauer, Marek

    2017-01-01

    Roč. 8, č. 1 (2017), č. článku 1731. ISSN 2041-1723 R&D Projects: GA ČR GA15-06989S Institutional support: RVO:61388955 ; RVO:68378050 Keywords : quantifying protein densities * membranes * single- molecule localization microscopy Subject RIV: CE - Biochemistry OBOR OECD: Biochemistry and molecular biology Impact factor: 12.124, year: 2016

  6. Increased levels of the multidrug resistance protein in lateral membranes of proliferating hepatocyte-derived cells

    NARCIS (Netherlands)

    Roelofsen, H; Vos, TA; Schippers, IJ; Kuipers, F; Moshage, H; Jansen, PLM; Muller, M

    Background & Aims: The multidrug resistance protein (MRP) functions as an organic anion efflux carrier. Recent studies suggest that hepatocytes contain two mrp homologues, named mrp1 and mrp2, localized on the lateral and canalicular membrane, respectively. The aim of this study was to evaluate the

  7. Pseudomonas aeruginosa Cell Membrane Protein Expression from Phenotypically Diverse Cystic Fibrosis Isolates Demonstrates Host-Specific Adaptations.

    Science.gov (United States)

    Kamath, Karthik Shantharam; Pascovici, Dana; Penesyan, Anahit; Goel, Apurv; Venkatakrishnan, Vignesh; Paulsen, Ian T; Packer, Nicolle H; Molloy, Mark P

    2016-07-01

    Pseudomonas aeruginosa is a Gram-negative, nosocomial, highly adaptable opportunistic pathogen especially prevalent in immuno-compromised cystic fibrosis (CF) patients. The bacterial cell surface proteins are important contributors to virulence, yet the membrane subproteomes of phenotypically diverse P. aeruginosa strains are poorly characterized. We carried out mass spectrometry (MS)-based proteome analysis of the membrane proteins of three novel P. aeruginosa strains isolated from the sputum of CF patients and compared protein expression to the widely used laboratory strain, PAO1. Microbes were grown in planktonic growth condition using minimal M9 media, and a defined synthetic lung nutrient mimicking medium (SCFM) limited passaging. Two-dimensional LC-MS/MS using iTRAQ labeling enabled quantitative comparisons among 3171 and 2442 proteins from the minimal M9 medium and in the SCFM, respectively. The CF isolates showed marked differences in membrane protein expression in comparison with PAO1 including up-regulation of drug resistance proteins (MexY, MexB, MexC) and down-regulation of chemotaxis and aerotaxis proteins (PA1561, PctA, PctB) and motility and adhesion proteins (FliK, FlgE, FliD, PilJ). Phenotypic analysis using adhesion, motility, and drug susceptibility assays confirmed the proteomics findings. These results provide evidence of host-specific microevolution of P. aeruginosa in the CF lung and shed light on the adaptation strategies used by CF pathogens.

  8. Variable opacity (Opa) outer membrane proteins account for the cell tropisms displayed by Neisseria gonorrhoeae for human leukocytes and epithelial cells.

    OpenAIRE

    Kupsch, E M; Knepper, B; Kuroki, T; Heuer, I; Meyer, T F

    1993-01-01

    Opacity proteins (Opa) of Neisseria gonorrhoeae, a family of variant outer membrane proteins implicated in pathogenesis, are subject to phase variation. In strain MS11, 11 different opa gene alleles have been identified, the expression of which can be turned on and off independently. Using a reverse genetic approach, we demonstrate that a single Opa protein variant of strain MS11, Opa50, enables gonococci to invade epithelial cells. The remaining variant Opa proteins show no, or very little, ...

  9. Identification and validation of T-cell epitopes in outer membrane protein (OMP) of Salmonella typhi.

    Science.gov (United States)

    Tanu, Arifur Rahman; Ashraf, Mohammad Arif; Hossain, Md Faruk; Ismail, Md; Shekhar, Hossain Uddin

    2014-01-01

    This study aims to design epitope-based peptides for the utility of vaccine development by targeting outer membrane protein F (Omp F), because two available licensed vaccines, live oral Ty21a and injectable polysaccharide, are 50% to 80% protective with a higher rate of side effects. Conventional vaccines take longer time for development and have less differentiation power between vaccinated and infected cells. On the other hand, Peptide-based vaccines present few advantages over other vaccines, such as stability of peptide, ease to manufacture, better storage, avoidance of infectious agents during manufacture, and different molecules can be linked with peptides to enhance their immunogenicity. Omp F is highly conserved and facilitates attachment and fusion of Salmonella typhi with host cells. Using various databases and tools, immune parameters of conserved sequences from Omp F of different isolates of Salmonella typhi were tested to predict probable epitopes. Binding analysis of the peptides with MHC molecules, epitopes conservancy, population coverage, and linear B cell epitope prediction were analyzed. Among all those predicted peptides, ESYTDMAPY epitope interacted with six MHC alleles and it shows highest amount of interaction compared to others. The cumulative population coverage for these epitopes as vaccine candidates was approximately 70%. Structural analysis suggested that epitope ESYTDMAPY fitted well into the epitope-binding groove of HLA-C*12:03, as this HLA molecule was common which interact with each and every predicted epitopes. So, this potential epitope may be linked with other molecules to enhance its immunogenicity and used for vaccine development.

  10. Integral Membrane Proteins and Bilayer Proteomics

    OpenAIRE

    Whitelegge, Julian P.

    2013-01-01

    Integral membrane proteins reside within the bilayer membranes that surround cells and organelles, playing critical roles in movement of molecules across them and the transduction of energy and signals. While their extreme amphipathicity presents technical challenges, biological mass spectrometry has been applied to all aspects of membrane protein chemistry and biology, including analysis of primary, secondary, tertiary and quaternary structure, as well as the dynamics that accompany function...

  11. Integral membrane proteins and bilayer proteomics.

    Science.gov (United States)

    Whitelegge, Julian P

    2013-03-05

    Integral membrane proteins reside within the bilayer membranes that surround cells and organelles, playing critical roles in movement of molecules across them and the transduction of energy and signals. While their extreme amphipathicity presents technical challenges, biological mass spectrometry has been applied to all aspects of membrane protein chemistry and biology, including analysis of primary, secondary, tertiary, and quaternary structures as well as the dynamics that accompany functional cycles and catalysis.

  12. Improved proliferation and differentiation capacity of human mesenchymal stromal cells cultured with basement-membrane extracellular matrix proteins.

    Science.gov (United States)

    Lindner, Ulrich; Kramer, Jan; Behrends, Jochen; Driller, Birgit; Wendler, Nils-Ole; Boehrnsen, Florian; Rohwedel, Jürgen; Schlenke, Peter

    2010-12-01

    In vitro cultured mesenchymal stromal cells (MSC) are characterized by a short proliferative lifespan, an increasing loss of proliferation capacity and progressive reduction of differentiation potential. Laminin-1, laminin-5, collagen IV and fibronectin are important constituents of the basement membrane extracellular matrix (ECM) that are involved in a variety of cellular activities, including cell attachment and motility. The in vitro proliferation capacity of MSC was significantly improved when the cells were incubated in the presence of basement membrane ECM proteins. For example, a mixture of proteins improved proliferation capacity 250-fold in comparison with standard conditions after five passages. Furthermore, in colony-forming unit-fibroblast (CFU-F) assays colony numbers and size were significantly extended. Blocking specific integrin cell-surface receptors, positive effects on the proliferation capacity of MSC were inhibited. Additionally, when MSC were co-cultivated with ECM proteins, cells maintained their multipotential differentiation capacity throughout many culture passages in comparison with cells cultivated on plastic. However, expansion of MSC on laminin-5 suppressed any subsequent chondrogenic differentiation. Our results suggest that expansion of bone marrow-derived MSC in the presence of ECM proteins is a powerful approach for generating large numbers of MSC, showing a prolonged capacity to differentiate into mesodermal cell lineages, with the exception of the lack of chondrogenesis by using laminin-5 coating.

  13. A hybrid total internal reflection fluorescence and optical tweezers microscope to study cell adhesion and membrane protein dynamics of single living cells

    NARCIS (Netherlands)

    Snijder-van As, M.I.; Rieger, B.; Joosten, B.; Subramaniam, Vinod; Figdor, Carl; Kanger, Johannes S.

    2009-01-01

    The dynamics of cell surface membrane proteins plays an important role in cell–cell interactions. The onset of the interaction is typically not precisely controlled by current techniques, making especially difficult the visualization of early-stage dynamics. We have developed a novel method where

  14. Characterising antimicrobial protein-membrane complexes

    International Nuclear Information System (INIS)

    Xun, Gloria; Dingley, Andrew; Tremouilhac, Pierre

    2009-01-01

    Full text: Antimicrobial proteins (AMPs) are host defence molecules that protect organisms from microbial infection. A number of hypotheses for AMP activity have been proposed which involve protein membrane interactions. However, there is a paucity of information describing AMP-membrane complexes in detail. The aim of this project is to characterise the interactions of amoebapore-A (APA-1) with membrane models using primarily solution-state NMR spectroscopy. APA-1 is an AMP which is regulated by a pH-dependent dimerisation event. Based on the atomic resolution solution structure of monomeric APA-1, it is proposed that this dimerisation is a prerequisite for ring-like hexameric pore formation. Due to the cytotoxicity of APA-1, we have developed a cell-free system to produce this protein. To facilitate our studies, we have adapted the cell-free system to isotope label APA-1. 13 C /15 N -enriched APA-1 sample was achieved and we have begun characterising APA-1 dimerisation and membrane interactions using NMR spectroscopy and other biochemical/biophysical methods. Neutron reflectometry is a surface-sensitive technique and therefore represents an ideal technique to probe how APA-1 interacts with membranes at the molecular level under different physiological conditions. Using Platypus, the pH-induced APA-1-membrane interactions should be detectable as an increase of the amount of protein adsorbed at the membrane surface and changes in the membrane properties. Specifically, detailed information of the structure and dimensions of the protein-membrane complex, the position and amount of the protein in the membrane, and the perturbation of the membrane phospholipids on protein incorporation can be extracted from the neutron reflectometry measurement. Such information will enable critical assessment of current proposed mechanisms of AMP activity in bacterial membranes and complement our NMR studies

  15. Characterization of the methotrexate transport pathway in murine L1210 leukemia cells: Involvement of a membrane receptor and a cytosolic protein

    International Nuclear Information System (INIS)

    Price, E.M.; Ratnam, M.; Rodeman, K.M.; Freisheim, J.H.

    1988-01-01

    A radioiodinated photoaffinity analogue of methotrexate, N α -(4-amino-4-deoxy-10-methyl-pteroyl)-N ε -(4-azidosalicylyl)-L-lysine (APA-ASA-Lys), was recently used to identify the plasma membrane derived binding protein involved in the transport of this folate antagonist into murine L1210 cells. The labeled protein has an apparent molecular weight of 46K-48K when analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, but no such labeling occurs in a methotrexate transport-defective cell line (L1210/R81). Labeling of the total cytosolic protein from disrupted cells, followed by electrophoresis and autoradiography, showed, among other proteins, a 21K band, corresponding to dihydrofolate reductase (DHFR), in both the parent and R81 cells and a 38K band only in the parent cells. However, when whole cells were UV irradiated at various times at 37 degree C following addition of radiolabeled APA-ASA-Lys, the 38K protein and DHFR were the only cytosolic proteins labeled in the parent cells, while the intact R81 cells showed no labeled cytosolic protein, since the photoprobe is not transported. Further, when the parent cells were treated with a pulse of radiolabeled photoprobe, followed by UV irradiation at different times at 37 degree C, the probe appeared sequentially on the 48K membrane protein and both the 38K cytosolic protein and dihydrofolate reductase. A 48K protein could be detected in both parent L1210 cells and the R81 cells on Western blots using antisera to a membrane folate binding protein from human placenta. These results suggest a vectorial transport of APA-ASA-Lys or methotrexate and reduced folate coenzymes into murine L1210 cells mediated by a 48K integral membrane protein and a 38K cytosolic or peripheral membrane protein. The 38K protein may help in the trafficking of reduced folate coenzymes, shuttling them to various cytosolic targets

  16. Association between RABV G Proteins Transported from the Perinuclear Space to the Cell Surface Membrane and N-Glycosylation of the Sequon Asn(204).

    Science.gov (United States)

    Hamamoto, Noriko; Uda, Akihiko; Tobiume, Minoru; Park, Chun-Ho; Noguchi, Akira; Kaku, Yoshihiro; Okutani, Akiko; Morikawa, Shigeru; Inoue, Satoshi

    2015-01-01

    In this study, G proteins of the rabies virus (RABV) Kyoto strain were detected in the cytoplasm but not distributed at the cell membrane of mouse neuroblastoma (MNA) cells. G proteins of CVS-26 were detected in both the cell membrane and perinuclear space of MNA cells. We found that N-glycosylation of street RABV G protein by the insertion of the sequon Asn(204) induced the transfer of RABV G proteins to the cell surface membrane. Fixed RABV budding from the plasma membrane has been found to depend not only on G protein but also on other structural proteins such as M protein. However, the differing N-glycosylation of G protein could be associated with the distinct budding and antigenic features of RABV in street and fixed viruses. Our study of the association of N-glycan of G protein at Asn(204) with the transport of RABV G protein to the cell surface membrane contributes to the understanding of the evolution of fixed virus from street virus, which in turn would help for determine the mechanism underlying RABV budding and enhanced host immune responses.

  17. High-protein-PUFA supplementation, red blood cell membranes, and plasma antioxidant activity in volleyball athletes.

    Science.gov (United States)

    Malaguti, Marco; Baldini, Marta; Angeloni, Cristina; Biagi, Pierluigi; Hrelia, Silvana

    2008-06-01

    The authors evaluated the role of a high-protein, low-calorie, polyunsaturated fatty-acid (PUFA) -supplemented diet on anthropometric parameters, erythrocyte-membrane fatty-acid composition, and plasma antioxidant defenses of nonprofessional volleyball athletes. The athletes were divided in two groups: One (n = 5) followed the Mediterranean diet, and the other (n = 6) followed a high-protein, low-calorie diet with a 3-g/day fish-oil supplementation. All the athletes had anthropometric measurements taken, both at the beginning and at the end of the study, which lasted for 2 months. Body-mass index and total body fat were significantly diminished in the second group, while they remained unchanged in the first. Plasma total antioxidant activity (TAA) was significantly increased in the plasma of both groups, with no differences between the groups, suggesting that physical activity, not the different diets, is the main contributor to the increase of plasma TAA. The second group showed a significant increase in erythrocyte-membrane PUFA content and in the unsaturation index value (UI) because of the fish-oil supplementation.A high-protein, low-carbohydrate, fish-oil-supplemented diet seems to be useful only when the aim of the diet is to obtain weight loss in a short-term period. The significant increase in the UI of erythrocyte membranes indicates the potential for harm, because a high intake of PUFA might increase susceptibility to lipid peroxidation not counterbalanced by a higher increase in TAA. Adherence to the Mediterranean diet seems to be the better choice.

  18. Modelling of proteins in membranes

    DEFF Research Database (Denmark)

    Sperotto, Maria Maddalena; May, S.; Baumgaertner, A.

    2006-01-01

    This review describes some recent theories and simulations of mesoscopic and microscopic models of lipid membranes with embedded or attached proteins. We summarize results supporting our understanding of phenomena for which the activities of proteins in membranes are expected to be significantly...

  19. Stochastic single-molecule dynamics of synaptic membrane protein domains

    Science.gov (United States)

    Kahraman, Osman; Li, Yiwei; Haselwandter, Christoph A.

    2016-09-01

    Motivated by single-molecule experiments on synaptic membrane protein domains, we use a stochastic lattice model to study protein reaction and diffusion processes in crowded membranes. We find that the stochastic reaction-diffusion dynamics of synaptic proteins provide a simple physical mechanism for collective fluctuations in synaptic domains, the molecular turnover observed at synaptic domains, key features of the single-molecule trajectories observed for synaptic proteins, and spatially inhomogeneous protein lifetimes at the cell membrane. Our results suggest that central aspects of the single-molecule and collective dynamics observed for membrane protein domains can be understood in terms of stochastic reaction-diffusion processes at the cell membrane.

  20. Human Immunodeficiency Virus Type 1 Nef protein modulates the lipid composition of virions and host cell membrane microdomains

    Directory of Open Access Journals (Sweden)

    Geyer Matthias

    2007-10-01

    Full Text Available Abstract Background The Nef protein of Human Immunodeficiency Viruses optimizes viral spread in the infected host by manipulating cellular transport and signal transduction machineries. Nef also boosts the infectivity of HIV particles by an unknown mechanism. Recent studies suggested a correlation between the association of Nef with lipid raft microdomains and its positive effects on virion infectivity. Furthermore, the lipidome analysis of HIV-1 particles revealed a marked enrichment of classical raft lipids and thus identified HIV-1 virions as an example for naturally occurring membrane microdomains. Since Nef modulates the protein composition and function of membrane microdomains we tested here if Nef also has the propensity to alter microdomain lipid composition. Results Quantitative mass spectrometric lipidome analysis of highly purified HIV-1 particles revealed that the presence of Nef during virus production from T lymphocytes enforced their raft character via a significant reduction of polyunsaturated phosphatidylcholine species and a specific enrichment of sphingomyelin. In contrast, Nef did not significantly affect virion levels of phosphoglycerolipids or cholesterol. The observed alterations in virion lipid composition were insufficient to mediate Nef's effect on particle infectivity and Nef augmented virion infectivity independently of whether virus entry was targeted to or excluded from membrane microdomains. However, altered lipid compositions similar to those observed in virions were also detected in detergent-resistant membrane preparations of virus producing cells. Conclusion Nef alters not only the proteome but also the lipid composition of host cell microdomains. This novel activity represents a previously unrecognized mechanism by which Nef could manipulate HIV-1 target cells to facilitate virus propagation in vivo.

  1. Flagellar membrane fusion and protein exchange in trypanosomes; a new form of cell-cell communication? [version 1; referees: 2 approved

    Directory of Open Access Journals (Sweden)

    Simon Imhof

    2016-04-01

    Full Text Available Diverse structures facilitate direct exchange of proteins between cells, including plasmadesmata in plants and tunnelling nanotubes in bacteria and higher eukaryotes.  Here we describe a new mechanism of protein transfer, flagellar membrane fusion, in the unicellular parasite Trypanosoma brucei. When fluorescently tagged trypanosomes were co-cultured, a small proportion of double-positive cells were observed. The formation of double-positive cells was dependent on the presence of extracellular calcium and was enhanced by placing cells in medium supplemented with fresh bovine serum. Time-lapse microscopy revealed that double-positive cells arose by bidirectional protein exchange in the absence of nuclear transfer.  Furthermore, super-resolution microscopy showed that this process occurred in ≤1 minute, the limit of temporal resolution in these experiments. Both cytoplasmic and membrane proteins could be transferred provided they gained access to the flagellum. Intriguingly, a component of the RNAi machinery (Argonaute was able to move between cells, raising the possibility that small interfering RNAs are transported as cargo. Transmission electron microscopy showed that shared flagella contained two axonemes and two paraflagellar rods bounded by a single membrane. In some cases flagellar fusion was partial and interactions between cells were transient. In other cases fusion occurred along the entire length of the flagellum, was stable for several hours and might be irreversible. Fusion did not appear to be deleterious for cell function: paired cells were motile and could give rise to progeny while fused. The motile flagella of unicellular organisms are related to the sensory cilia of higher eukaryotes, raising the possibility that protein transfer between cells via cilia or flagella occurs more widely in nature.

  2. Expression and deposition of basement membrane proteins by brain capillary endothelial cells in a primary murine model of the blood-brain barrier

    DEFF Research Database (Denmark)

    Thomsen, Maj Schneider; Birkelund, Svend; Larsen, Annette Burkhart

    2016-01-01

    of the present study was to create four different in vitro constructs of the murine BBB to characterise if the expression and secretion of basement membrane proteins by the murine brain capillary endothelial cells (mBCECs) was affected by co-culturing with pericytes, mixed glial cells, or both. Primary m...... membrane, and astrocyte endfeet. To study the interaction of the different cells of the BBB, construction of in vitro BBB models is valuable. However, the modulation and contribution of the cells of the BBB to the synthesis of basement membrane proteins in vitro is not fully elaborated. Thus, the aim......, and immunofluorescent labelling were used. The mBCECs were found to express major basement membrane proteins in vitro and increased expression of laminin α5 and collagen IV α1 was correlated to the addition of BBB inducing factors (hydrocortisone, Ro20-1724, pCPT-cAMP). Co-culturing of the mBCECs with pericytes, mixed...

  3. Red cell autoantibodies characterized by competitive inhibition of iodine 125 Rh alloantibody binding and by immunoprecipitation of membrane proteins

    International Nuclear Information System (INIS)

    Pierce, S.W.; Victoria, E.J.; Masouredis, S.P.

    1990-01-01

    The relationship between determinants recognized by warm-type immunoglobulin G red cell autoantibodies and the Rh antigens was characterized by autoantibody competitive inhibition of iodine 125 Rh alloantibody binding and autoantibody immunoprecipitation of iodine 125 red blood cell membrane proteins. The majority of blood donor autoantibody recognized epitopes that are closely related to Rh antigens as determined by competitive inhibition studies. Eighteen of 20 (90%) autoantibodies inhibited anti-Rh(c) binding, 15 inhibited anti-Rh(E), 5 inhibited anti-Rh(D), and only 2 failed to inhibit any of the three Rh alloantibodies tested. Autoantibodies that inhibited anti-Rh(D) also inhibited anti-Rh(c) and anti-Rh(E) and all those that inhibited anti-Rh(E) also inhibited anti-Rh(c). Autoantibodies that inhibited all three Rh alloantibodies immunoprecipitated 30 kd membrane polypeptides, as did two of the three autoantibodies that inhibited only anti-Rh(c) and anti-Rh(E). One autoantibody in this group and two autoantibodies that inhibited only anti-Rh(c), as well as an autoantibody that did not inhibit any of the Rh alloantibodies, immunoprecipitated only a single membrane polypeptide identified as band 3. The majority of normal donor red blood cell autoantibodies inhibited the binding of Rh alloantibodies, which indicates that they either bound to the Rh polypeptides or to epitopes on band 3 that were closely associated with the Rh complex

  4. Protein profiles of hatchery egg shell membrane.

    Science.gov (United States)

    Rath, N C; Liyanage, R; Makkar, S K; Lay, J O

    2016-01-01

    Eggshells which consist largely of calcareous outer shell and shell membranes, constitute a significant part of poultry hatchery waste. The shell membranes (ESM) not only contain proteins that originate from egg whites but also from the developing embryos and different contaminants of microbial and environmental origins. As feed supplements, during post hatch growth, the hatchery egg shell membranes (HESM) have shown potential for imparting resistance of chickens to endotoxin stress and exert positive health effects. Considering that these effects are mediated by the bioactive proteins and peptides present in the membrane, the objective of the study was to identify the protein profiles of hatchery eggshell membranes (HESM). Hatchery egg shell membranes were extracted with acidified methanol and a guanidine hydrochloride buffer then subjected to reduction/alkylation, and trypsin digestion. The methanol extract was additionally analyzed by matrix assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF-MS). The tryptic digests were analyzed by liquid chromatography and tandem mass spectrometry (LC-MS-MS) to identify the proteins. Our results showed the presence of several proteins that are inherent and abundant in egg white such as, ovalbumin, ovotransferrin, ovocleidin-116, and lysozyme, and several proteins associated with cytoskeletal, cell signaling, antimicrobial, and catalytic functions involving carbohydrate, nucleic acid, and protein metabolisms. There were some blood derived proteins most likely originating from the embryos and several other proteins identified with different aerobic, anaerobic, gram positive, gram negative, soil, and marine bacterial species some commensals and others zoonotic. The variety of bioactive proteins, particularly the cell signaling and enzymatic proteins along with the diverse microbial proteins, make the HESM suitable for nutritional and biological application to improve post hatch immunity of poultry.

  5. Marked increase in rat red blood cell membrane protein glycosylation by one-month treatment with a cafeteria diet

    Directory of Open Access Journals (Sweden)

    Laia Oliva

    2015-07-01

    Full Text Available Background and Objectives. Glucose, an aldose, spontaneously reacts with protein amino acids yielding glycosylated proteins. The compounds may reorganize to produce advanced glycosylation products, which regulatory importance is increasingly being recognized. Protein glycosylation is produced without the direct intervention of enzymes and results in the loss of function. Glycosylated plasma albumin, and glycosylated haemoglobin are currently used as index of mean plasma glucose levels, since higher glucose availability results in higher glycosylation rates. In this study we intended to detect the early changes in blood protein glycosylation elicited by an obesogenic diet.Experimental Design. Since albumin is in constant direct contact with plasma glucose, as are the red blood cell (RBC membranes, we analyzed their degree or glycosylation in female and male rats, either fed a standard diet or subjected to a hyper-energetic self-selected cafeteria diet for 30 days. This model produces a small increase in basal glycaemia and a significant increase in body fat, leaving the animals in the initial stages of development of metabolic syndrome. We also measured the degree of glycosylation of hemoglobin, and the concentration of glucose in contact with this protein, that within the RBC. Glycosylation was measured by colorimetric estimation of the hydroxymethylfurfural liberated from glycosyl residues by incubation with oxalate.Results. Plasma glucose was higher in cafeteria diet and in male rats, both independent effects. However, there were no significant differences induced by sex or diet in either hemoglobin or plasma proteins. Purified RBC membranes showed a marked effect of diet: higher glycosylation in cafeteria rats, which was more marked in females (not in controls. In any case, the number of glycosyl residues per molecule were higher in hemoglobin than in plasma proteins (after correction for molecular weight. The detected levels of glucose in

  6. A cell-free method for expressing and reconstituting membrane proteins enables functional characterization of the plant receptor-like protein kinase FERONIA.

    Science.gov (United States)

    Minkoff, Benjamin B; Makino, Shin-Ichi; Haruta, Miyoshi; Beebe, Emily T; Wrobel, Russell L; Fox, Brian G; Sussman, Michael R

    2017-04-07

    There are more than 600 receptor-like kinases (RLKs) in Arabidopsis , but due to challenges associated with the characterization of membrane proteins, only a few have known biological functions. The plant RLK FERONIA is a peptide receptor and has been implicated in plant growth regulation, but little is known about its molecular mechanism of action. To investigate the properties of this enzyme, we used a cell-free wheat germ-based expression system in which mRNA encoding FERONIA was co-expressed with mRNA encoding the membrane scaffold protein variant MSP1D1. With the addition of the lipid cardiolipin, assembly of these proteins into nanodiscs was initiated. FERONIA protein kinase activity in nanodiscs was higher than that of soluble protein and comparable with other heterologously expressed protein kinases. Truncation experiments revealed that the cytoplasmic juxtamembrane domain is necessary for maximal FERONIA activity, whereas the transmembrane domain is inhibitory. An ATP analogue that reacts with lysine residues inhibited catalytic activity and labeled four lysines; mutagenesis demonstrated that two of these, Lys-565 and Lys-663, coordinate ATP in the active site. Mass spectrometric phosphoproteomic measurements further identified phosphorylation sites that were examined using phosphomimetic mutagenesis. The results of these experiments are consistent with a model in which kinase-mediated phosphorylation within the C-terminal region is inhibitory and regulates catalytic activity. These data represent a step further toward understanding the molecular basis for the protein kinase catalytic activity of FERONIA and show promise for future characterization of eukaryotic membrane proteins. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  7. Transmembrane protein sorting driven by membrane curvature

    Science.gov (United States)

    Strahl, H.; Ronneau, S.; González, B. Solana; Klutsch, D.; Schaffner-Barbero, C.; Hamoen, L. W.

    2015-11-01

    The intricate structure of prokaryotic and eukaryotic cells depends on the ability to target proteins to specific cellular locations. In most cases, we have a poor understanding of the underlying mechanisms. A typical example is the assembly of bacterial chemoreceptors at cell poles. Here we show that the classical chemoreceptor TlpA of Bacillus subtilis does not localize according to the consensus stochastic nucleation mechanism but accumulates at strongly curved membrane areas generated during cell division. This preference was confirmed by accumulation at non-septal curved membranes. Localization appears to be an intrinsic property of the protein complex and does not rely on chemoreceptor clustering, as was previously shown for Escherichia coli. By constructing specific amino-acid substitutions, we demonstrate that the preference for strongly curved membranes arises from the curved shape of chemoreceptor trimer of dimers. These findings demonstrate that the intrinsic shape of transmembrane proteins can determine their cellular localization.

  8. Analysis of Membrane Protein Topology in the Plant Secretory Pathway.

    Science.gov (United States)

    Guo, Jinya; Miao, Yansong; Cai, Yi

    2017-01-01

    Topology of membrane proteins provides important information for the understanding of protein function and intermolecular associations. Integrate membrane proteins are generally transported from endoplasmic reticulum (ER) to Golgi and downstream compartments in the plant secretory pathway. Here, we describe a simple method to study membrane protein topology along the plant secretory pathway by transiently coexpressing a fluorescent protein (XFP)-tagged membrane protein and an ER export inhibitor protein, ARF1 (T31N), in tobacco BY-2 protoplast. By fractionation, microsome isolation, and trypsin digestion, membrane protein topology could be easily detected by either direct confocal microscopy imaging or western-blot analysis using specific XFP antibodies. A similar strategy in determining membrane protein topology could be widely adopted and applied to protein analysis in a broad range of eukaryotic systems, including yeast cells and mammalian cells.

  9. The membrane protein melanoma cell adhesion molecule (MCAM) is a novel tumor marker that stimulates tumorigenesis in hepatocellular carcinoma.

    Science.gov (United States)

    Wang, J; Tang, X; Weng, W; Qiao, Y; Lin, J; Liu, W; Liu, R; Ma, L; Yu, W; Yu, Y; Pan, Q; Sun, F

    2015-11-19

    Yes-associated protein (YAP) is overexpressed and has an oncogenic role in hepatocellular carcinoma (HCC). However, whether membrane protein can serve not only as a tumor marker that reflects YAP function but also as a therapeutic target that stimulates tumorigenesis in HCC remains unknown. Here we report that the membrane protein melanoma cell adhesion molecule (MCAM) was under positive regulation by YAP and was highly elevated in HCC cells. Within the MCAM promoter, we found the presence of a cAMP Response Element (CRE; -32 to -25 nt), which is conserved among species and is essential for YAP- and CREB-dependent regulation. Moreover, the interaction between CREB and YAP at the CRE site was dependent on PTPIY-WW domain interactions. However, MCAM expression was low and could not be regulated by YAP in breast and colon cancer cells because of the low levels of the acetyltransferase p300. In HCC cells, high levels of p300 facilitated the binding of YAP to the MCAM promoter, which in turn enhanced histone acetylation and polymerase II recruitment through the dissociation of the deacetylase Sirt1. These results suggest that MCAM is an HCC-specific target of YAP. In clinical serum samples, we found that the serum levels of MCAM were highly elevated in patients with HCC compared with healthy controls and with patients with cirrhosis, hepatitis, colon cancer and breast cancer. MCAM levels were shown to be a slightly better indicator than serum alpha-fetoprotein for predicting HCC. We further demonstrated that MCAM is essential for the survival and transformation of HCC. Mechanistically, MCAM induced translation initiation and the transcriptional activities of c-Jun/c-Fos. In addition, AKT activation had an essential role in the MCAM-promoted binding of eukaryotic initiation factor 4E to c-Jun/c-Fos mRNA. In conclusion, we demonstrated that MCAM may be a potential tumor marker and therapeutic target for the diagnosis and treatment of HCC.

  10. Decidual-secreted factors alter invasive trophoblast membrane and secreted proteins implying a role for decidual cell regulation of placentation.

    Directory of Open Access Journals (Sweden)

    Ellen Melaleuca Menkhorst

    Full Text Available Inadequate or inappropriate implantation and placentation during the establishment of human pregnancy is thought to lead to first trimester miscarriage, placental insufficiency and other obstetric complications. To create the placental blood supply, specialized cells, the 'extravillous trophoblast' (EVT invade through the differentiated uterine endometrium (the decidua to engraft and remodel uterine spiral arteries. We hypothesized that decidual factors would regulate EVT function by altering the production of EVT membrane and secreted factors. We used a proteomics approach to identify EVT membrane and secreted proteins regulated by decidual cell factors. Human endometrial stromal cells were decidualized in vitro by treatment with estradiol (10(-8 M, medroxyprogesterone acetate (10(-7 M and cAMP (0.5 mM for 14 days. Conditioned media (CM was collected on day 2 (non-decidualized CM and 14 (decidualized CM of treatment. Isolated primary EVT cultured on Matrigel™ were treated with media control, non-decidualized or decidualized CM for 16 h. EVT CM was fractionated for proteins <30 kDa using size-exclusion affinity nanoparticles (SEAN before trypsin digestion and HPLC-MS/MS. 43 proteins produced by EVT were identified; 14 not previously known to be expressed in the placenta and 12 which had previously been associated with diseases of pregnancy including preeclampsia. Profilin 1, lysosome associated membrane glycoprotein 1 (LAMP1, dipeptidyl peptidase 1 (DPP1/cathepsin C and annexin A2 expression by interstitial EVT in vivo was validated by immunhistochemistry. Decidual CM regulation in vitro was validated by western blotting: decidualized CM upregulated profilin 1 in EVT CM and non-decidualized CM upregulated annexin A2 in EVT CM and pro-DPP1 in EVT cell lysate. Here, non-decidualized factors induced protease expression by EVT suggesting that non-decidualized factors may induce a pro-inflammatory cascade. Preeclampsia is a pro

  11. Cooperation of decay-accelerating factor and membrane cofactor protein in regulating survival of human cervical cancer cells

    International Nuclear Information System (INIS)

    Gao, Ling-Juan; Guo, Shu-Yu; Cai, You-Qun; Gu, Ping-Qing; Su, Ya-Juan; Gong, Hui; Liu, Yun; Chen, Chen

    2009-01-01

    Decay-accelerating factor (DAF) and membrane cofactor protein (MCP) are the key molecules involved in cell protection against autologus complement, which restricts the action of complement at critical stages of the cascade reaction. The cooperative effect of DAF and MCP on the survival of human cervical cancer cell (ME180) has not been demonstrated. In this study we applied, for the first time, short hairpin RNA (shRNA) to knock down the expression of the DAF and MCP with the aim of exploiting complement more effectively for tumor cell damage. Meanwhile, we investigated the cooperative effects of DAF and MCP on the viability and migration, moreover the proliferation of ME180 cell. The results showed that shRNA inhibition of DAF and MCP expression enhanced complement-dependent cytolysis (CDC) up to 39% for MCP and up to 36% for DAF, and the combined inhibition of both regulators yielded further additive effects in ME180 cells. Thus, the activities of DAF and MCP, when present together, are greater than the sum of the two protein individually. These data indicated that combined DAF and MCP shRNA described in this study may offer an additional alternative to improve the efficacy of antibody-and complement-based cancer immunotherapy

  12. Major Intrinsic Proteins in Biomimetic Membranes

    DEFF Research Database (Denmark)

    Helix Nielsen, Claus

    2010-01-01

    will generally have finite permeabilities to both electrolytes and non-electrolytes. The feasibility of a biomimetic MIP device thus depends on the relative transport contribution from both protein and biomimetic support matrix. Also the biomimetic matrix must be encapsulated in order to protect it and make....../separation technology, a unique class of membrane transport proteins is especially interesting the major intrinsic proteins (MIPs). Generally, MIPs conduct water molecules and selected solutes in and out of the cell while preventing the passage of other solutes, a property critical for the conservation of the cells...... it sufficiently stable in a final application. Here, I specifically discuss the feasibility of developing osmotic biomimetic MIP membranes, but the technical issues are of general concern in the design of biomimetic membranes capable of supporting selective transmembrane fluxes....

  13. Membrane Cells for Brine Electrolysis.

    Science.gov (United States)

    Tingle, M.

    1982-01-01

    Membrane cells were developed as alternatives to mercury and diaphragm cells for the electrolysis of brine. Compares the three types of cells, focusing on the advantages and disadvantages of membrane cells. (JN)

  14. Cationic amphipathic peptides accumulate sialylated proteins and lipids in the plasma membrane of eukaryotic host cells

    OpenAIRE

    Weghuber, Julian; Aichinger, Michael C.; Brameshuber, Mario; Wieser, Stefan; Ruprecht, Verena; Plochberger, Birgit; Madl, Josef; Horner, Andreas; Reipert, Siegfried; Lohner, Karl; Henics, Tamas; Schuetz, Gerhard J

    2011-01-01

    Cationic antimicrobial peptides (CAMPs) selectively target bacterial membranes by electrostatic interactions with negatively charged lipids. It turned out that for inhibition of microbial growth a high CAMP membrane concentration is required, which can be realized by the incorporation of hydrophobic groups within the peptide. Increasing hydrophobicity, however, reduces the CAMP selectivity for bacterial over eukaryotic host membranes, thereby causing the risk of detrimental side-effects. In t...

  15. Achiral Mannich-Base Curcumin Analogs Induce Unfolded Protein Response and Mitochondrial Membrane Depolarization in PANC-1 Cells

    Directory of Open Access Journals (Sweden)

    Gábor J. Szebeni

    2017-10-01

    Full Text Available Achiral Mannich-type curcumin analogs have been synthetized and assayed for their cytotoxic activity. The anti-proliferative and cytotoxic activity of curcuminoids has been tested on human non-small-cell lung carcinoma (A549, hepatocellular carcinoma (HepG2 and pancreatic cancer cell line (PANC-1. Based on the highest anti-proliferative activity nine drug candidates were further tested and proved to cause phosphatidylserine exposure as an early sign of apoptosis. Curcumin analogs with the highest apoptotic activity were selected for mechanistic studies in the most sensitive PANC-1 cells. Cytotoxic activity was accompanied by cytostatic effect since curcumin and analogs treatment led to G0/G1 cell cycle arrest. Moreover, cytotoxic effect could be also detected via the accumulation of curcuminoids in the endoplasmic reticulum (ER and the up-regulation of ER stress-related unfolded protein response (UPR genes: HSPA5, ATF4, XBP1, and DDIT3. The activated UPR induced mitochondrial membrane depolarization, caspase-3 activation and subsequent DNA breakdown in PANC-1 cells. Achiral curcumin analogs, C509, C521 and C524 possessed superior, 40-times more potent cytotoxic activity compared to natural dihydroxy-dimetoxycurcumin in PANC-1 cells.

  16. Nanodisc-solubilized membrane protein library reflects the membrane proteome.

    Science.gov (United States)

    Marty, Michael T; Wilcox, Kyle C; Klein, William L; Sligar, Stephen G

    2013-05-01

    The isolation and identification of unknown membrane proteins offers the prospect of discovering new pharmaceutical targets and identifying key biochemical receptors. However, interactions between membrane protein targets and soluble ligands are difficult to study in vitro due to the insolubility of membrane proteins in non-detergent systems. Nanodiscs, nanoscale discoidal lipid bilayers encircled by a membrane scaffold protein belt, have proven to be an effective platform to solubilize membrane proteins and have been used to study a wide variety of purified membrane proteins. This report details the incorporation of an unbiased population of membrane proteins from Escherichia coli membranes into Nanodiscs. This solubilized membrane protein library (SMPL) forms a soluble in vitro model of the membrane proteome. Since Nanodiscs contain isolated proteins or small complexes, the SMPL is an ideal platform for interactomics studies and pull-down assays of membrane proteins. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of the protein population before and after formation of the Nanodisc library indicates that a large percentage of the proteins are incorporated into the library. Proteomic identification of several prominent bands demonstrates the successful incorporation of outer and inner membrane proteins into the Nanodisc library.

  17. Nanodisc-solubilized membrane protein library reflects the membrane proteome

    OpenAIRE

    Marty, Michael T.; Wilcox, Kyle C.; Klein, William L.; Sligar, Stephen G.

    2013-01-01

    The isolation and identification of unknown membrane proteins offers the prospect of discovering new pharmaceutical targets and identifying key biochemical receptors. However, interactions between membrane protein targets and soluble ligands are difficult to study in vitro due to the insolubility of membrane proteins in non-detergent systems. Nanodiscs, nanoscale discoidal lipid bilayers encircled by a membrane scaffold protein belt, have proven to be an effective platform to solubilize membr...

  18. Intrinsically disordered proteins drive membrane curvature.

    Science.gov (United States)

    Busch, David J; Houser, Justin R; Hayden, Carl C; Sherman, Michael B; Lafer, Eileen M; Stachowiak, Jeanne C

    2015-07-24

    Assembly of highly curved membrane structures is essential to cellular physiology. The prevailing view has been that proteins with curvature-promoting structural motifs, such as wedge-like amphipathic helices and crescent-shaped BAR domains, are required for bending membranes. Here we report that intrinsically disordered domains of the endocytic adaptor proteins, Epsin1 and AP180 are highly potent drivers of membrane curvature. This result is unexpected since intrinsically disordered domains lack a well-defined three-dimensional structure. However, in vitro measurements of membrane curvature and protein diffusivity demonstrate that the large hydrodynamic radii of these domains generate steric pressure that drives membrane bending. When disordered adaptor domains are expressed as transmembrane cargo in mammalian cells, they are excluded from clathrin-coated pits. We propose that a balance of steric pressure on the two surfaces of the membrane drives this exclusion. These results provide quantitative evidence for the influence of steric pressure on the content and assembly of curved cellular membrane structures.

  19. Efficient overproduction of membrane proteins in Lactococcus lactis requires the cell envelope stress sensor/regulator couple CesSR

    NARCIS (Netherlands)

    Pinto, Joao P C; Kuipers, Oscar P; Marreddy, Ravi K R; Poolman, Bert; Kok, Jan

    2011-01-01

    BACKGROUND: Membrane proteins comprise an important class of molecules whose study is largely frustrated by several intrinsic constraints, such as their hydrophobicity and added requirements for correct folding. Additionally, the complexity of the cellular mechanisms that are required to insert

  20. Solubilization of lipids and membrane proteins into nanodiscs : Mode of action and applications of SMA copolymers

    NARCIS (Netherlands)

    Scheidelaar, S.

    2016-01-01

    Cell membranes separate the inside and outside of cells. Membrane proteins in the cell membrane control the traffic of molecules across the membrane and are therefore targets for a lot of drugs: about 50 % of all approved drugs target a membrane protein! Unfortunately, scientists only know little

  1. Stratum, a Homolog of the Human GEF Mss4, Partnered with Rab8, Controls the Basal Restriction of Basement Membrane Proteins in Epithelial Cells

    Directory of Open Access Journals (Sweden)

    Olivier Devergne

    2017-02-01

    Full Text Available The basement membrane (BM, a sheet of extracellular matrix lining the basal side of epithelia, is essential for epithelial cell function and integrity, yet the mechanisms that control the basal restriction of BM proteins are poorly understood. In epithelial cells, a specialized pathway is dedicated to restrict the deposition of BM proteins basally. Here, we report the identification of a factor in this pathway, a homolog of the mammalian guanine nucleotide exchange factor (GEF Mss4, which we have named Stratum. The loss of Stratum leads to the missecretion of BM proteins at the apical side of the cells, forming aberrant layers in close contact with the plasma membrane. We found that Rab8GTPase acts downstream of Stratum in this process. Altogether, our results uncover the importance of this GEF/Rab complex in specifically coordinating the basal restriction of BM proteins, a critical process for the establishment and maintenance of epithelial cell polarity.

  2. Rhesus lymphocryptovirus latent membrane protein 2A activates β-catenin signaling and inhibits differentiation in epithelial cells

    International Nuclear Information System (INIS)

    Siler, Catherine A.; Raab-Traub, Nancy

    2008-01-01

    Rhesus lymphocryptovirus (LCV) is a γ-herpesvirus closely related to Epstein-Barr virus (EBV). The rhesus latent membrane protein 2A (LMP2A) is highly homologous to EBV LMP2A. EBV LMP2A activates the phosphatidylinositol 3-kinase (PI3K) and β-catenin signaling pathways in epithelial cells and affects differentiation. In the present study, the biochemical and biological properties of rhesus LMP2A in epithelial cells were investigated. The expression of rhesus LMP2A in epithelial cells induced Akt activation, GSK3β inactivation and accumulation of β-catenin in the cytoplasm and nucleus. The nuclear translocation, but not accumulation of β-catenin was dependent on Akt activation. Rhesus LMP2A also impaired epithelial cell differentiation; however, this process was not dependent upon Akt activation. A mutant rhesus LMP2A lacking six transmembrane domains functioned similarly to wild-type rhesus LMP2A indicating that the full number of transmembrane domains is not required for effects on β-catenin or cell differentiation. These results underscore the similarity of LCV to EBV and the suitability of the macaque as an animal model for studying EBV pathogenesis

  3. SPAK and OSR1 Sensitive Cell Membrane Protein Abundance and Activity of KCNQ1/E1 K+ Channels

    Directory of Open Access Journals (Sweden)

    Bernat Elvira

    2015-11-01

    Full Text Available Background/Aims: KCNQ1/E1 channels are expressed in diverse tissues and serve a variety of functions including endolymph secretion in the inner ear, cardiac repolarization, epithelial transport and cell volume regulation. Kinases involved in regulation of epithelial transport and cell volume include SPAK (SPS1-related proline/alanine-rich kinase and OSR1 (oxidative stress-responsive kinase 1, which are under control of WNK (with-no-K[Lys] kinases. The present study explored whether KCNQ1/E1 channels are regulated by SPAK and/or OSR1. Methods: cRNA encoding KCNQ1/E1 was injected into Xenopus oocytes with or without additional injection of cRNA encoding wild-type SPAK, constitutively active T233ESPAK, WNK insensitive T233ASPAK, catalytically inactive D212ASPAK, wild-type OSR1, constitutively active T185EOSR1, WNK insensitive T185AOSR1 and catalytically inactive D164AOSR1. Voltage gated K+ channel activity was quantified utilizing dual electrode voltage clamp and KCNQ1/E1 channel protein abundance in the cell membrane utilizing chemiluminescence of KCNQ1/E1 containing an extracellular Flag tag epitope (KCNQ1-Flag/E1. Results: KCNQ1/E1 activity and KCNQ1-Flag/E1 protein abundance were significantly enhanced by wild-type SPAK and T233ESPAK, but not by T233ASPAK and D212ASPAK. Similarly, KCNQ1/E1 activity and KCNQ1-Flag/E1 protein abundance were significantly increased by wild-type OSR1 and T185EOSR1, but not by T185AOSR1 and D164AOSR1. Conclusions: SPAK and OSR1 participate in the regulation of KCNQ1/E1 protein abundance and activity.

  4. SPAK and OSR1 Sensitive Cell Membrane Protein Abundance and Activity of KCNQ1/E1 K+ Channels.

    Science.gov (United States)

    Elvira, Bernat; Warsi, Jamshed; Fezai, Myriam; Munoz, Carlos; Lang, Florian

    2015-01-01

    KCNQ1/E1 channels are expressed in diverse tissues and serve a variety of functions including endolymph secretion in the inner ear, cardiac repolarization, epithelial transport and cell volume regulation. Kinases involved in regulation of epithelial transport and cell volume include SPAK (SPS1-related proline/alanine-rich kinase) and OSR1 (oxidative stress-responsive kinase 1), which are under control of WNK (with-no-K[Lys]) kinases. The present study explored whether KCNQ1/E1 channels are regulated by SPAK and/or OSR1. cRNA encoding KCNQ1/E1 was injected into Xenopus oocytes with or without additional injection of cRNA encoding wild-type SPAK, constitutively active T233ESPAK, WNK insensitive T233ASPAK, catalytically inactive D212ASPAK, wild-type OSR1, constitutively active T185EOSR1, WNK insensitive T185AOSR1 and catalytically inactive D164AOSR1. Voltage gated K+ channel activity was quantified utilizing dual electrode voltage clamp and KCNQ1/E1 channel protein abundance in the cell membrane utilizing chemiluminescence of KCNQ1/E1 containing an extracellular Flag tag epitope (KCNQ1-Flag/E1). KCNQ1/E1 activity and KCNQ1-Flag/E1 protein abundance were significantly enhanced by wild-type SPAK and T233ESPAK, but not by T233ASPAK and D212ASPAK. Similarly, KCNQ1/E1 activity and KCNQ1-Flag/E1 protein abundance were significantly increased by wild-type OSR1 and T185EOSR1, but not by T185AOSR1 and D164AOSR1. SPAK and OSR1 participate in the regulation of KCNQ1/E1 protein abundance and activity. © 2015 S. Karger AG, Basel.

  5. Fuel cell membrane humidification

    Science.gov (United States)

    Wilson, Mahlon S.

    1999-01-01

    A polymer electrolyte membrane fuel cell assembly has an anode side and a cathode side separated by the membrane and generating electrical current by electrochemical reactions between a fuel gas and an oxidant. The anode side comprises a hydrophobic gas diffusion backing contacting one side of the membrane and having hydrophilic areas therein for providing liquid water directly to the one side of the membrane through the hydrophilic areas of the gas diffusion backing. In a preferred embodiment, the hydrophilic areas of the gas diffusion backing are formed by sewing a hydrophilic thread through the backing. Liquid water is distributed over the gas diffusion backing in distribution channels that are separate from the fuel distribution channels.

  6. The effect of protein-protein and protein-membrane interactions on membrane fouling in ultrafiltration

    NARCIS (Netherlands)

    Huisman, I.H.; Prádanos, P.; Hernández, A.

    2000-01-01

    It was studied how protein-protein and protein-membrane interactions influence the filtration performance during the ultrafiltration of protein solutions over polymeric membranes. This was done by measuring flux, streaming potential, and protein transmission during filtration of bovine serum albumin

  7. Cell Membrane Coating Nanotechnology.

    Science.gov (United States)

    Fang, Ronnie H; Kroll, Ashley V; Gao, Weiwei; Zhang, Liangfang

    2018-03-27

    Nanoparticle-based therapeutic, prevention, and detection modalities have the potential to greatly impact how diseases are diagnosed and managed in the clinic. With the wide range of nanomaterials available, the rational design of nanocarriers on an application-specific basis has become increasingly commonplace. Here, a comprehensive overview is provided on an emerging platform: cell-membrane-coating nanotechnology. As a fundamental unit of biology, cells carry out a wide range of functions, including the remarkable ability to interface and interact with their surrounding environment. Instead of attempting to replicate such functions via synthetic techniques, researchers are now directly leveraging naturally derived cell membranes as a means of bestowing nanoparticles with enhanced biointerfacing capabilities. This top-down technique is facile, highly generalizable, and has the potential to greatly augment existing nanocarriers. Further, the introduction of a natural membrane substrate onto nanoparticles surfaces has enabled additional applications beyond those traditionally associated with nanomedicine. Despite its relative youth, there exists an impressive body of literature on cell membrane coating, which is covered here in detail. Overall, there is still significant room for development, as researchers continue to refine existing workflows while finding new and exciting applications that can take advantage of this developing technology. © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  8. Co-expression of foreign proteins tethered to HIV-1 envelope glycoprotein on the cell surface by introducing an intervening second membrane-spanning domain.

    Directory of Open Access Journals (Sweden)

    Hongyun Wang

    Full Text Available The envelope glycoprotein (Env of human immunodeficiency virus type I (HIV-1 mediates membrane fusion. To analyze the mechanism of HIV-1 Env-mediated membrane fusion, it is desirable to determine the expression level of Env on the cell surface. However, the quantification of Env by immunological staining is often hampered by the diversity of HIV-1 Env and limited availability of universal antibodies that recognize different Envs with equal efficiency. To overcome this problem, here we linked a tag protein called HaloTag at the C-terminus of HIV-1 Env. To relocate HaloTag to the cell surface, we introduced a second membrane-spanning domain (MSD between Env and HaloTag. The MSD of transmembrane protease serine 11D, a type II transmembrane protein, successfully relocated HaloTag to the cell surface. The surface level of Env can be estimated indirectly by staining HaloTag with a specific membrane-impermeable fluorescent ligand. This tagging did not compromise the fusogenicity of Env drastically. Furthermore, fusogenicity of Env was preserved even after the labeling with the ligands. We have also found that an additional foreign peptide or protein such as C34 or neutralizing single-chain variable fragment (scFv can be linked to the C-terminus of the HaloTag protein. Using these constructs, we were able to determine the required length of C34 and critical residues of neutralizing scFv for blocking membrane fusion, respectively.

  9. Variable-order fractional MSD function to describe the evolution of protein lateral diffusion ability in cell membranes

    Science.gov (United States)

    Yin, Deshun; Qu, Pengfei

    2018-02-01

    Protein lateral diffusion is considered anomalous in the plasma membrane. And this diffusion is related to membrane microstructure. In order to better describe the property of protein lateral diffusion and find out the inner relationship between protein lateral diffusion and membrane microstructure, this article applies variable-order fractional mean square displacement (f-MSD) function for characterizing the anomalous diffusion. It is found that the variable order can reflect the evolution of diffusion ability. The results of numerical simulation demonstrate variable-order f-MSD function can predict the tendency of anomalous diffusion during the process of confined diffusion. It is also noted that protein lateral diffusion ability during the processes of confined and hop diffusion can be split into three parts. In addition, the comparative analyses reveal that the variable order is related to the confinement-domain size and microstructure of compartment boundary too.

  10. Focus on Membrane Differentiation and Membrane Domains in the Prokaryotic Cell

    NARCIS (Netherlands)

    Boekema, Egbert J.; Scheffers, Dirk-Jan; van Bezouwen, Laura S.; Bolhuis, Henk; Folea, I. Mihaela

    2013-01-01

    A summary is presented of membrane differentiation in the prokaryotic cell, with an emphasis on the organization of proteins in the plasma/cell membrane. Many species belonging to the Eubacteria and Archaea have special membrane domains and/or membrane proliferation, which are vital for different

  11. Ligand binding to G protein-coupled receptors in tethered cell membranes

    DEFF Research Database (Denmark)

    Martinez, Karen L.; Meyer, Bruno H.; Hovius, Ruud

    2003-01-01

    G protein-coupled receptors (GPCRs) constitute a large class of seven transmembrane proteins, which bind selectively agonists or antagonists with important consequences for cellular signaling and function. Comprehension of the molecular details of ligand binding is important for the understanding...

  12. Engineering Lipid Bilayer Membranes for Protein Studies

    Science.gov (United States)

    Khan, Muhammad Shuja; Dosoky, Noura Sayed; Williams, John Dalton

    2013-01-01

    Lipid membranes regulate the flow of nutrients and communication signaling between cells and protect the sub-cellular structures. Recent attempts to fabricate artificial systems using nanostructures that mimic the physiological properties of natural lipid bilayer membranes (LBM) fused with transmembrane proteins have helped demonstrate the importance of temperature, pH, ionic strength, adsorption behavior, conformational reorientation and surface density in cellular membranes which all affect the incorporation of proteins on solid surfaces. Much of this work is performed on artificial templates made of polymer sponges or porous materials based on alumina, mica, and porous silicon (PSi) surfaces. For example, porous silicon materials have high biocompatibility, biodegradability, and photoluminescence, which allow them to be used both as a support structure for lipid bilayers or a template to measure the electrochemical functionality of living cells grown over the surface as in vivo. The variety of these media, coupled with the complex physiological conditions present in living systems, warrant a summary and prospectus detailing which artificial systems provide the most promise for different biological conditions. This study summarizes the use of electrochemical impedance spectroscopy (EIS) data on artificial biological membranes that are closely matched with previously published biological systems using both black lipid membrane and patch clamp techniques. PMID:24185908

  13. Pattern formation by curvature-inducing proteins on spherical membranes

    Science.gov (United States)

    Agudo-Canalejo, Jaime; Golestanian, Ramin

    2017-12-01

    Spatial organisation is a hallmark of all living cells, and recreating it in model systems is a necessary step in the creation of synthetic cells. It is therefore of both fundamental and practical interest to better understand the basic mechanisms underlying spatial organisation in cells. In this work, we use a continuum model of membrane and protein dynamics to study the behaviour of curvature-inducing proteins on membranes of spherical shape, such as living cells or lipid vesicles. We show that the interplay between curvature energy, entropic forces, and the geometric constraints on the membrane can result in the formation of patterns of highly-curved/protein-rich and weakly-curved/protein-poor domains on the membrane. The spontaneous formation of such patterns can be triggered either by an increase in the average density of curvature-inducing proteins, or by a relaxation of the geometric constraints on the membrane imposed by the membrane tension or by the tethering of the membrane to a rigid cell wall or cortex. These parameters can also be tuned to select the size and number of the protein-rich domains that arise upon pattern formation. The very general mechanism presented here could be related to protein self-organisation in many biological processes, ranging from (proto)cell division to the formation of membrane rafts.

  14. Latent membrane protein 1 deletion mutants accumulate in reed-sternberg cells of human immunodeficiency virus-related Hodgkin's lymphoma.

    Science.gov (United States)

    Guidoboni, Massimo; Ponzoni, Maurilio; Caggiari, Laura; Lettini, Antonia A; Vago, Luca; De Re, Valli; Gloghini, Annunziata; Zancai, Paola; Carbone, Antonino; Boiocchi, Mauro; Dolcetti, Riccardo

    2005-02-01

    The origin and biological significance of deletions at the 3' end of the Epstein-Barr virus (EBV)-encoded latent membrane protein 1 (LMP-1) gene are still controversial. We herein demonstrate that LMP-1 deletion mutants are highly associated with human immunodeficiency virus-related Hodgkin's lymphoma (HIV-HL) of Italian patients (29 of 31 cases; 93.5%), a phenomenon that is not due to a peculiar distribution of EBV strains in this area. In fact, although HIV-HL patients are infected by multiple EBV variants, we demonstrate that LMP-1 deletion mutants preferentially accumulate within neoplastic tissues. Subcloning and sequencing of the 3' LMP-1 ends of two HIV-HL genes in which both variants were present showed the presence of molecular signatures suggestive of a likely derivation of the LMP-1 deletion mutant from a nondeletion ancestor. This phenomenon likely occurs within tumor cells in vivo, as shown by the detection of both LMP-1 variants in single microdissected Reed-Sternberg cells, and may at least in part explain the high prevalence of LMP-1 deletions associated with HIV-HL.

  15. Peripheral myelin protein 22 alters membrane architecture

    Science.gov (United States)

    Mittendorf, Kathleen F.; Marinko, Justin T.; Hampton, Cheri M.; Ke, Zunlong; Hadziselimovic, Arina; Schlebach, Jonathan P.; Law, Cheryl L.; Li, Jun; Wright, Elizabeth R.; Sanders, Charles R.; Ohi, Melanie D.

    2017-01-01

    Peripheral myelin protein 22 (PMP22) is highly expressed in myelinating Schwann cells of the peripheral nervous system. PMP22 genetic alterations cause the most common forms of Charcot-Marie-Tooth disease (CMTD), which is characterized by severe dysmyelination in the peripheral nerves. However, the functions of PMP22 in Schwann cell membranes remain unclear. We demonstrate that reconstitution of purified PMP22 into lipid vesicles results in the formation of compressed and cylindrically wrapped protein-lipid vesicles that share common organizational traits with compact myelin of peripheral nerves in vivo. The formation of these myelin-like assemblies depends on the lipid-to-PMP22 ratio, as well as on the PMP22 extracellular loops. Formation of the myelin-like assemblies is disrupted by a CMTD-causing mutation. This study provides both a biochemical assay for PMP22 function and evidence that PMP22 directly contributes to membrane organization in compact myelin. PMID:28695207

  16. A role for protein kinase C in the regulation of membrane fluidity and Ca²(+) flux at the endoplasmic reticulum and plasma membranes of HEK293 and Jurkat cells.

    Science.gov (United States)

    Chen, Lihong; Meng, Qingli; Jing, Xian; Xu, Pingxiang; Luo, Dali

    2011-02-01

    Protein kinase C (PKC) plays a prominent role in the regulation of a variety of cellular functions, including Ca²(+) signalling. In HEK293 and Jurkat cells, the Ca²(+) release and Ca²(+) uptake stimulated by several different activators were attenuated by activation of PKC with phorbol myristate acetate (PMA) or 1-oleoyl-2-acetyl-sn-glycerol (OAG) and potentiated by PKC inhibition with Gö6983 or knockdown of PKCα or PKCβ using shRNA. Immunostaining and Western blotting analyses revealed that PKCα and PKCβII accumulated at the plasma membrane (PM) and that these isoforms, along with PKCβI, also translocated to the endoplasmic reticulum (ER) upon activation with PMA. Measurements of membrane fluidity showed that, like the cell membrane stabilizers bovine serum albumin (BSA) and ursodeoxycholate (UDCA), PMA and OAG significantly reduced the fluidity of both the PM and ER membranes; these effects were blocked in PKC-knockdown cells. Interestingly, both BSA and UDCA inhibited the Ca²(+) responses to agonists to the same extent as PMA, whereas Tween 20, which increases membrane fluidity, raised the internal Ca²(+) concentration. Thus, activation of PKC induces both translocation of PKC to the PM and ER membranes and downregulation of membrane fluidity, thereby negatively modulating Ca²(+) flux. Copyright © 2010 Elsevier Inc. All rights reserved.

  17. Identification and characterization of stable membrane protein complexes

    NARCIS (Netherlands)

    Spelbrink, R.E.J.

    2007-01-01

    Many membrane proteins exist as oligomers. Such oligomers play an important role in a broad variety of cellular processes such as ion transport, energy transduction, osmosensing and cell wall synthesis. We developed an electrophoresis-based method of identifying oligomeric membrane proteins that are

  18. Proton exchange membrane fuel cells

    CERN Document Server

    Qi, Zhigang

    2013-01-01

    Preface Proton Exchange Membrane Fuel CellsFuel CellsTypes of Fuel CellsAdvantages of Fuel CellsProton Exchange Membrane Fuel CellsMembraneCatalystCatalyst LayerGas Diffusion MediumMicroporous LayerMembrane Electrode AssemblyPlateSingle CellStackSystemCell Voltage Monitoring Module (CVM)Fuel Supply Module (FSM)Air Supply Module (ASM)Exhaust Management Module (EMM)Heat Management Module (HMM)Water Management Module (WMM)Internal Power Supply Module (IPM)Power Conditioning Module (PCM)Communications Module (COM)Controls Module (CM)SummaryThermodynamics and KineticsTheoretical EfficiencyVoltagePo

  19. Novel proteins, putative membrane transporters, and an integrated metabolic network are revealed by quantitative proteomic analysis of Arabidopsis cell culture peroxisomes.

    Science.gov (United States)

    Eubel, Holger; Meyer, Etienne H; Taylor, Nicolas L; Bussell, John D; O'Toole, Nicholas; Heazlewood, Joshua L; Castleden, Ian; Small, Ian D; Smith, Steven M; Millar, A Harvey

    2008-12-01

    Peroxisomes play key roles in energy metabolism, cell signaling, and plant development. A better understanding of these important functions will be achieved with a more complete definition of the peroxisome proteome. The isolation of peroxisomes and their separation from mitochondria and other major membrane systems have been significant challenges in the Arabidopsis (Arabidopsis thaliana) model system. In this study, we present new data on the Arabidopsis peroxisome proteome obtained using two new technical advances that have not previously been applied to studies of plant peroxisomes. First, we followed density gradient centrifugation with free-flow electrophoresis to improve the separation of peroxisomes from mitochondria. Second, we used quantitative proteomics to identify proteins enriched in the peroxisome fractions relative to mitochondrial fractions. We provide evidence for peroxisomal localization of 89 proteins, 36 of which have not previously been identified in other analyses of Arabidopsis peroxisomes. Chimeric green fluorescent protein constructs of 35 proteins have been used to confirm their localization in peroxisomes or to identify endoplasmic reticulum contaminants. The distribution of many of these peroxisomal proteins between soluble, membrane-associated, and integral membrane locations has also been determined. This core peroxisomal proteome from nonphotosynthetic cultured cells contains a proportion of proteins that cannot be predicted to be peroxisomal due to the lack of recognizable peroxisomal targeting sequence 1 (PTS1) or PTS2 signals. Proteins identified are likely to be components in peroxisome biogenesis, beta-oxidation for fatty acid degradation and hormone biosynthesis, photorespiration, and metabolite transport. A considerable number of the proteins found in peroxisomes have no known function, and potential roles of these proteins in peroxisomal metabolism are discussed. This is aided by a metabolic network analysis that reveals a

  20. Self-assembling peptide and protein nanodiscs for studies of membrane proteins

    DEFF Research Database (Denmark)

    Midtgaard, Søren Roi

    investigations of membrane proteins by traditional X-ray crystallography have proved a difficult challenge, and a surprisingly small amount of membrane proteins has been crystalized so far. This implies that development of lipoproteins as a platform for studying membrane proteins is much needed. In this thesis......Particles containing both lipids and proteins (so-called lipoproteins) are vital to study. They are selfassembling particles that, in the human body, are responsible for the transport of lipids and cholesterol. Due to the increasing problems of obesity and related illnesses in the world, obtaining...... for working with lipoprotein particles are their potential in the study membrane proteins. Membrane proteins are responsible for most of the transport in and out of cells and signaling between cells. As an example G-protein coupled receptors, a class of membrane proteins, are the third largest class...

  1. Molecular Characterization of a Novel Family of Trypanosoma cruzi Surface Membrane Proteins (TcSMP) Involved in Mammalian Host Cell Invasion.

    Science.gov (United States)

    Martins, Nadini Oliveira; Souza, Renata Torres de; Cordero, Esteban Mauricio; Maldonado, Danielle Cortez; Cortez, Cristian; Marini, Marjorie Mendes; Ferreira, Eden Ramalho; Bayer-Santos, Ethel; Almeida, Igor Correia de; Yoshida, Nobuko; Silveira, José Franco da

    2015-11-01

    The surface coat of Trypanosoma cruzi is predominantly composed of glycosylphosphatidylinositol-anchored proteins, which have been extensively characterized. However, very little is known about less abundant surface proteins and their role in host-parasite interactions. Here, we described a novel family of T. cruzi surface membrane proteins (TcSMP), which are conserved among different T. cruzi lineages and have orthologs in other Trypanosoma species. TcSMP genes are densely clustered within the genome, suggesting that they could have originated by tandem gene duplication. Several lines of evidence indicate that TcSMP is a membrane-spanning protein located at the cellular surface and is released into the extracellular milieu. TcSMP exhibited the key elements typical of surface proteins (N-terminal signal peptide or signal anchor) and a C-terminal hydrophobic sequence predicted to be a trans-membrane domain. Immunofluorescence of live parasites showed that anti-TcSMP antibodies clearly labeled the surface of all T. cruzi developmental forms. TcSMP peptides previously found in a membrane-enriched fraction were identified by proteomic analysis in membrane vesicles as well as in soluble forms in the T. cruzi secretome. TcSMP proteins were also located intracellularly likely associated with membrane-bound structures. We demonstrated that TcSMP proteins were capable of inhibiting metacyclic trypomastigote entry into host cells. TcSMP bound to mammalian cells and triggered Ca2+ signaling and lysosome exocytosis, events that are required for parasitophorous vacuole biogenesis. The effects of TcSMP were of lower magnitude compared to gp82, the major adhesion protein of metacyclic trypomastigotes, suggesting that TcSMP may play an auxiliary role in host cell invasion. We hypothesized that the productive interaction of T. cruzi with host cells that effectively results in internalization may depend on diverse adhesion molecules. In the metacyclic forms, the signaling induced by

  2. Structure and properties of cell membranes. Volume 3: Methodology and properties of membranes

    International Nuclear Information System (INIS)

    Benga, G.

    1985-01-01

    This book covers the topics: Quantum chemical approach to study the mechanisms of proton translocation across membranes through protein molecules; monomolecular films as biomembrane models; planar lipid bilayers in relation to biomembranes; relation of liposomes to cell membranes; reconstitution of membrane transport systems; structure-function relationships in cell membranes as revealed by X-ray techniques; structure-function relationships in cell membranes as revealed by spin labeling ESR; structure and dynamics of cell membranes as revealed by NMR techniques; the effect of dietary lipids on the composition and properties of biological membranes and index

  3. Protein nanocoatings on synthetic polymeric nanofibrous membranes designed as carriers for skin cells

    Czech Academy of Sciences Publication Activity Database

    Bačáková, Markéta; Pajorová, Julie; Stránská, D.; Hadraba, Daniel; Lopot, F.; Riedel, Tomáš; Brynda, Eduard; Žaloudková, Margit; Bačáková, Lucie

    2017-01-01

    Roč. 12, Feb (2017), s. 1143-1160 E-ISSN 1178-2013 R&D Projects: GA ČR(CZ) GBP108/12/G108 Institutional support: RVO:67985823 ; RVO:67985891 ; RVO:61389013 Keywords : skin-tissue engineering * nanocoating * nanofibers * skin cell s * fibrin * collagen Subject RIV: EI - Biotechnology ; Bionics; EI - Biotechnology ; Bionics (UMCH-V); EI - Biotechnology ; Bionics (USMH-B) OBOR OECD: Technologies involving the manipulation of cell s, tissues, organs or the whole organism (assisted reproduction); Biomaterials (as related to medical implants, devices, sensors) (UMCH-V); Health-related biotechnology (USMH-B) Impact factor: 4.300, year: 2016

  4. A framework for protein and membrane interactions

    Directory of Open Access Journals (Sweden)

    Giorgio Bacci

    2009-11-01

    Full Text Available We introduce the BioBeta Framework, a meta-model for both protein-level and membrane-level interactions of living cells. This formalism aims to provide a formal setting where to encode, compare and merge models at different abstraction levels; in particular, higher-level (e.g. membrane activities can be given a formal biological justification in terms of low-level (i.e., protein interactions. A BioBeta specification provides a protein signature together a set of protein reactions, in the spirit of the kappa-calculus. Moreover, the specification describes when a protein configuration triggers one of the only two membrane interaction allowed, that is "pinch" and "fuse". In this paper we define the syntax and semantics of BioBeta, analyse its properties, give it an interpretation as biobigraphical reactive systems, and discuss its expressivity by comparing with kappa-calculus and modelling significant examples. Notably, BioBeta has been designed after a bigraphical metamodel for the same purposes. Hence, each instance of the calculus corresponds to a bigraphical reactive system, and vice versa (almost. Therefore, we can inherith the rich theory of bigraphs, such as the automatic construction of labelled transition systems and behavioural congruences.

  5. Cell-free expression, purification, and membrane reconstitution for NMR studies of the nonstructural protein 4B from hepatitis C virus

    Energy Technology Data Exchange (ETDEWEB)

    Fogeron, Marie-Laure [Université de Lyon, Institut de Biologie et Chimie des Protéines, Bases Moléculaires et Structurales des Systèmes Infectieux, Labex Ecofect, UMR 5086 CNRS (France); Jirasko, Vlastimil; Penzel, Susanne [ETH Zurich, Physical Chemistry (Switzerland); Paul, David [Heidelberg University, Department of Infectious Diseases, Molecular Virology (Germany); Montserret, Roland; Danis, Clément; Lacabanne, Denis; Badillo, Aurélie [Université de Lyon, Institut de Biologie et Chimie des Protéines, Bases Moléculaires et Structurales des Systèmes Infectieux, Labex Ecofect, UMR 5086 CNRS (France); Gouttenoire, Jérôme; Moradpour, Darius [University of Lausanne, Division of Gastroenterology and Hepatology, Centre Hospitalier Universitaire Vaudois (Switzerland); Bartenschlager, Ralf [Heidelberg University, Department of Infectious Diseases, Molecular Virology (Germany); Penin, François [Université de Lyon, Institut de Biologie et Chimie des Protéines, Bases Moléculaires et Structurales des Systèmes Infectieux, Labex Ecofect, UMR 5086 CNRS (France); Meier, Beat H., E-mail: beme@ethz.ch [ETH Zurich, Physical Chemistry (Switzerland); and others

    2016-06-15

    We describe the expression of the hepatitis C virus nonstructural protein 4B (NS4B), which is an integral membrane protein, in a wheat germ cell-free system, the subsequent purification and characterization of NS4B and its insertion into proteoliposomes in amounts sufficient for multidimensional solid-state NMR spectroscopy. First spectra of the isotopically [{sup 2}H,{sup 13}C,{sup 15}N]-labeled protein are shown to yield narrow {sup 13}C resonance lines and a proper, predominantly α-helical fold. Clean residue-selective leucine, isoleucine and threonine-labeling is demonstrated. These results evidence the suitability of the wheat germ-produced integral membrane protein NS4B for solid-state NMR. Still, the proton linewidth under fast magic angle spinning is broader than expected for a perfect sample and possible causes are discussed.

  6. Neutrons describe ectoine effects on water H-bonding and hydration around a soluble protein and a cell membrane

    Science.gov (United States)

    Zaccai, Giuseppe; Bagyan, Irina; Combet, Jérôme; Cuello, Gabriel J.; Demé, Bruno; Fichou, Yann; Gallat, François-Xavier; Galvan Josa, Victor M.; von Gronau, Susanne; Haertlein, Michael; Martel, Anne; Moulin, Martine; Neumann, Markus; Weik, Martin; Oesterhelt, Dieter

    2016-08-01

    Understanding adaptation to extreme environments remains a challenge of high biotechnological potential for fundamental molecular biology. The cytosol of many microorganisms, isolated from saline environments, reversibly accumulates molar concentrations of the osmolyte ectoine to counterbalance fluctuating external salt concentrations. Although they have been studied extensively by thermodynamic and spectroscopic methods, direct experimental structural data have, so far, been lacking on ectoine-water-protein interactions. In this paper, in vivo deuterium labeling, small angle neutron scattering, neutron membrane diffraction and inelastic scattering are combined with neutron liquids diffraction to characterize the extreme ectoine-containing solvent and its effects on purple membrane of H. salinarum and E. coli maltose binding protein. The data reveal that ectoine is excluded from the hydration layer at the membrane surface and does not affect membrane molecular dynamics, and prove a previous hypothesis that ectoine is excluded from a monolayer of dense hydration water around the soluble protein. Neutron liquids diffraction to atomic resolution shows how ectoine enhances the remarkable properties of H-bonds in water—properties that are essential for the proper organization, stabilization and dynamics of biological structures.

  7. The ER membrane protein complex is a transmembrane domain insertase

    Science.gov (United States)

    Guna, Alina; Volkmar, Norbert; Christianson, John C.; Hegde, Ramanujan S.

    2018-01-01

    Insertion of proteins into membranes is an essential cellular process. The extensive biophysical and topological diversity of membrane proteins necessitates multiple insertion pathways that remain incompletely defined. Here, we found that known membrane insertion pathways fail to effectively engage tail-anchored membrane proteins with moderately hydrophobic transmembrane domains. These proteins are instead shielded in the cytosol by calmodulin. Dynamic release from calmodulin allowed sampling of the endoplasmic reticulum (ER), where the conserved ER membrane protein complex (EMC) was shown to be essential for efficient insertion in vitro and in cells. Purified EMC in synthetic liposomes catalyzed insertion of its substrates in a reconstituted system. Thus, EMC is a transmembrane domain insertase, a function that may explain its widely pleiotropic membrane-associated phenotypes across organisms. PMID:29242231

  8. Bilayer-thickness-mediated interactions between integral membrane proteins.

    Science.gov (United States)

    Kahraman, Osman; Koch, Peter D; Klug, William S; Haselwandter, Christoph A

    2016-04-01

    Hydrophobic thickness mismatch between integral membrane proteins and the surrounding lipid bilayer can produce lipid bilayer thickness deformations. Experiment and theory have shown that protein-induced lipid bilayer thickness deformations can yield energetically favorable bilayer-mediated interactions between integral membrane proteins, and large-scale organization of integral membrane proteins into protein clusters in cell membranes. Within the continuum elasticity theory of membranes, the energy cost of protein-induced bilayer thickness deformations can be captured by considering compression and expansion of the bilayer hydrophobic core, membrane tension, and bilayer bending, resulting in biharmonic equilibrium equations describing the shape of lipid bilayers for a given set of bilayer-protein boundary conditions. Here we develop a combined analytic and numerical methodology for the solution of the equilibrium elastic equations associated with protein-induced lipid bilayer deformations. Our methodology allows accurate prediction of thickness-mediated protein interactions for arbitrary protein symmetries at arbitrary protein separations and relative orientations. We provide exact analytic solutions for cylindrical integral membrane proteins with constant and varying hydrophobic thickness, and develop perturbative analytic solutions for noncylindrical protein shapes. We complement these analytic solutions, and assess their accuracy, by developing both finite element and finite difference numerical solution schemes. We provide error estimates of our numerical solution schemes and systematically assess their convergence properties. Taken together, the work presented here puts into place an analytic and numerical framework which allows calculation of bilayer-mediated elastic interactions between integral membrane proteins for the complicated protein shapes suggested by structural biology and at the small protein separations most relevant for the crowded membrane

  9. The cell-free integration of a polytopic mitochondrial membrane protein into liposomes occurs cotranslationally and in a lipid-dependent manner.

    Directory of Open Access Journals (Sweden)

    Ashley R Long

    Full Text Available The ADP/ATP Carrier (AAC is the most abundant transporter of the mitochondrial inner membrane. The central role that this transporter plays in cellular energy production highlights the importance of understanding its structure, function, and the basis of its pathologies. As a means of preparing proteoliposomes for the study of membrane proteins, several groups have explored the use of cell-free translation systems to facilitate membrane protein integration directly into preformed unilamellar vesicles without the use of surfactants. Using AAC as a model, we report for the first time the detergent-free reconstitution of a mitochondrial inner membrane protein into liposomes using a wheat germ-based in vitro translation system. Using a host of independent approaches, we demonstrate the efficient integration of AAC into vesicles with an inner membrane-mimetic lipid composition and, more importantly, that the integrated AAC is functionally active in transport. By adding liposomes at different stages of the translation reaction, we show that this direct integration is obligatorily cotranslational, and by synthesizing stable ribosome-bound nascent chain intermediates, we show that the nascent AAC polypeptide interacts with lipid vesicles while ribosome-bound. Finally, we show that the presence of the phospholipid cardiolipin in the liposomes specifically enhances AAC translation rate as well as the efficiency of vesicle association and integration. In light of these results, the possible mechanisms of liposome-assisted membrane protein integration during cell-free translation are discussed with respect to the mode of integration and the role of specific lipids.

  10. Proteins and Peptides in Biomimetic Polymeric Membranes

    DEFF Research Database (Denmark)

    Perez, Alfredo Gonzalez

    2013-01-01

    This chapter discusses recent advances and the main advantages of block copolymers for functional membrane protein reconstitution in biomimetic polymeric membranes. A rational approach to the reconstitution of membrane proteins in a functional form can be addressed by a more holistic view by using...

  11. Covisualization in living onion cells of putative integrin, putative spectrin, actin, putative intermediate filaments, and other proteins at the cell membrane and in an endomembrane sheath

    Science.gov (United States)

    Reuzeau, C.; Doolittle, K. W.; McNally, J. G.; Pickard, B. G.; Evans, M. L. (Principal Investigator)

    1997-01-01

    Covisualizations with wide-field computational optical-sectioning microscopy of living epidermal cells of the onion bulb scale have evidenced two major new cellular features. First, a sheath of cytoskeletal elements clads the endomembrane system. Similar elements clad the inner faces of punctate plasmalemmal sites interpreted as plasmalemmal control centers. One component of the endomembrane sheath and plasmalemmal control center cladding is anti-genicity-recognized by two injected antibodies against animal spectrin. Immunoblots of separated epidermal protein also showed bands recognized by these antibodies. Injected phalloidin identified F-actin with the same cellular distribution pattern, as did antibodies against intermediate-filament protein and other cytoskeletal elements known from animal cells. Injection of general protein stains demonstrated the abundance of endomembrane sheath protein. Second, the endomembrane system, like the plasmalemmal puncta, contains antigen recognized by an anti-beta 1 integrin injected into the cytoplasm. Previously, immunoblots of separated epidermal protein were shown to have a major band recognized both by this antibody prepared against a peptide representing the cytosolic region of beta 1 integrin and an antibody against the matrix region of beta 1 integrin. The latter antiboby also identified puncta at the external face of protoplasts. It is proposed that integrin and associated transmembrane proteins secure the endomembrane sheath and transmit signals between it and the lumen or matrix of the endoplasmic reticulum and organellar matrices. This function is comparable to that proposed for such transmembrane linkers in the plasmalemmal control centers, which also appear to bind cytoskeleton and a host of related molecules and transmit signals between them and the wall matrix. It is at the plasmalemmal control centers that the endoplasmic reticulum, a major component of the endomembrane system, attaches to the plasma membrane.

  12. Bacteriophage membrane protein P9 as a fusion partner for the efficient expression of membrane proteins in Escherichia coli.

    Science.gov (United States)

    Jung, Yuna; Jung, Hyeim; Lim, Dongbin

    2015-12-01

    Despite their important roles and economic values, studies of membrane proteins have been hampered by the difficulties associated with obtaining sufficient amounts of protein. Here, we report a novel membrane protein expression system that uses the major envelope protein (P9) of phage φ6 as an N-terminal fusion partner. Phage membrane protein P9 facilitated the synthesis of target proteins and their integration into the Escherichia coli cell membrane. This system was used to produce various multi-pass transmembrane proteins, including G-protein-coupled receptors, transporters, and ion channels of human origin. Green fluorescent protein fusion was used to confirm the correct folding of the expressed proteins. Of the 14 membrane proteins tested, eight were highly expressed, three were moderately expressed, and three were barely expressed in E. coli. Seven of the eight highly expressed proteins could be purified after extraction with the mild detergent lauryldimethylamine-oxide. Although a few proteins have previously been developed as fusion partners to augment membrane protein production, we believe that the major envelope protein P9 described here is better suited to the efficient expression of eukaryotic transmembrane proteins in E. coli. Copyright © 2015 Elsevier Inc. All rights reserved.

  13. Site-directed fluorescence labeling reveals a revised N-terminal membrane topology and functional periplasmic residues in the Escherichia coli cell division protein FtsK.

    Science.gov (United States)

    Berezuk, Alison M; Goodyear, Mara; Khursigara, Cezar M

    2014-08-22

    In Escherichia coli, FtsK is a large integral membrane protein that coordinates chromosome segregation and cell division. The N-terminal domain of FtsK (FtsKN) is essential for division, and the C terminus (FtsKC) is a well characterized DNA translocase. Although the function of FtsKN is unknown, it is suggested that FtsK acts as a checkpoint to ensure DNA is properly segregated before septation. This may occur through modulation of protein interactions between FtsKN and other division proteins in both the periplasm and cytoplasm; thus, a clear understanding of how FtsKN is positioned in the membrane is required to characterize these interactions. The membrane topology of FtsKN was initially determined using site-directed reporter fusions; however, questions regarding this topology persist. Here, we report a revised membrane topology generated by site-directed fluorescence labeling. The revised topology confirms the presence of four transmembrane segments and reveals a newly identified periplasmic loop between the third and fourth transmembrane domains. Within this loop, four residues were identified that, when mutated, resulted in the appearance of cellular voids. High resolution transmission electron microscopy of these voids showed asymmetric division of the cytoplasm in the absence of outer membrane invagination or visible cell wall ingrowth. This uncoupling reveals a novel role for FtsK in linking cell envelope septation events and yields further evidence for FtsK as a critical checkpoint of cell division. The revised topology of FtsKN also provides an important platform for future studies on essential interactions required for this process. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

  14. Artificial membranes for membrane protein purification, functionality and structure studies.

    Science.gov (United States)

    Parmar, Mayuriben J; Lousa, Carine De Marcos; Muench, Stephen P; Goldman, Adrian; Postis, Vincent L G

    2016-06-15

    Membrane proteins represent one of the most important targets for pharmaceutical companies. Unfortunately, technical limitations have long been a major hindrance in our understanding of the function and structure of such proteins. Recent years have seen the refinement of classical approaches and the emergence of new technologies that have resulted in a significant step forward in the field of membrane protein research. This review summarizes some of the current techniques used for studying membrane proteins, with overall advantages and drawbacks for each method. © 2016 The Author(s). published by Portland Press Limited on behalf of the Biochemical Society.

  15. Herpes Simplex Virus 1 Glycoprotein M and the Membrane-Associated Protein UL11 Are Required for Virus-Induced Cell Fusion and Efficient Virus Entry

    Science.gov (United States)

    Kim, In-Joong; Chouljenko, Vladimir N.; Walker, Jason D.

    2013-01-01

    Herpes simplex virus 1 (HSV-1) facilitates virus entry into cells and cell-to-cell spread by mediating fusion of the viral envelope with cellular membranes and fusion of adjacent cellular membranes. Although virus strains isolated from herpetic lesions cause limited cell fusion in cell culture, clinical herpetic lesions typically contain large syncytia, underscoring the importance of cell-to-cell fusion in virus spread in infected tissues. Certain mutations in glycoprotein B (gB), gK, UL20, and other viral genes drastically enhance virus-induced cell fusion in vitro and in vivo. Recent work has suggested that gB is the sole fusogenic glycoprotein, regulated by interactions with the viral glycoproteins gD, gH/gL, and gK, membrane protein UL20, and cellular receptors. Recombinant viruses were constructed to abolish either gM or UL11 expression in the presence of strong syncytial mutations in either gB or gK. Virus-induced cell fusion caused by deletion of the carboxyl-terminal 28 amino acids of gB or the dominant syncytial mutation in gK (Ala to Val at amino acid 40) was drastically reduced in the absence of gM. Similarly, syncytial mutations in either gB or gK did not cause cell fusion in the absence of UL11. Neither the gM nor UL11 gene deletion substantially affected gB, gC, gD, gE, and gH glycoprotein synthesis and expression on infected cell surfaces. Two-way immunoprecipitation experiments revealed that the membrane protein UL20, which is found as a protein complex with gK, interacted with gM while gM did not interact with other viral glycoproteins. Viruses produced in the absence of gM or UL11 entered into cells more slowly than their parental wild-type virus strain. Collectively, these results indicate that gM and UL11 are required for efficient membrane fusion events during virus entry and virus spread. PMID:23678175

  16. Kinetics and Thermodynamics of Membrane Protein Folding

    Directory of Open Access Journals (Sweden)

    Ernesto A. Roman

    2014-03-01

    Full Text Available Understanding protein folding has been one of the great challenges in biochemistry and molecular biophysics. Over the past 50 years, many thermodynamic and kinetic studies have been performed addressing the stability of globular proteins. In comparison, advances in the membrane protein folding field lag far behind. Although membrane proteins constitute about a third of the proteins encoded in known genomes, stability studies on membrane proteins have been impaired due to experimental limitations. Furthermore, no systematic experimental strategies are available for folding these biomolecules in vitro. Common denaturing agents such as chaotropes usually do not work on helical membrane proteins, and ionic detergents have been successful denaturants only in few cases. Refolding a membrane protein seems to be a craftsman work, which is relatively straightforward for transmembrane β-barrel proteins but challenging for α-helical membrane proteins. Additional complexities emerge in multidomain membrane proteins, data interpretation being one of the most critical. In this review, we will describe some recent efforts in understanding the folding mechanism of membrane proteins that have been reversibly refolded allowing both thermodynamic and kinetic analysis. This information will be discussed in the context of current paradigms in the protein folding field.

  17. Identification of Murine B-Cell and T-Cell Epitopes of Escherichia coli Outer Membrane Protein F with Synthetic Polypeptides

    Science.gov (United States)

    Williams, Kristina M.; Bigley, Elmer C.; Raybourne, Richard B.

    2000-01-01

    The major pore-forming outer membrane proteins (Omps) of gram-negative bacteria demonstrate numerous immunomodulating properties and are involved in the virulence of pathogenic strains. Because Escherichia coli OmpF is the best-characterized porin in terms of structural and functional characteristics, in vitro B-cell and T-cell responses to this porin in six different strains of mice were analyzed. Mice were immunized with purified OmpF trimers or overlapping synthetic polypeptides (20-mers) spanning the entire 340-amino-acid sequence of the OmpF monomer. T-cell proliferative responses and immunoglobulin G antibody responses to native OmpF and the peptide analogues were determined. For each strain, patterns of T-cell proliferation were similar regardless of whether native OmpF or synthetic peptides were inoculated, although all strains recognized one or more cryptic determinants. Mice exhibited several haplotype-specific responses, but genetically permissive epitopes were also identified. Four peptides (75-94, 265-284, 295-314, and 305-324) elicited strong T-cell proliferative responses from all strains of mice when mice were presensitized with native OmpF or a homologous peptide. In general, 10 or fewer peptides were recognized by sera from mice immunized with native OmpF or synthetic peptides, and most sera from peptide-immunized mice reacted poorly with the native protein. Four peptides spanning amino acids 45 to 64, 95 to 114, 115 to 134, and 275 to 294 were recognized by sera from all strains immunized with native OmpF but not by sera from peptide-immunized mice. Peptides 245-264 and 305-324 were universally recognized by sera from peptide-immunized mice, but these sera reacted weakly or were negative when tested against the native protein. Based on the pattern of cytokine secretion by proliferating T cells, immunization with native OmpF polarizes T helper cells toward development of a TH1 response. T-cell and B-cell responses have been investigated based on

  18. Export of a Toxoplasma gondii rhoptry neck protein complex at the host cell membrane to form the moving junction during invasion.

    Directory of Open Access Journals (Sweden)

    Sébastien Besteiro

    2009-02-01

    Full Text Available One of the most conserved features of the invasion process in Apicomplexa parasites is the formation of a moving junction (MJ between the apex of the parasite and the host cell membrane that moves along the parasite and serves as support to propel it inside the host cell. The MJ was, up to a recent period, completely unknown at the molecular level. Recently, proteins originated from two distinct post-Golgi specialised secretory organelles, the micronemes (for AMA1 and the neck of the rhoptries (for RON2/RON4/RON5 proteins, have been shown to form a complex. AMA1 and RON4 in particular, have been localised to the MJ during invasion. Using biochemical approaches, we have identified RON8 as an additional member of the complex. We also demonstrated that all RON proteins are present at the MJ during invasion. Using metabolic labelling and immunoprecipitation, we showed that RON2 and AMA1 were able to interact in the absence of the other members. We also discovered that all MJ proteins are subjected to proteolytic maturation during trafficking to their respective organelles and that they could associate as non-mature forms in vitro. Finally, whereas AMA1 has previously been shown to be inserted into the parasite membrane upon secretion, we demonstrated, using differential permeabilization and loading of RON-specific antibodies into the host cell, that the RON complex is targeted to the host cell membrane, where RON4/5/8 remain associated with the cytoplasmic face. Globally, these results point toward a model of MJ organization where the parasite would be secreting and inserting interacting components on either side of the MJ, both at the host and at its own plasma membranes.

  19. Isomeric Detergent Comparison for Membrane Protein Stability

    DEFF Research Database (Denmark)

    Cho, Kyung Ho; Hariharan, Parameswaran; Mortensen, Jonas S.

    2016-01-01

    Membrane proteins encapsulated by detergent micelles are widely used for structural study. Because of their amphipathic property, detergents have the ability to maintain protein solubility and stability in an aqueous medium. However, conventional detergents have serious limitations in their scope...... and utility, particularly for eukaryotic membrane proteins and membrane protein complexes. Thus, a number of new agents have been devised; some have made significant contributions to membrane protein structural studies. However, few detergent design principles are available. In this study, we prepared meta....../stability of the membrane proteins. We propose that interplay between the hydrophile–lipophile balance (HLB) and alkyl chain length is of central importance for high detergent efficacy. In addition, differences in inter-alkyl-chain distance between the isomers influence the ability of the detergents to stabilise membrane...

  20. Pepper pathogenesis-related protein 4c is a plasma membrane-localized cysteine protease inhibitor that is required for plant cell death and defense signaling.

    Science.gov (United States)

    Kim, Nak Hyun; Hwang, Byung Kook

    2015-01-01

    Xanthomonas campestris pv. vesicatoria (Xcv) type III effector AvrBsT triggers programmed cell death (PCD) and activates the hypersensitive response (HR) in plants. Here, we isolated and identified the plasma membrane localized pathogenesis-related (PR) protein 4c gene (CaPR4c) from pepper (Capsicum annuum) leaves undergoing AvrBsT-triggered HR cell death. CaPR4c encodes a protein with a signal peptide and a Barwin domain. Recombinant CaPR4c protein expressed in Escherichia coli exhibited cysteine protease-inhibitor activity and ribonuclease (RNase) activity. Subcellular localization analyses revealed that CaPR4c localized to the plasma membrane in plant cells. CaPR4c expression was rapidly and specifically induced by avirulent Xcv (avrBsT) infection. Transient expression of CaPR4c caused HR cell death in pepper leaves, which was accompanied by enhanced accumulation of H2 O2 and significant induction of some defense-response genes. Deletion of the signal peptide from CaPR4c abolished the induction of HR cell death, indicating a requirement for plasma membrane localization of CaPR4c for HR cell death. CaPR4c silencing in pepper disrupted both basal and AvrBsT-triggered resistance responses, and enabled Xcv proliferation in infected leaves. H2 O2 accumulation, cell-death induction, and defense-response gene expression were distinctly reduced in CaPR4c-silenced pepper. CaPR4c overexpression in transgenic Arabidopsis plants conferred greater resistance against infection by Pseudomonas syringae pv. tomato and Hyaloperonospora arabidopsidis. These results collectively suggest that CaPR4c plays an important role in plant cell death and defense signaling. © 2014 The Authors The Plant Journal © 2014 John Wiley & Sons Ltd.

  1. The Protein 4.1 family: hub proteins in animals for organizing membrane proteins.

    Science.gov (United States)

    Baines, Anthony J; Lu, Hui-Chun; Bennett, Pauline M

    2014-02-01

    Proteins of the 4.1 family are characteristic of eumetazoan organisms. Invertebrates contain single 4.1 genes and the Drosophila model suggests that 4.1 is essential for animal life. Vertebrates have four paralogues, known as 4.1R, 4.1N, 4.1G and 4.1B, which are additionally duplicated in the ray-finned fish. Protein 4.1R was the first to be discovered: it is a major mammalian erythrocyte cytoskeletal protein, essential to the mechanochemical properties of red cell membranes because it promotes the interaction between spectrin and actin in the membrane cytoskeleton. 4.1R also binds certain phospholipids and is required for the stable cell surface accumulation of a number of erythrocyte transmembrane proteins that span multiple functional classes; these include cell adhesion molecules, transporters and a chemokine receptor. The vertebrate 4.1 proteins are expressed in most tissues, and they are required for the correct cell surface accumulation of a very wide variety of membrane proteins including G-Protein coupled receptors, voltage-gated and ligand-gated channels, as well as the classes identified in erythrocytes. Indeed, such large numbers of protein interactions have been mapped for mammalian 4.1 proteins, most especially 4.1R, that it appears that they can act as hubs for membrane protein organization. The range of critical interactions of 4.1 proteins is reflected in disease relationships that include hereditary anaemias, tumour suppression, control of heartbeat and nervous system function. The 4.1 proteins are defined by their domain structure: apart from the spectrin/actin-binding domain they have FERM and FERM-adjacent domains and a unique C-terminal domain. Both the FERM and C-terminal domains can bind transmembrane proteins, thus they have the potential to be cross-linkers for membrane proteins. The activity of the FERM domain is subject to multiple modes of regulation via binding of regulatory ligands, phosphorylation of the FERM associated domain and

  2. Efficient Activation of Human T Cells of Both CD4 and CD8 Subsets by Urease-Deficient Recombinant Mycobacterium bovis BCG That Produced a Heat Shock Protein 70-M. tuberculosis-Derived Major Membrane Protein II Fusion Protein

    Science.gov (United States)

    Mukai, Tetsu; Tsukamoto, Yumiko; Maeda, Yumi; Tamura, Toshiki

    2014-01-01

    For the purpose of obtaining Mycobacterium bovis bacillus Calmette-Guérin (BCG) capable of activating human naive T cells, urease-deficient BCG expressing a fusion protein composed of Mycobacterium tuberculosis-derived major membrane protein II (MMP-II) and heat shock protein 70 (HSP70) of BCG (BCG-DHTM) was produced. BCG-DHTM secreted the HSP70-MMP-II fusion protein and effectively activated human monocyte-derived dendritic cells (DCs) by inducing phenotypic changes and enhanced cytokine production. BCG-DHTM-infected DCs activated naive T cells of both CD4 and naive CD8 subsets, in an antigen (Ag)-dependent manner. The T cell activation induced by BCG-DHTM was inhibited by the pretreatment of DCs with chloroquine. The naive CD8+ T cell activation was mediated by the transporter associated with antigen presentation (TAP) and the proteosome-dependent cytosolic cross-priming pathway. Memory CD8+ T cells and perforin-producing effector CD8+ T cells were efficiently produced from the naive T cell population by BCG-DHTM stimulation. Single primary infection with BCG-DHTM in C57BL/6 mice efficiently produced T cells responsive to in vitro secondary stimulation with HSP70, MMP-II, and M. tuberculosis-derived cytosolic protein and inhibited the multiplication of subsequently aerosol-challenged M. tuberculosis more efficiently than did vector control BCG. These results indicate that the introduction of MMP-II and HSP70 into urease-deficient BCG may be useful for improving BCG for control of tuberculosis. PMID:24152387

  3. Novel Tripod Amphiphiles for Membrane Protein Analysis

    DEFF Research Database (Denmark)

    Chae, Pil Seok; Kruse, Andrew C; Gotfryd, Kamil

    2013-01-01

    . The use of a detergent or other amphipathic agents is required to overcome the intrinsic incompatibility between the large lipophilic surfaces displayed by the membrane proteins in their native forms and the polar solvent molecules. Here, we introduce new tripod amphiphiles displaying favourable......Integral membrane proteins play central roles in controlling the flow of information and molecules across membranes. Our understanding of membrane protein structures and functions, however, is seriously limited, mainly due to difficulties in handling and analysing these proteins in aqueous solution...

  4. Cell invasion through basement membrane

    OpenAIRE

    Morrissey, Meghan A; Hagedorn, Elliott J; Sherwood, David R

    2013-01-01

    Cell invasion through basement membrane is an essential part of normal development and physiology, and occurs during the pathological progression of human inflammatory diseases and cancer. F-actin-rich membrane protrusions, called invadopodia, have been hypothesized to be the “drill bits” of invasive cells, mediating invasion through the dense, highly cross-linked basement membrane matrix. Though studied in vitro for over 30 y, invadopodia function in vivo has remained elusive. We have recent...

  5. Model cell membranes

    DEFF Research Database (Denmark)

    Günther-Pomorski, Thomas; Nylander, Tommy; Cardenas Gomez, Marite

    2014-01-01

    The high complexity of biological membranes has motivated the development and application of a wide range of model membrane systems to study biochemical and biophysical aspects of membranes in situ under well defined conditions. The aim is to provide fundamental understanding of processes control...

  6. Membrane phosphorylation and nerve cell function

    International Nuclear Information System (INIS)

    Baer, P.R.

    1982-01-01

    This thesis deals with the phosphorylation of membrane components. In part I a series of experiments is described using the hippocampal slice as a model system. In part II a different model system - cultured hybrid cells - is used to study protein and lipid phosphorylation, influenced by incubation with neuropeptides. In part III in vivo and in vitro studies are combined to study protein phosphorylation after neuroanatomical lesions. In a section of part II (Page 81-90) labelling experiments of the membrane inositol-phospholipids are described. 32 P-ATP was used to label phospholipids in intact hybrid cells, and short incubations were found to be the most favourable. (C.F.)

  7. Analysis of Protein-Membrane Interactions

    DEFF Research Database (Denmark)

    Kemmer, Gerdi Christine

    interactions between proteins and lipids. First, interactions of soluble proteins with membranes and specific lipids were studied, using two proteins: Annexin V and Tma1. The protein was first subjected to a lipid/protein overlay assay to identify candidate interaction partners in a fast and efficient way...

  8. POLYMER ELECTROLYTE MEMBRANE FUEL CELLS

    DEFF Research Database (Denmark)

    2001-01-01

    A method for preparing polybenzimidazole or polybenzimidazole blend membranes and fabricating gas diffusion electrodes and membrane-electrode assemblies is provided for a high temperature polymer electrolyte membrane fuel cell. Blend polymer electrolyte membranes based on PBI and various thermopl......A method for preparing polybenzimidazole or polybenzimidazole blend membranes and fabricating gas diffusion electrodes and membrane-electrode assemblies is provided for a high temperature polymer electrolyte membrane fuel cell. Blend polymer electrolyte membranes based on PBI and various...... thermoplastic polymers for high temperature polymer electrolyte fuel cells have also been developed. Miscible blends are used for solution casting of polymer membranes (solid electrolytes). High conductivity and enhanced mechanical strength were obtained for the blend polymer solid electrolytes...... electrolyte membrane by hot-press. The fuel cell can operate at temperatures up to at least 200 °C with hydrogen-rich fuel containing high ratios of carbon monoxide such as 3 vol% carbon monoxide or more, compared to the carbon monoxide tolerance of 10-20 ppm level for Nafion$m(3)-based polymer electrolyte...

  9. Phenotypic effects of membrane protein overexpression in Saccharomyces cerevisiae

    Science.gov (United States)

    Melén, Karin; Blomberg, Anders; von Heijne, Gunnar

    2006-07-01

    Large-scale protein overexpression phenotype screens provide an important complement to the more common gene knockout screens. Here, we have targeted the so far poorly understood Saccharomyces cerevisiae membrane proteome and report growth phenotypes for a strain collection overexpressing 600 C-terminally tagged integral membrane proteins grown both under normal and three different stress conditions. Although overexpression of most membrane proteins reduce the growth rate in synthetic defined medium, we identify a large number of proteins that, when overexpressed, confer specific resistance to various stress conditions. Our data suggest that regulation of glycosylphosphatidylinositol anchor biosynthesis and the Na+/K+ homeostasis system constitute major downstream targets of the yeast PKA/RAS pathway and point to a possible connection between the early secretory pathway and the cells' response to oxidative stress. We also have quantified the expression levels for >550 membrane proteins, facilitating the choice of well expressing proteins for future functional and structural studies. caffeine | paraquat | salt tolerance | yeast

  10. A unifying mechanism accounts for sensing of membrane curvature by BAR domains, amphipathic helices and membrane-anchored proteins

    DEFF Research Database (Denmark)

    Bhatia, Vikram Kjøller; Hatzakis, Nikos; Stamou, Dimitrios

    2010-01-01

    itself. We thus anticipate that membrane curvature will promote the redistribution of proteins that are anchored in membranes through any type of hydrophobic moiety, a thesis that broadens tremendously the implications of membrane curvature for protein sorting, trafficking and signaling in cell biology....

  11. Latent Membrane Protein 1 Deletion Mutants Accumulate in Reed-Sternberg Cells of Human Immunodeficiency Virus-Related Hodgkin's Lymphoma

    OpenAIRE

    Guidoboni, Massimo; Ponzoni, Maurilio; Caggiari, Laura; Lettini, Antonia A.; Vago, Luca; De Re, Valli; Gloghini, Annunziata; Zancai, Paola; Carbone, Antonino; Boiocchi, Mauro; Dolcetti, Riccardo

    2005-01-01

    The origin and biological significance of deletions at the 3′ end of the Epstein-Barr virus (EBV)-encoded latent membrane protein 1 (LMP-1) gene are still controversial. We herein demonstrate that LMP-1 deletion mutants are highly associated with human immunodeficiency virus-related Hodgkin's lymphoma (HIV-HL) of Italian patients (29 of 31 cases; 93.5%), a phenomenon that is not due to a peculiar distribution of EBV strains in this area. In fact, although HIV-HL patients are infected by multi...

  12. Outer membrane protein antigens of Moraxella bovis.

    Science.gov (United States)

    Ostle, A G; Rosenbusch, R F

    1986-07-01

    Outer membranes were isolated from bovine isolates and type strains of Moraxella bovis, M phenylpyruvica, M lacunata, and M ovis by sodium N lauroyl sarcosinate extraction and differential centrifugation. Analysis of outer membranes from these organisms by sodium dodecyl sulfate-polyacrylamide-gel electrophoresis revealed that all M bovis isolates shared a common polypeptide pattern that was readily distinguishable from other Moraxella spp. Nine major outer membrane protein bands were identified by sodium dodecyl sulfate-polyacrylamide-gel electrophoresis analysis of M bovis. Immunoblotting of protein antigens of M bovis revealed several outer membrane proteins that seemed to be common antigens of all M bovis isolates.

  13. Investigation of free fatty acid associated recombinant membrane receptor protein expression in HEK293 cells using Raman spectroscopy, calcium imaging, and atomic force microscopy.

    Science.gov (United States)

    Lin, Juqiang; Xu, Han; Wu, Yangzhe; Tang, Mingjie; McEwen, Gerald D; Liu, Pin; Hansen, Dane R; Gilbertson, Timothy A; Zhou, Anhong

    2013-02-05

    G-protein-coupled receptor 120 (GPR120) is a previously orphaned G-protein-coupled receptor that apparently functions as a sensor for dietary fat in the gustatory and digestive systems. In this study, a cDNA sequence encoding a doxycycline (Dox)-inducible mature peptide of GPR120 was inserted into an expression vector and transfected in HEK293 cells. We measured Raman spectra of single HEK293 cells as well as GPR120-expressing HEK293-GPR120 cells at a 48 h period following the additions of Dox at several concentrations. We found that the spectral intensity of HEK293-GPR120 cells is dependent upon the dose of Dox, which correlates with the accumulation of GPR120 protein in the cells. However, the amount of the fatty acid activated changes in intracellular calcium (Ca(2+)) as measured by ratiometric calcium imaging was not correlated with Dox concentration. Principal components analysis (PCA) of Raman spectra reveals that the spectra from different treatments of HEK293-GPR120 cells form distinct, completely separated clusters with the receiver operating characteristic (ROC) area of 1, while those spectra for the HEK293 cells form small overlap clusters with the ROC area of 0.836. It was also found that expression of GPR120 altered the physiochemical and biomechanical properties of the parental cell membrane surface, which was quantitated by atomic force microscopy (AFM). These findings demonstrate that the combination of Raman spectroscopy, calcium imaging, and AFM may provide new tools in noninvasive and quantitative monitoring of membrane receptor expression induced alterations in the biophysical and signaling properties of single living cells.

  14. APPL proteins FRET at the BAR: direct observation of APPL1 and APPL2 BAR domain-mediated interactions on cell membranes using FRET microscopy.

    Directory of Open Access Journals (Sweden)

    Heidi J Chial

    2010-08-01

    Full Text Available Human APPL1 and APPL2 are homologous RAB5 effectors whose binding partners include a diverse set of transmembrane receptors, signaling proteins, and phosphoinositides. APPL proteins associate dynamically with endosomal membranes and are proposed to function in endosome-mediated signaling pathways linking the cell surface to the cell nucleus. APPL proteins contain an N-terminal Bin/Amphiphysin/Rvs (BAR domain, a central pleckstrin homology (PH domain, and a C-terminal phosphotyrosine binding (PTB domain. Previous structural and biochemical studies have shown that the APPL BAR domains mediate homotypic and heterotypic APPL-APPL interactions and that the APPL1 BAR domain forms crescent-shaped dimers. Although previous studies have shown that APPL minimal BAR domains associate with curved cell membranes, direct interaction between APPL BAR domains on cell membranes in vivo has not been reported.Herein, we used a laser-scanning confocal microscope equipped with a spectral detector to carry out fluorescence resonance energy transfer (FRET experiments with cyan fluorescent protein/yellow fluorescent protein (CFP/YFP FRET donor/acceptor pairs to examine interactions between APPL minimal BAR domains at the subcellular level. This comprehensive approach enabled us to evaluate FRET levels in a single cell using three methods: sensitized emission, standard acceptor photobleaching, and sequential acceptor photobleaching. We also analyzed emission spectra to address an outstanding controversy regarding the use of CFP donor/YFP acceptor pairs in FRET acceptor photobleaching experiments, based on reports that photobleaching of YFP converts it into a CFP-like species.All three methods consistently showed significant FRET between APPL minimal BAR domain FRET pairs, indicating that they interact directly in a homotypic (i.e., APPL1-APPL1 and APPL2-APPL2 and heterotypic (i.e., APPL1-APPL2 manner on curved cell membranes. Furthermore, the results of our experiments

  15. Membrane Proteins Are Dramatically Less Conserved than Water-Soluble Proteins across the Tree of Life.

    Science.gov (United States)

    Sojo, Victor; Dessimoz, Christophe; Pomiankowski, Andrew; Lane, Nick

    2016-11-01

    Membrane proteins are crucial in transport, signaling, bioenergetics, catalysis, and as drug targets. Here, we show that membrane proteins have dramatically fewer detectable orthologs than water-soluble proteins, less than half in most species analyzed. This sparse distribution could reflect rapid divergence or gene loss. We find that both mechanisms operate. First, membrane proteins evolve faster than water-soluble proteins, particularly in their exterior-facing portions. Second, we demonstrate that predicted ancestral membrane proteins are preferentially lost compared with water-soluble proteins in closely related species of archaea and bacteria. These patterns are consistent across the whole tree of life, and in each of the three domains of archaea, bacteria, and eukaryotes. Our findings point to a fundamental evolutionary principle: membrane proteins evolve faster due to stronger adaptive selection in changing environments, whereas cytosolic proteins are under more stringent purifying selection in the homeostatic interior of the cell. This effect should be strongest in prokaryotes, weaker in unicellular eukaryotes (with intracellular membranes), and weakest in multicellular eukaryotes (with extracellular homeostasis). We demonstrate that this is indeed the case. Similarly, we show that extracellular water-soluble proteins exhibit an even stronger pattern of low homology than membrane proteins. These striking differences in conservation of membrane proteins versus water-soluble proteins have important implications for evolution and medicine. © The Author 2016. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.

  16. Lipid Directed Intrinsic Membrane Protein Segregation

    DEFF Research Database (Denmark)

    Hansen, Jesper S.; Thompson, James R.; Helix Nielsen, Claus

    2013-01-01

    We demonstrate a new approach for direct reconstitution of membrane proteins during giant vesicle formation. We show that it is straightforward to create a tissue-like giant vesicle film swelled with membrane protein using aquaporin SoPIP2;1 as an illustration. These vesicles can also be easily h...

  17. Outer membrane proteins of pathogenic spirochetes

    OpenAIRE

    Cullen, Paul A.; Haake, David A.; Adler, Ben

    2004-01-01

    Pathogenic spirochetes are the causative agents of several important diseases including syphilis, Lyme disease, leptospirosis, swine dysentery, periodontal disease and some forms of relapsing fever. Spirochetal bacteria possess two membranes and the proteins present in the outer membrane are at the site of interaction with host tissue and the immune system. This review describes the current knowledge in the field of spirochetal outer membrane protein (OMP) biology. What is known concerning bi...

  18. GPR30, the non-classical membrane G protein related estrogen receptor, is overexpressed in human seminoma and promotes seminoma cell proliferation.

    Directory of Open Access Journals (Sweden)

    Nicolas Chevalier

    Full Text Available BACKGROUND: Testicular germ cell tumours are the most frequent cancer of young men with an increasing incidence all over the world. Pathogenesis and reasons of this increase remain unknown but epidemiological and clinical data have suggested that fetal exposure to environmental endocrine disruptors (EEDs with estrogenic effects, could participate to testicular germ cell carcinogenesis. However, these EEDs (like bisphenol A are often weak ligands for classical nuclear estrogen receptors. Several research groups recently showed that the non classical membrane G-protein coupled estrogen receptor (GPER/GPR30 mediates the effects of estrogens and several xenoestrogens through rapid non genomic activation of signal transduction pathways in various human estrogen dependent cancer cells (breast, ovary, endometrium. The aim of this study was to demonstrate that GPER was overexpressed in testicular tumours and was able to trigger JKT-1 seminoma cell proliferation. RESULTS: We report here for the first time a complete morphological and functional characterization of GPER in normal and malignant human testicular germ cells. In normal adult human testes, GPER was expressed by somatic (Sertoli cells and germ cells (spermatogonia and spermatocytes. GPER was exclusively overexpressed in seminomas, the most frequent testicular germ cell cancer, localized at the cell membrane and triggered a proliferative effect on JKT-1 cells in vitro, which was completely abolished by G15 (a GPER selective antagonist and by siRNA invalidation. CONCLUSION: These results demonstrate that GPER is expressed by human normal adult testicular germ cells, specifically overexpressed in seminoma tumours and able to trigger seminoma cell proliferation in vitro. It should therefore be considered rather than classical ERs when xeno-estrogens or other endocrine disruptors are assessed in testicular germ cell cancers. It may also represent a prognosis marker and/or a therapeutic target for

  19. GPR30, the Non-Classical Membrane G Protein Related Estrogen Receptor, Is Overexpressed in Human Seminoma and Promotes Seminoma Cell Proliferation

    Science.gov (United States)

    Chevalier, Nicolas; Vega, Aurélie; Bouskine, Adil; Siddeek, Bénazir; Michiels, Jean-François; Chevallier, Daniel; Fénichel, Patrick

    2012-01-01

    Background Testicular germ cell tumours are the most frequent cancer of young men with an increasing incidence all over the world. Pathogenesis and reasons of this increase remain unknown but epidemiological and clinical data have suggested that fetal exposure to environmental endocrine disruptors (EEDs) with estrogenic effects, could participate to testicular germ cell carcinogenesis. However, these EEDs (like bisphenol A) are often weak ligands for classical nuclear estrogen receptors. Several research groups recently showed that the non classical membrane G-protein coupled estrogen receptor (GPER/GPR30) mediates the effects of estrogens and several xenoestrogens through rapid non genomic activation of signal transduction pathways in various human estrogen dependent cancer cells (breast, ovary, endometrium). The aim of this study was to demonstrate that GPER was overexpressed in testicular tumours and was able to trigger JKT-1 seminoma cell proliferation. Results We report here for the first time a complete morphological and functional characterization of GPER in normal and malignant human testicular germ cells. In normal adult human testes, GPER was expressed by somatic (Sertoli cells) and germ cells (spermatogonia and spermatocytes). GPER was exclusively overexpressed in seminomas, the most frequent testicular germ cell cancer, localized at the cell membrane and triggered a proliferative effect on JKT-1 cells in vitro, which was completely abolished by G15 (a GPER selective antagonist) and by siRNA invalidation. Conclusion These results demonstrate that GPER is expressed by human normal adult testicular germ cells, specifically overexpressed in seminoma tumours and able to trigger seminoma cell proliferation in vitro. It should therefore be considered rather than classical ERs when xeno-estrogens or other endocrine disruptors are assessed in testicular germ cell cancers. It may also represent a prognosis marker and/or a therapeutic target for seminomas. PMID

  20. Fast and efficient protein purification using membrane adsorber systems.

    Science.gov (United States)

    Suck, Kirstin; Walter, Johanna; Menzel, Frauke; Tappe, Alexander; Kasper, Cornelia; Naumann, Claudia; Zeidler, Robert; Scheper, Thomas

    2006-02-10

    The purification of proteins from complex cell culture samples is an essential step in proteomic research. Traditional chromatographic methods often require several steps resulting in time consuming and costly procedures. In contrast, protein purification via membrane adsorbers offers the advantage of fast and gentle but still effective isolation. In this work, we present a new method for purification of proteins from crude cell extracts via membrane adsorber based devices. This isolation procedure utilises the membranes favourable pore structure allowing high flow rates without causing high back pressure. Therefore, shear stress to fragile structures is avoided. In addition, mass transfer takes place through convection rather than diffusion, thus allowing very rapid separation processes. Based on this membrane adsorber technology the separation of two model proteins, human serum albumin (HSA) and immungluboline G (IgG) is shown. The isolation of human growth hormone (hGH) from chinese hamster ovary (CHO) cell culture supernatant was performed using a cation exchange membrane. The isolation of the enzyme penicillin acylase from the crude Escherichia coli supernatant was achieved using an anion exchange spin column within one step at a considerable purity. In summary, the membrane adsorber devices have proven to be suitable tools for the purification of proteins from different complex cell culture samples.

  1. Cell envelope of Bordetella pertussis: immunological and biochemical analyses and characterization of a major outer membrane porin protein

    International Nuclear Information System (INIS)

    Armstrong, S.K.

    1986-01-01

    Surface molecules of Bordetella pertussis which may be important in metabolism, pathogenesis, and immunity to whooping cough were examined using cell fractionation and 125 I cell surface labeling. Antigenic envelope proteins were examined by immunofluorescence microscopy and Western blotting procedures using monoclonal antibodies and convalescent sera. A surface protein with a high M/sub r/, missing in a mutant lacking the filamentous hemagglutinin, was identified in virulent Bordetella pertussis but was absent in virulent B. pertussis strains. At least three envelope proteins were found only in virulent B. pertussis strains and were absent or diminished in avirulent and most phenotypically modulated strains. Transposon-induced mutants unable to produce hemolysin, dermonecrotic toxin, pertussis toxin, and filamentous hemagglutinin also lacked these three envelope proteins, confirming that virulence-associated envelope proteins were genetically regulated with other virulence-associated traits. Two dimensional gel electrophoresis revealed at least five heat modifiable proteins which migrated as higher or lower M/sub r/ moieties if solubilized at 25 0 C instead of 100 0 C

  2. A haemolytic syndrome associated with the complete absence of red cell membrane protein 4.2 in two Tunisian siblings.

    Science.gov (United States)

    Ghanem, A; Pothier, B; Marechal, J; Ducluzeau, M T; Morle, L; Alloisio, N; Feo, C; Ben Abdeladhim, A; Fattoum, S; Delaunay, J

    1990-07-01

    We report on the complete absence of protein 4.2 in two Tunisian siblings. The propositus presented with a haemolytic anaemia that evolved in an intermittent fashion until she was cured by splenectomy. Her red cells had a normal morphology, as well as normal deformability upon osmotic gradient ektacytometry. SDS-polyacrylamide gel electrophoresis failed to reveal any protein 4.2. Using anti-protein 4.2 polyclonal antibodies. Western blots were also unable to detect protein 4.2. Preparation of inside out vesicles resulted in no detectable loss of ankyrin. The propositus's sister presented with a haemolytic anaemia but had not undergone splenectomy; she showed the same biochemical features. The two cases presented of missing protein 4.2 are the first ones to be described outside the Japanese population. Considered as homozygotes for some defect that must alter the protein 4.2 gene itself, they exemplify a unique syndrome pertaining neither to elliptocytosis nor to spherocytosis, at least not closely. The parents, who are first cousins and whom we regarded as heterozygotes, were clinically and morphologically normal; they had a normal content of protein 4.2. Therefore, the 4.2 (-) haemolytic anaemia appears as entirely recessive.

  3. Kinetics of B Cell responses to Plasmodium falciparum erythrocyte membrane protein 1 in Ghanaian women naturally exposed to malaria parasites

    DEFF Research Database (Denmark)

    Ampomah, Paulina; Stevenson, Liz; Ofori, Michael F

    2014-01-01

    acquisition of clinical protection takes years to develop, but it probably involves a range of immune-evasive parasite features, not least of which are PfEMP1 polymorphism and clonal variation. Parasite-induced subversion of immunological memory and expansion of "atypical" memory B cells may also contribute......Naturally acquired protective immunity to Plasmodium falciparum malaria takes years to develop. It relies mainly on Abs, particularly IgG specific for Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) proteins on the infected erythrocyte surface. It is only partially understood why...... confirmed earlier reports of high atypical memory B cell frequencies among residents of P. falciparum-endemic areas, and indicated an additional effect of pregnancy. Our study provides new knowledge regarding immunity to P. falciparum malaria and underpins efforts to develop PfEMP1-based vaccines against...

  4. Performance of a novel sieving matrix of poly(vinyl alcohol)/acrylamide copolymer in electrophoretic separations of high molecular weight proteins from red cell membrane.

    Science.gov (United States)

    Matte, Alessandro; Sola, Laura; Chiari, Marcella; Tomelleri, Carlo; Consonni, Roberto; Turrini, Franco; Franceschi, Lucia De

    2014-04-01

    The analysis of high molecular weight (HMW) proteins from complex mixtures is still a challenge in proteomics. This work introduces a novel hydrogel obtained by the copolymerization of an allyl-PVA derivative with acrylamide and bisacrylamide and applies this matrix to the electrophoretic separation of HMW proteins. By inducing gelation of polyacrylamide in the presence of variable amounts of allyl-PVA, it is possible to control and vary the average gel porosity. This gel is easy to produce and handle and offers the advantage of being highly mechanically resistant and macroporous. The new matrix was tested in mono-dimensional separations of complex protein mixtures extracted from red cell membranes with different detergents. The improved performance of this macroporous matrix allowed to identify new proteins by MS and immunoblot analysis using specific antibodies. In particular, the resolution of proteins ranging in size between 97 and 279 kDa was greatly improved here compared to standard polyacrylamide gels, suggesting that this matrix can be a useful tool in routine analysis of HMW proteins in cell biology. © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  5. Apoptosis induced by lipid-associated membrane proteins from Mycoplasma hyopneumoniae in a porcine lung epithelial cell line with the involvement of caspase 3 and the MAPK pathway.

    Science.gov (United States)

    Ni, B; Bai, F F; Wei, Y; Liu, M J; Feng, Z X; Xiong, Q Y; Hua, L Z; Shao, G Q

    2015-09-25

    Lipid-associated membrane proteins (LAMPs) are important in the pathogenicity of the Mycoplasma genus of bacteria. We investigated whether Mycoplasma hyopneumoniae LAMPs have pathogenic potential by inducing apoptosis in a St. Jude porcine lung epithelial cell line (SJPL). LAMPs from a pathogenic strain of M. hyopneumoniae (strain 232) were used in the research. Our investigation made use of diamidino-phenylindole (DAPI) and acridine orange/ethidium bromide (AO/EB) staining, terminal dexynucleotidyl transferase (TdT)-mediated dUTP nick end labeling (TUNEL) analysis, and Annexin-V-propidium iodide staining. After LAMP treatment for 24 h, typical changes were induced, chromosomes were concentrated, apoptotic bodies were observed, the 3'-OH groups of cleaved genomes were exposed, and the percentage of apoptotic cells reached 36.5 ± 11.66%. Caspase 3 and caspase 8 were activated and cytochrome c (cyt c) was released from the mitochondria into the cytoplasm; poly ADP ribose polymerase (PARP) was digested into two fragments; p38 mitogen-activated protein kinase (MAPK) was phosphorylated; and the expression of pro-apoptosis protein Bax increased while the anti-apoptosis protein Bcl-2 decreased. LAMPs also stimulated SJPL cells to produce nitric oxide (NO) and superoxide. This study demonstrated that LAMPs from M. hyopneumoniae can induce apoptosis in SJPL cells through the activation of caspase 3, caspase 8, cyt c, Bax, and p38 MAPK, thereby contributing to our understanding of the pathogenesis of M. hyopneumoniae, which should improve the treatment of M. hyopneumoniae infections.

  6. Hybrid membranes for fuel cells

    Science.gov (United States)

    Bochkareva, S. S.; Shashkina, S. S.

    2018-01-01

    Fuel cells are a very efficient, reliable, durable, and environmentally friendly energy source. Membranes for fuel cells were developed based on nitrogen-containing high-molecular compounds and organic–inorganic composites. Their electrical conductivities were measured. The influence of a silicon block of composites on the proton exchange properties of membranes was proved.The comparative characterization of the studied materials was performed.

  7. A Cysteine-Rich Protein Kinase Associates with a Membrane Immune Complex and the Cysteine Residues Are Required for Cell Death.

    Science.gov (United States)

    Yadeta, Koste A; Elmore, James M; Creer, Athena Y; Feng, Baomin; Franco, Jessica Y; Rufian, Jose Sebastian; He, Ping; Phinney, Brett; Coaker, Gitta

    2017-01-01

    Membrane-localized proteins perceive and respond to biotic and abiotic stresses. We performed quantitative proteomics on plasma membrane-enriched samples from Arabidopsis (Arabidopsis thaliana) treated with bacterial flagellin. We identified multiple receptor-like protein kinases changing in abundance, including cysteine (Cys)-rich receptor-like kinases (CRKs) that are up-regulated upon the perception of flagellin. CRKs possess extracellular Cys-rich domains and constitute a gene family consisting of 46 members in Arabidopsis. The single transfer DNA insertion lines CRK28 and CRK29, two CRKs induced in response to flagellin perception, did not exhibit robust alterations in immune responses. In contrast, silencing of multiple bacterial flagellin-induced CRKs resulted in enhanced susceptibility to pathogenic Pseudomonas syringae, indicating functional redundancy in this large gene family. Enhanced expression of CRK28 in Arabidopsis increased disease resistance to P. syringae Expression of CRK28 in Nicotiana benthamiana induced cell death, which required intact extracellular Cys residues and a conserved kinase active site. CRK28-mediated cell death required the common receptor-like protein kinase coreceptor BAK1. CRK28 associated with BAK1 as well as the activated FLAGELLIN-SENSING2 (FLS2) immune receptor complex. CRK28 self-associated as well as associated with the closely related CRK29. These data support a model where Arabidopsis CRKs are synthesized upon pathogen perception, associate with the FLS2 complex, and coordinately act to enhance plant immune responses. © 2017 American Society of Plant Biologists. All Rights Reserved.

  8. Quantitative proteomics of fractionated membrane and lumen exosome proteins from isogenic metastatic and non-metastatic bladder cancer cells reveal differential expression of EMT factors

    DEFF Research Database (Denmark)

    Jeppesen, Dennis Kjølhede; Nawrocki, Arkadiusz; Jensen, Steffen Grann

    2014-01-01

    T24 without metastatic capacity and its two isogenic derivate cell lines SLT4 and FL3, which form metastases in the lungs and liver of mice, respectively. Cultivation in CLAD1000 bioreactors rather than conventional culture flasks resulted in a 13-16-fold increased exosome yield and facilitated......, with changes in cellular mRNA expression. Our proteomic approach may help identification of proteins in the membrane and lumen of exosomes potentially involved in the metastatic process. This article is protected by copyright. All rights reserved....

  9. Cell invasion through basement membrane

    Science.gov (United States)

    Morrissey, Meghan A; Hagedorn, Elliott J; Sherwood, David R

    2013-01-01

    Cell invasion through basement membrane is an essential part of normal development and physiology, and occurs during the pathological progression of human inflammatory diseases and cancer. F-actin-rich membrane protrusions, called invadopodia, have been hypothesized to be the “drill bits” of invasive cells, mediating invasion through the dense, highly cross-linked basement membrane matrix. Though studied in vitro for over 30 y, invadopodia function in vivo has remained elusive. We have recently discovered that invadopodia breach basement membrane during anchor cell invasion in C. elegans, a genetically and visually tractable in vivo invasion event. Further, we found that the netrin receptor DCC localizes to the initial site of basement membrane breach and directs invasion through a single gap in the matrix. In this commentary, we examine how the dynamics and structure of AC-invadopodia compare with in vitro invadopodia and how the netrin receptor guides invasion through a single basement membrane breach. We end with a discussion of our surprising result that the anchor cell pushes the basement membrane aside, instead of completely dissolving it through proteolysis, and provide some ideas for how proteases and physical displacement may work together to ensure efficient and robust invasion. PMID:24778942

  10. Phytochemicals perturb membranes and promiscuously alter protein function.

    Science.gov (United States)

    Ingólfsson, Helgi I; Thakur, Pratima; Herold, Karl F; Hobart, E Ashley; Ramsey, Nicole B; Periole, Xavier; de Jong, Djurre H; Zwama, Martijn; Yilmaz, Duygu; Hall, Katherine; Maretzky, Thorsten; Hemmings, Hugh C; Blobel, Carl; Marrink, Siewert J; Koçer, Armağan; Sack, Jon T; Andersen, Olaf S

    2014-08-15

    A wide variety of phytochemicals are consumed for their perceived health benefits. Many of these phytochemicals have been found to alter numerous cell functions, but the mechanisms underlying their biological activity tend to be poorly understood. Phenolic phytochemicals are particularly promiscuous modifiers of membrane protein function, suggesting that some of their actions may be due to a common, membrane bilayer-mediated mechanism. To test whether bilayer perturbation may underlie this diversity of actions, we examined five bioactive phenols reported to have medicinal value: capsaicin from chili peppers, curcumin from turmeric, EGCG from green tea, genistein from soybeans, and resveratrol from grapes. We find that each of these widely consumed phytochemicals alters lipid bilayer properties and the function of diverse membrane proteins. Molecular dynamics simulations show that these phytochemicals modify bilayer properties by localizing to the bilayer/solution interface. Bilayer-modifying propensity was verified using a gramicidin-based assay, and indiscriminate modulation of membrane protein function was demonstrated using four proteins: membrane-anchored metalloproteases, mechanosensitive ion channels, and voltage-dependent potassium and sodium channels. Each protein exhibited similar responses to multiple phytochemicals, consistent with a common, bilayer-mediated mechanism. Our results suggest that many effects of amphiphilic phytochemicals are due to cell membrane perturbations, rather than specific protein binding.

  11. Inhibition of latent membrane protein 1 impairs the growth and tumorigenesis of latency II Epstein-Barr virus-transformed T cells.

    Science.gov (United States)

    Ndour, Papa Alioune; Brocqueville, Guillaume; Ouk, Tan-Sothéa; Goormachtigh, Gautier; Morales, Olivier; Mougel, Alexandra; Bertout, Julie; Melnyk, Oleg; Fafeur, Véronique; Feuillard, Jean; Coll, Jean; Adriaenssens, Eric

    2012-04-01

    Epstein-Barr virus (EBV) is a common human herpesvirus. Infection with EBV is associated with several human malignancies in which the virus expresses a set of latent proteins, among which is latent membrane protein 1 (LMP1). LMP1 is able to transform numerous cell types and is considered the main oncogenic protein of EBV. The mechanism of action is based on mimicry of activated members of the tumor necrosis factor (TNF) receptor superfamily, through the ability of LMP1 to bind similar adapters and to activate signaling pathways. We previously generated two unique models: a monocytic cell line and a lymphocytic (NC5) cell line immortalized by EBV that expresses the type II latency program. Here we generated LMP1 dominant negative forms (DNs), based on fusion between green fluorescent protein (GFP) and transformation effector site 1 (TES1) or TES2 of LMP1. Then we generated cell lines conditionally expressing these DNs. These DNs inhibit NF-κB and Akt pathways, resulting in the impairment of survival processes and increased apoptosis in these cell lines. This proapoptotic effect is due to reduced interaction of LMP1 with specific adapters and the recruitment of these adapters to DNs, which enable the generation of an apoptotic complex involving TRADD, FADD, and caspase 8. Similar results were obtained with cell lines displaying a latency III program in which LMP1-DNs decrease cell viability. Finally, we prove that synthetic peptides display similar inhibitory effects in EBV-infected cells. DNs derived from LMP1 could be used to develop therapeutic approaches for malignant diseases associated with EBV.

  12. Immediate-Early Protein ME53 Forms Foci and Colocalizes with GP64 and the Major Capsid Protein VP39 at the Cell Membranes of Autographa californica Multiple Nucleopolyhedrovirus-Infected Cells ▿ †

    Science.gov (United States)

    de Jong, Jondavid; Theilmann, David A.; Arif, Basil M.; Krell, Peter J.

    2011-01-01

    me53 is an immediate-early/late gene found in all lepidopteran baculoviruses sequenced to date. Deletion of me53 results in a greater-than-1,000-fold reduction in budded-virus production in tissue culture (J. de Jong, B. M. Arif, D. A. Theilmann, and P. J. Krell, J. Virol. 83:7440-7448, 2009). We investigated the localization of ME53 using an ME53 construct fused to green fluorescent protein (GFP). ME53:GFP adopted a primarily cytoplasmic distribution at early times postinfection and a primarily nuclear distribution at late times postinfection. Additionally, at late times ME53:GFP formed distinct foci at the cell periphery. These foci colocalized with the major envelope fusion protein GP64 and frequently with VP39 capsid protein, suggesting that these cell membrane regions may represent viral budding sites. Deletion of vp39 did not influence the distribution of ME53:GFP; however, deletion of gp64 abolished ME53:GFP foci at the cell periphery, implying an association between ME53 and GP64. Despite the association of ME53 and GP64, ME53 fractionated with the nucleocapsid only after budded-virus fractionation. Together these findings suggest that ME53 may be providing a scaffold that bridges the viral envelope and nucleocapsid. PMID:21775466

  13. Contribution of aquaporin 9 and multidrug resistance-associated protein 2 to differential sensitivity to arsenite between primary cultured chorion and amnion cells prepared from human fetal membranes

    International Nuclear Information System (INIS)

    Yoshino, Yuta; Yuan, Bo; Kaise, Toshikazu; Takeichi, Makoto; Tanaka, Sachiko; Hirano, Toshihiko; Kroetz, Deanna L.; Toyoda, Hiroo

    2011-01-01

    Arsenic trioxide (arsenite, As III ) has shown a remarkable clinical efficacy, whereas its side effects are still a serious concern. Therefore, it is critical to understand the effects of As III on human-derived normal cells for revealing the mechanisms underlying these side effects. We examined the effects of As III on primary cultured chorion (C) and amnion (A) cells prepared from human fetal membranes. A significant dose-dependent As III -mediated cytotoxicity was observed in the C-cells accompanied with an increase of lactate dehydrogenase (LDH) release. Higher concentrations of As III were required for the A-cells to show cytotoxicity and LDH release, suggesting that the C-cells were more sensitive to As III than the A-cells. The expression levels of aquaporin 9 (AQP9) were approximately 2 times higher in the C-cells than those in the A-cells. Both intracellular arsenic accumulation and its cytotoxicity in the C-cells were significantly abrogated by sorbitol, a competitive AQP9 inhibitor, in a dose-dependent manner. The protein expression levels of multidrug resistance-associated protein (MRP) 2 were downregulated by As III in the C-cells, but not in the A-cells. No significant differences in the expression levels of MRP1 were observed between C- and A-cells. The protein expression of P-glycoprotein (P-gp) was hardly detected in both cells, although a detectable amount of its mRNA was observed. Cyclosporine A, a broad-spectrum inhibitor for ABC transporters, and MK571, a MRP inhibitor, but not PGP-4008, a P-gp specific inhibitor, potently sensitized both cells to As III -mediated cytotoxicity. These results suggest that AQP9 and MRP2 are involved in controlling arsenic accumulation in these normal cells, which then contribute to differential sensitivity to As III cytotoxicity between these cells. -- Highlights: ► Examination of effect of As III on primary cultured chorion (C) and amnion (A) cells. ► Dose-dependent As III -mediated cytotoxicity in C-cells

  14. Contribution of aquaporin 9 and multidrug resistance-associated protein 2 to differential sensitivity to arsenite between primary cultured chorion and amnion cells prepared from human fetal membranes

    Energy Technology Data Exchange (ETDEWEB)

    Yoshino, Yuta [Department of Clinical Molecular Genetics, School of Pharmacy, Tokyo University of Pharmacy and Life Sciences, 1432-1 Horinouchi, Hachioji, Tokyo 192-0392 (Japan); Yuan, Bo, E-mail: yuanbo@toyaku.ac.jp [Department of Clinical Molecular Genetics, School of Pharmacy, Tokyo University of Pharmacy and Life Sciences, 1432-1 Horinouchi, Hachioji, Tokyo 192-0392 (Japan); Department of Bioengineering and Therapeutic Sciences, University of California San Francisco, 1550 4th St, RH584E Box 2911 San Francisco, CA 94158-2911 (United States); Kaise, Toshikazu [Laboratory of Environmental Chemodynamics, School of Life Sciences, Tokyo University of Pharmacy and Life Sciences, 1432-1 Horinouchi, Hachioji, Tokyo 192-0392 (Japan); Takeichi, Makoto [Yoneyama Maternity Hospital, 2-12 Shin-machi, Hachioji, Tokyo 192-0065 (Japan); Tanaka, Sachiko; Hirano, Toshihiko [Department of Clinical Pharmacology, School of Pharmacy, Tokyo University of Pharmacy and Life Sciences, 1432-1 Horinouchi, Hachioji, Tokyo 192-0392 (Japan); Kroetz, Deanna L. [Department of Bioengineering and Therapeutic Sciences, University of California San Francisco, 1550 4th St, RH584E Box 2911 San Francisco, CA 94158-2911 (United States); Toyoda, Hiroo [Department of Clinical Molecular Genetics, School of Pharmacy, Tokyo University of Pharmacy and Life Sciences, 1432-1 Horinouchi, Hachioji, Tokyo 192-0392 (Japan)

    2011-12-15

    Arsenic trioxide (arsenite, As{sup III}) has shown a remarkable clinical efficacy, whereas its side effects are still a serious concern. Therefore, it is critical to understand the effects of As{sup III} on human-derived normal cells for revealing the mechanisms underlying these side effects. We examined the effects of As{sup III} on primary cultured chorion (C) and amnion (A) cells prepared from human fetal membranes. A significant dose-dependent As{sup III}-mediated cytotoxicity was observed in the C-cells accompanied with an increase of lactate dehydrogenase (LDH) release. Higher concentrations of As{sup III} were required for the A-cells to show cytotoxicity and LDH release, suggesting that the C-cells were more sensitive to As{sup III} than the A-cells. The expression levels of aquaporin 9 (AQP9) were approximately 2 times higher in the C-cells than those in the A-cells. Both intracellular arsenic accumulation and its cytotoxicity in the C-cells were significantly abrogated by sorbitol, a competitive AQP9 inhibitor, in a dose-dependent manner. The protein expression levels of multidrug resistance-associated protein (MRP) 2 were downregulated by As{sup III} in the C-cells, but not in the A-cells. No significant differences in the expression levels of MRP1 were observed between C- and A-cells. The protein expression of P-glycoprotein (P-gp) was hardly detected in both cells, although a detectable amount of its mRNA was observed. Cyclosporine A, a broad-spectrum inhibitor for ABC transporters, and MK571, a MRP inhibitor, but not PGP-4008, a P-gp specific inhibitor, potently sensitized both cells to As{sup III}-mediated cytotoxicity. These results suggest that AQP9 and MRP2 are involved in controlling arsenic accumulation in these normal cells, which then contribute to differential sensitivity to As{sup III} cytotoxicity between these cells. -- Highlights: Black-Right-Pointing-Pointer Examination of effect of As{sup III} on primary cultured chorion (C) and amnion

  15. The impact of physiological crowding on the diffusivity of membrane bound proteins.

    Science.gov (United States)

    Houser, Justin R; Busch, David J; Bell, David R; Li, Brian; Ren, Pengyu; Stachowiak, Jeanne C

    2016-02-21

    Diffusion of transmembrane and peripheral membrane-bound proteins within the crowded cellular membrane environment is essential to diverse biological processes including cellular signaling, endocytosis, and motility. Nonetheless we presently lack a detailed understanding of the influence of physiological levels of crowding on membrane protein diffusion. Utilizing quantitative in vitro measurements, here we demonstrate that the diffusivities of membrane bound proteins follow a single linearly decreasing trend with increasing membrane coverage by proteins. This trend holds for homogenous protein populations across a range of protein sizes and for heterogeneous mixtures of proteins of different sizes, such that protein diffusivity is controlled by the total coverage of the surrounding membrane. These results demonstrate that steric exclusion within the crowded membrane environment can fundamentally limit the diffusive rate of proteins, regardless of their size. In cells this "speed limit" could be modulated by changes in local membrane coverage, providing a mechanism for tuning the rate of molecular interaction and assembly.

  16. The structure and function of the urokinase receptor, a membrane protein governing plasminogen activation on the cell surface

    DEFF Research Database (Denmark)

    Behrendt, N; Rønne, E; Danø, K

    1995-01-01

    PA receptor, uPAR, is a cell-surface protein which plays an important role in the localization and regulation of these processes. In the present article a number of established conclusions concerning the structure and function of uPAR are presented, and in addition various models are discussed which might...... domain is directly involved in the molecular contact with uPA. The receptor binds uPA as well as its proenzyme, pro-uPA, in such a manner that the activation cascade can occur directly on the cell surface. Furthermore, the activation rates are enhanced relative to the situation in solution, probably due...... between uPAR and uPA. The growing knowledge on the structure and function of uPAR which is a result of protein chemical analyses, functional studies and analyses of other, interacting components, should help to obtain a better understanding of the regulation of extracellular proteolysis. In conjunction...

  17. Compact halo-ligand-conjugated quantum dots for multicolored single-molecule imaging of overcrowding GPCR proteins on cell membranes.

    Science.gov (United States)

    Komatsuzaki, Akihito; Ohyanagi, Tatsuya; Tsukasaki, Yoshikazu; Miyanaga, Yukihiro; Ueda, Masahiro; Jin, Takashi

    2015-03-25

    To detect single molecules within the optical diffraction limit (< ca. 200 nm), a multicolored imaging technique is developed using Halo-ligand conjugated quantum dots (Halo-QDs; <6 nm in diameter). Using three types of Halo-QDs, multicolored single-molecule fluorescence imaging of GPCR proteins in Dictyostelium cells is achieved. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  18. Protein and DNA technologies for functional expression of membrane-associated cytochromes P450 in bacterial cell factories

    DEFF Research Database (Denmark)

    Vazquez Albacete, Dario

    The heavy dependence and massive consumption of fossil fuels by humans is changing our environment very rapidly. Some of the side effects of industrial activity include the pollution of the natural resources we rely on, and the reduction of biodiversity. Some chemicals found in nature exhibit great....... In most of biosynthetic pathways leading to these chemicals the cytochrome P450 enzyme family (P450s) is responsible for their final functionalization. However, the membrane-bound nature of P450s, makes their expression in microbial hosts a challenge. In order to meet the global demand for these natural......450 engineering guidelines and serves as platform to improve performance of microbial cells, thereby boosting recombinant production of complex plant P450-derived biochemicals. The knowledge generated, could guide future reconstruction of functional plant metabolic pathways leading to high valuable...

  19. Loss of lysosomal membrane protein NCU-G1 in mice results in spontaneous liver fibrosis with accumulation of lipofuscin and iron in Kupffer cells

    Directory of Open Access Journals (Sweden)

    Xiang Y. Kong

    2014-03-01

    Full Text Available Human kidney predominant protein, NCU-G1, is a highly conserved protein with an unknown biological function. Initially described as a nuclear protein, it was later shown to be a bona fide lysosomal integral membrane protein. To gain insight into the physiological function of NCU-G1, mice with no detectable expression of this gene were created using a gene-trap strategy, and Ncu-g1gt/gt mice were successfully characterized. Lysosomal disorders are mainly caused by lack of or malfunctioning of proteins in the endosomal-lysosomal pathway. The clinical symptoms vary, but often include liver dysfunction. Persistent liver damage activates fibrogenesis and, if unremedied, eventually leads to liver fibrosis/cirrhosis and death. We demonstrate that the disruption of Ncu-g1 results in spontaneous liver fibrosis in mice as the predominant phenotype. Evidence for an increased rate of hepatic cell death, oxidative stress and active fibrogenesis were detected in Ncu-g1gt/gt liver. In addition to collagen deposition, microscopic examination of liver sections revealed accumulation of autofluorescent lipofuscin and iron in Ncu-g1gt/gt Kupffer cells. Because only a few transgenic mouse models have been identified with chronic liver injury and spontaneous liver fibrosis development, we propose that the Ncu-g1gt/gt mouse could be a valuable new tool in the development of novel treatments for the attenuation of fibrosis due to chronic liver damage.

  20. Revolutionizing membrane protein overexpression in bacteria

    NARCIS (Netherlands)

    Schlegel, Susan; Klepsch, Mirjam; Gialama, Dimitra; Wickstrom, David; Slotboom, Dirk Jan; de Gier, Jan-Willem; Wickström, David

    The bacterium Escherichia coli is the most widely used expression host for overexpression trials of membrane proteins. Usually, different strains, culture conditions and expression regimes are screened for to identify the optimal overexpression strategy. However, yields are often not satisfactory,

  1. Protein receptor-independent plasma membrane remodeling by HAMLET

    DEFF Research Database (Denmark)

    Nadeem, Aftab; Sanborn, Jeremy; Gettel, Douglas L.

    2015-01-01

    A central tenet of signal transduction in eukaryotic cells is that extra-cellular ligands activate specific cell surface receptors, which orchestrate downstream responses. This "protein-centric" view is increasingly challenged by evidence for the involvement of specialized membrane domains in sig...

  2. Monocrotaline pyrrole-induced megalocytosis of lung and breast epithelial cells: Disruption of plasma membrane and Golgi dynamics and an enhanced unfolded protein response

    International Nuclear Information System (INIS)

    Mukhopadhyay, Somshuvra; Shah, Mehul; Patel, Kirit; Sehgal, Pravin B.

    2006-01-01

    The pyrrolizidine alkaloid monocrotaline (MCT) initiates pulmonary hypertension by inducing a 'megalocytosis' phenotype in target pulmonary arterial endothelial, smooth muscle and Type II alveolar epithelial cells. In cultured endothelial cells, a single exposure to the pyrrolic derivative of monocrotaline (MCTP) results in large cells with enlarged endoplasmic reticulum (ER) and Golgi and increased vacuoles. However, these cells fail to enter mitosis. Largely based upon data from endothelial cells, we proposed earlier that a disruption of the trafficking and mitosis-sensor functions of the Golgi (the 'Golgi blockade' hypothesis) may represent the subcellular mechanism leading to MCTP-induced megalocytosis. In the present study, we investigated the applicability of the Golgi blockade hypothesis to epithelial cells. MCTP induced marked megalocytosis in cultures of lung A549 and breast MCF-7 cells. This was associated with a change in the distribution of the cis-Golgi scaffolding protein GM130 from a discrete juxtanuclear localization to a circumnuclear distribution consistent with an anterograde block of GM130 trafficking to/through the Golgi. There was also a loss of plasma membrane caveolin-1 and E-cadherin, cortical actin together with a circumnuclear accumulation of clathrin heavy chain (CHC) and α-tubulin. Flotation analyses revealed losses/alterations in the association of caveolin-1, E-cadherin and CHC with raft microdomains. Moreover, megalocytosis was accompanied by an enhanced unfolded protein response (UPR) as evidenced by nuclear translocation of Ire1α and glucose regulated protein 58 (GRP58/ER-60/ERp57) and a circumnuclear accumulation of PERK kinase and protein disulfide isomerase (PDI). These data further support the hypothesis that an MCTP-induced Golgi blockade and enhanced UPR may represent the subcellular mechanism leading to enlargement of ER and Golgi and subsequent megalocytosis

  3. Roles of membrane trafficking in plant cell wall dynamics

    Directory of Open Access Journals (Sweden)

    Kazuo eEbine

    2015-10-01

    Full Text Available The cell wall is one of the characteristic components of plant cells. The cell wall composition differs among cell types and is modified in response to various environmental conditions. To properly generate and modify the cell wall, many proteins are transported to the plasma membrane or extracellular space through membrane trafficking, which is one of the key protein transport mechanisms in eukaryotic cells. Given the diverse composition and functions of the cell wall in plants, the transport of the cell wall components and proteins that are involved in cell wall-related events could be specialized for each cell type, i.e., the machinery for cell wall biogenesis, modification, and maintenance could be transported via different trafficking pathways. In this review, we summarize the recent progress in the current understanding of the roles and mechanisms of membrane trafficking in plant cells and focus on the biogenesis and regulation of the cell wall.

  4. Membrane Shape Instability Induced by Protein Crowding.

    Science.gov (United States)

    Chen, Zhiming; Atefi, Ehsan; Baumgart, Tobias

    2016-11-01

    Peripheral proteins can bend membranes through several different mechanisms, including scaffolding, wedging, oligomerization, and crowding. The crowding effect in particular has received considerable attention recently, in part because it is a colligative mechanism-implying that it could, in principle, be explored by any peripheral protein. Here we sought to clarify to what extent this mechanism is exploited by endocytic accessory proteins. We quantitatively investigate membrane curvature generation by means of a GUV shape stability assay. We found that the amount of crowding required to induce membrane curvature is correlated with membrane tension. Importantly, we also revealed that at the same membrane tension, the crowding mechanism requires far higher protein coverage to induce curvature changes compared to those observed for the endophilin BAR domain, serving here as an example of an endocytic accessory protein. Our results are important for the design of membrane-targeted biosensors as well as the understanding of mechanisms of biological membrane shaping. Copyright © 2016 Biophysical Society. Published by Elsevier Inc. All rights reserved.

  5. Current strategies for protein production and purification enabling membrane protein structural biology.

    Science.gov (United States)

    Pandey, Aditya; Shin, Kyungsoo; Patterson, Robin E; Liu, Xiang-Qin; Rainey, Jan K

    2016-12-01

    Membrane proteins are still heavily under-represented in the protein data bank (PDB), owing to multiple bottlenecks. The typical low abundance of membrane proteins in their natural hosts makes it necessary to overexpress these proteins either in heterologous systems or through in vitro translation/cell-free expression. Heterologous expression of proteins, in turn, leads to multiple obstacles, owing to the unpredictability of compatibility of the target protein for expression in a given host. The highly hydrophobic and (or) amphipathic nature of membrane proteins also leads to challenges in producing a homogeneous, stable, and pure sample for structural studies. Circumventing these hurdles has become possible through the introduction of novel protein production protocols; efficient protein isolation and sample preparation methods; and, improvement in hardware and software for structural characterization. Combined, these advances have made the past 10-15 years very exciting and eventful for the field of membrane protein structural biology, with an exponential growth in the number of solved membrane protein structures. In this review, we focus on both the advances and diversity of protein production and purification methods that have allowed this growth in structural knowledge of membrane proteins through X-ray crystallography, nuclear magnetic resonance (NMR) spectroscopy, and cryo-electron microscopy (cryo-EM).

  6. Mutation of the dengue virus type 2 envelope protein heparan sulfate binding sites or the domain III lateral ridge blocks replication in Vero cells prior to membrane fusion

    International Nuclear Information System (INIS)

    Roehrig, John T.; Butrapet, Siritorn; Liss, Nathan M.; Bennett, Susan L.; Luy, Betty E.; Childers, Thomas; Boroughs, Karen L.; Stovall, Janae L.; Calvert, Amanda E.; Blair, Carol D.; Huang, Claire Y.-H.

    2013-01-01

    Using an infectious cDNA clone we engineered seven mutations in the putative heparan sulfate- and receptor-binding motifs of the envelope protein of dengue virus serotype 2, strain 16681. Four mutant viruses, KK122/123EE, E202K, G304K, and KKK305/307/310EEE, were recovered following transfection of C6/36 cells. A fifth mutant, KK291/295EE, was recovered from C6/36 cells with a compensatory E295V mutation. All mutants grew in and mediated fusion of virus-infected C6/36 cells, but three of the mutants, KK122/123EE, E202K, G304K, did not grow in Vero cells without further modification. Two Vero cell lethal mutants, KK291/295EV and KKK307/307/310EEE, failed to replicate in DC-SIGN-transformed Raji cells and did not react with monoclonal antibodies known to block DENV attachment to Vero cells. Additionally, both mutants were unable to initiate negative-strand vRNA synthesis in Vero cells by 72 h post-infection, suggesting that the replication block occurred prior to virus-mediated membrane fusion. - Highlights: • Heparan sulfate- and receptor-binding motifs of DENV2 envelope protein were mutated. • Four mutant viruses were isolated—all could fuse C6/36 cells. • Two of these mutants were lethal in Vero cells without further modification. • Lethal mutations were KK291/295EV and KKK305/307/310EEE. • Cell attachment was implicated as the replication block for both mutants

  7. Mutation of the dengue virus type 2 envelope protein heparan sulfate binding sites or the domain III lateral ridge blocks replication in Vero cells prior to membrane fusion

    Energy Technology Data Exchange (ETDEWEB)

    Roehrig, John T., E-mail: jtr1@cdc.gov [Division of Vector-Borne Diseases, Centers for Disease Control and Prevention, Fort Collins, CO 80521 (United States); Butrapet, Siritorn; Liss, Nathan M. [Division of Vector-Borne Diseases, Centers for Disease Control and Prevention, Fort Collins, CO 80521 (United States); Bennett, Susan L. [Arthropod-borne and Infectious Diseases Laboratory, Department of Microbiology, Immunology, and Pathology, Colorado State University, Fort Collins, CO 80523 (United States); Luy, Betty E.; Childers, Thomas; Boroughs, Karen L.; Stovall, Janae L.; Calvert, Amanda E. [Division of Vector-Borne Diseases, Centers for Disease Control and Prevention, Fort Collins, CO 80521 (United States); Blair, Carol D. [Arthropod-borne and Infectious Diseases Laboratory, Department of Microbiology, Immunology, and Pathology, Colorado State University, Fort Collins, CO 80523 (United States); Huang, Claire Y.-H. [Division of Vector-Borne Diseases, Centers for Disease Control and Prevention, Fort Collins, CO 80521 (United States)

    2013-07-05

    Using an infectious cDNA clone we engineered seven mutations in the putative heparan sulfate- and receptor-binding motifs of the envelope protein of dengue virus serotype 2, strain 16681. Four mutant viruses, KK122/123EE, E202K, G304K, and KKK305/307/310EEE, were recovered following transfection of C6/36 cells. A fifth mutant, KK291/295EE, was recovered from C6/36 cells with a compensatory E295V mutation. All mutants grew in and mediated fusion of virus-infected C6/36 cells, but three of the mutants, KK122/123EE, E202K, G304K, did not grow in Vero cells without further modification. Two Vero cell lethal mutants, KK291/295EV and KKK307/307/310EEE, failed to replicate in DC-SIGN-transformed Raji cells and did not react with monoclonal antibodies known to block DENV attachment to Vero cells. Additionally, both mutants were unable to initiate negative-strand vRNA synthesis in Vero cells by 72 h post-infection, suggesting that the replication block occurred prior to virus-mediated membrane fusion. - Highlights: • Heparan sulfate- and receptor-binding motifs of DENV2 envelope protein were mutated. • Four mutant viruses were isolated—all could fuse C6/36 cells. • Two of these mutants were lethal in Vero cells without further modification. • Lethal mutations were KK291/295EV and KKK305/307/310EEE. • Cell attachment was implicated as the replication block for both mutants.

  8. Diffusion of Integral Membrane Proteins in Protein-Rich Membranes

    Czech Academy of Sciences Publication Activity Database

    Javanainen, M.; Martinez-Seara, Hector; Metzler, R.; Vattulainen, I.

    2017-01-01

    Roč. 8, č. 17 (2017), s. 4308-4313 ISSN 1948-7185 R&D Projects: GA ČR(CZ) GBP208/12/G016 Institutional support: RVO:61388963 Keywords : giant unilamellar vesicles * single-molecule tracking * lipid bilayer membranes Subject RIV: CF - Physical ; Theoretical Chemistry OBOR OECD: Physical chemistry Impact factor: 9.353, year: 2016

  9. Annexin A4 and A6 induce membrane curvature and constriction during cell membrane repair

    DEFF Research Database (Denmark)

    Boye, Theresa Louise; Maeda, Kenji; Pezeshkian, Weria

    2017-01-01

    that annexin A4 binds to artificial membranes and generates curvature force initiated from free edges, whereas annexin A6 induces constriction force. In cells, plasma membrane injury and Ca2+ influx recruit annexin A4 to the vicinity of membrane wound edges where its homo-trimerization leads to membrane......Efficient cell membrane repair mechanisms are essential for maintaining membrane integrity and thus for cell life. Here we show that the Ca2+- and phospholipid-binding proteins annexin A4 and A6 are involved in plasma membrane repair and needed for rapid closure of micron-size holes. We demonstrate...... curvature near the edges. We propose that curvature force is utilized together with annexin A6-mediated constriction force to pull the wound edges together for eventual fusion. We show that annexin A4 can counteract various plasma membrane disruptions including holes of several micrometers indicating...

  10. Tubulation of class II MHC compartments is microtubule dependent and involves multiple endolysosomal membrane proteins in primary dendritic cells.

    Science.gov (United States)

    Vyas, Jatin M; Kim, You-Me; Artavanis-Tsakonas, Katerina; Love, J Christopher; Van der Veen, Annemarthe G; Ploegh, Hidde L

    2007-06-01

    Immature dendritic cells (DCs) capture exogenous Ags in the periphery for eventual processing in endolysosomes. Upon maturation by TLR agonists, DCs deliver peptide-loaded class II MHC molecules from these compartments to the cell surface via long tubular structures (endolysosomal tubules). The nature and rules that govern the movement of these DC compartments are unknown. In this study, we demonstrate that the tubules contain multiple proteins including the class II MHC molecules and LAMP1, a lysosomal resident protein, as well as CD63 and CD82, members of the tetraspanin family. Endolysosomal tubules can be stained with acidotropic dyes, indicating that they are extensions of lysosomes. However, the proper trafficking of class II MHC molecules themselves is not necessary for endolysosomal tubule formation. DCs lacking MyD88 can also form endolysosomal tubules, demonstrating that MyD88-dependent TLR activation is not necessary for the formation of this compartment. Endolysosomal tubules in DCs exhibit dynamic and saltatory movement, including bidirectional travel. Measured velocities are consistent with motor-based movement along microtubules. Indeed, nocodazole causes the collapse of endolysosomal tubules. In addition to its association with microtubules, endolysosomal tubules follow the plus ends of microtubules as visualized in primary DCs expressing end binding protein 1 (EB1)-enhanced GFP.

  11. Single channel analysis of membrane proteins in artificial bilayer membranes.

    Science.gov (United States)

    Bartsch, Philipp; Harsman, Anke; Wagner, Richard

    2013-01-01

    The planar lipid bilayer technique is a powerful experimental approach for electrical single channel recordings of pore-forming membrane proteins in a chemically well-defined and easily modifiable environment. Here we provide a general survey of the basic materials and procedures required to set up a robust bilayer system and perform electrophysiological single channel recordings of reconstituted proteins suitable for the in-depth characterization of their functional properties.

  12. Efficient preparation and analysis of membrane and membrane protein systems

    Czech Academy of Sciences Publication Activity Database

    Javanainen, M.; Martinez-Seara, Hector

    2016-01-01

    Roč. 1858, č. 10 (2016), s. 2468-2482 ISSN 0005-2736 Institutional support: RVO:61388963 Keywords : tools and software * membrane building * protein insertion * molecular dynamics * lipid bilayer Subject RIV: CF - Physical ; Theoretical Chemistry Impact factor: 3.498, year: 2016

  13. Quantitative measurement of cell membrane receptor internalization by the nanoluciferase reporter: Using the G protein-coupled receptor RXFP3 as a model.

    Science.gov (United States)

    Liu, Yu; Song, Ge; Shao, Xiao-Xia; Liu, Ya-Li; Guo, Zhan-Yun

    2015-02-01

    Nanoluciferase (NanoLuc) is a newly developed small luciferase reporter with the brightest bioluminescence to date. In the present work, we developed NanoLuc as a sensitive bioluminescent reporter to measure quantitatively the internalization of cell membrane receptors, based on the pH dependence of the reporter activity. The G protein-coupled receptor RXFP3, the cognate receptor of relaxin-3/INSL7, was used as a model receptor. We first generated stable HEK293T cells that inducibly coexpressed a C-terminally NanoLuc-tagged human RXFP3 and a C-terminally enhanced green fluorescent protein (EGFP)-tagged human RXFP3. The C-terminal EGFP-tag and NanoLuc-tag had no detrimental effects on the ligand-binding potency and intracellular trafficking of RXFP3. Based on the fluorescence of the tagged EGFP reporter, the ligand-induced RXFP3 internalization was visualized directly under a fluorescence microscope. Based on the bioluminescence of the tagged NanoLuc reporter, the ligand-induced RXFP3 internalization was measured quantitatively by a convenient bioluminescent assay. Coexpression of an EGFP-tagged inactive [E141R]RXFP3 had no detrimental effect on the ligand-binding potency and ligand-induced internalization of the NanoLuc-tagged wild-type RXFP3, suggesting that the mutant RXFP3 and wild-type RXFP3 worked independently. The present bioluminescent internalization assay could be extended to other G protein-coupled receptors and other cell membrane receptors to study ligand-receptor and receptor-receptor interactions. Copyright © 2014 Elsevier B.V. All rights reserved.

  14. The endocytic pathways of a secretory granule membrane protein in HEK293 cells: PAM and EGF traverse a dynamic multivesicular body network together.

    Science.gov (United States)

    Bäck, Nils; Kanerva, Kristiina; Kurutihalli, Vishwanatha; Yanik, Andrew; Ikonen, Elina; Mains, Richard E; Eipper, Betty A

    2017-08-01

    Peptidylglycine α-amidating monooxygenase (PAM) is highly expressed in neurons and endocrine cells, where it catalyzes one of the final steps in the biosynthesis of bioactive peptides. PAM is also expressed in unicellular organisms such as Chlamydomonas reinhardtii, which do not store peptides in secretory granules. As for other granule membrane proteins, PAM is retrieved from the cell surface and returned to the trans-Golgi network. This pathway involves regulated entry of PAM into multivesicular body intralumenal vesicles (ILVs). The aim of this study was defining the endocytic pathways utilized by PAM in cells that do not store secretory products in granules. Using stably transfected HEK293 cells, endocytic trafficking of PAM was compared to that of the mannose 6-phosphate (MPR) and EGF (EGFR) receptors, established markers for the endosome to trans-Golgi network and degradative pathways, respectively. As in neuroendocrine cells, PAM internalized by HEK293 cells accumulated in the trans-Golgi network. Based on surface biotinylation, >70% of the PAM on the cell surface was recovered intact after a 4h chase and soluble, bifunctional PAM was produced. Endosomes containing PAM generally contained both EGFR and MPR and ultrastructural analysis confirmed that all three cargos accumulated in ILVs. PAM containing multivesicular bodies made frequent dynamic tubular contacts with younger and older multivesicular bodies. Frequent dynamic contacts were observed between lysosomes and PAM containing early endosomes and multivesicular bodies. The ancient ability of PAM to localize to ciliary membranes, which release bioactive ectosomes, may be related to its ability to accumulate in ILVs and exosomes. Copyright © 2017 Elsevier GmbH. All rights reserved.

  15. Membrane proteins PmpG and PmpH are major constituents of Chlamydia trachomatis L2 outer membrane complex

    DEFF Research Database (Denmark)

    Mygind, Per H; Christiansen, Gunna; Roepstorff, P

    2000-01-01

    The outer membrane complex of Chlamydia is involved in the initial adherence and ingestion of Chlamydia by the host cell. In order to identify novel proteins in the outer membrane of Chlamydia trachomatis L2, proteins were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis. By....... By silver staining of the protein profile, a major protein doublet of 100-110 kDa was detected. In-gel tryptic digestion and matrix-assisted laser desorption/ionization mass spectrometry identified these proteins as the putative outer membrane proteins PmpG and PmpH....

  16. Overcoming barriers to membrane protein structure determination

    NARCIS (Netherlands)

    Bill, Roslyn M.; Henderson, Peter J. F.; Iwata, So; Kunji, Edmund R. S.; Michel, Hartmut; Neutze, Richard; Newstead, Simon; Poolman, Bert; Tate, Christopher G.; Vogel, Horst

    After decades of slow progress, the pace of research on membrane protein structures is beginning to quicken thanks to various improvements in technology, including protein engineering and microfocus X-ray diffraction. Here we review these developments and, where possible, highlight generic new

  17. An overview of membrane transport proteins in Saccharomyces cerevisiae.

    Science.gov (United States)

    Andre, B

    1995-12-01

    All eukaryotic cells contain a wide variety of proteins embedded in the plasma and internal membranes, which ensure transmembrane solute transport. It is now established that a large proportion of these transport proteins can be grouped into families apparently conserved throughout organisms. This article presents the data of an in silicio analysis aimed at establishing a preliminary classification of membrane transport proteins in Saccharomyces cerevisiae. This analysis was conducted at a time when about 65% of all yeast genes were available in public databases. In addition to approximately 60 transport proteins whose function was at least partially known, approximately 100 deduced protein sequences of unknown function display significant sequence similarity to membrane transport proteins characterized in yeast and/or other organisms. While some protein families have been well characterized by classical genetic experimental approaches, others have largely if not totally escaped characterization. The proteins revealed by this in silicio analysis also include a putative K+ channel, proteins similar to aquaporins of plant and animal origin, proteins similar to Na+-solute symporters, a protein very similar to electroneural cation-chloride cotransporters, and a putative Na+-H+ antiporter. A new research area is anticipated: the functional analysis of many transport proteins whose existence was revealed by genome sequencing.

  18. Proteins of the kidney microvillar membrane

    International Nuclear Information System (INIS)

    Booth, A.G.; Kenny, A.J.

    1980-01-01

    Two methods were used to label pig kidney microvillar membrane proteins from the luminal and cytoplasmic surfaces of closed membrane vesicles. The first method was lactoperoxidase-catalysed radioiodination. Lactoperoxidase and glucose oxidase were positioned inside or outside the vesicles, iodination being initiated by adding glucose and 125 I. After electrophoresis of the proteins, asymmetric labelling patterns on radioautographs were observed. However the major disadvantage of this method was the high degree of intramembrane labelling of the fatty acid chains of membrane lipids. The second method overcame this disadvantage. A new hydophilic photoreagent, 3,5-di( 125 I)iodo-4-azidobenzenesulphonate, was transported by a Na + -dependent system into microvillar vesicles, thus permitting labelling from either side of the membrane when the vesicles were photolysed. The activity of several microvillar peptidases survived the labelling reaction and they could be identified in the immunoprecipitates after resolution of the detergent-solubilized membrane proteins by crossed-immunoelectrophoresis. Treatment with papain converted the detergent-solubilized form of susceptible enzymes into the proteinase-solubilized form. Radioautography established that aminopeptidases M and A, dipeptidyl peptidase IV and neutral endopeptidase were transmembrane proteins. This novel approach may be applicable to the topological investigation of other complex membranes. (author)

  19. The coronavirus spike protein : mechanisms of membrane fusion and virion incorporation

    NARCIS (Netherlands)

    Bosch, B.J.

    2004-01-01

    The coronavirus spike protein is a membrane-anchored glycoprotein responsible for virus-cell attachment and membrane fusion, prerequisites for a successful virus infection. In this thesis, two aspects are described regarding the molecular biology of the coronavirus spike protein: its membrane fusion

  20. Disruption of an M. tuberculosis membrane protein causes a magnesium-dependent cell division defect and failure to persist in mice.

    Directory of Open Access Journals (Sweden)

    Nichole Goodsmith

    2015-02-01

    Full Text Available The identification of Mycobacterium tuberculosis genes necessary for persistence in vivo provides insight into bacterial biology as well as host defense strategies. We show that disruption of M. tuberculosis membrane protein PerM (Rv0955 resulted in an IFN-γ-dependent persistence defect in chronic mouse infection despite the mutant's near normal growth during acute infection. The perM mutant required increased magnesium for replication and survival; incubation in low magnesium media resulted in cell elongation and lysis. Transcriptome analysis of the perM mutant grown in reduced magnesium revealed upregulation of cell division and cell wall biosynthesis genes, and live cell imaging showed PerM accumulation at the division septa in M. smegmatis. The mutant was acutely sensitive to β-lactam antibiotics, including specific inhibitors of cell division-associated peptidoglycan transpeptidase FtsI. Together, these data implicate PerM as a novel player in mycobacterial cell division and pathogenesis, and are consistent with the hypothesis that immune activation deprives M. tuberculosis of magnesium.

  1. Cell membrane structures during exocytosis.

    Science.gov (United States)

    Savigny, Pascale; Evans, John; McGrath, Kathryn M

    2007-08-01

    Exocytosis is a key biological process that controls the neurotransmission and release of hormones from cells. In endocrine cells, hormones are packed into secretory vesicles and released into the extracellular environment via openings in the plasma membrane, a few hundred nanometers wide, which form as a result of fusion of the membranes of the granule and cell. The complex processes and dynamics that result in the formation of the fusion pore, as well as its structure, remain scantly understood. A number of different exocytosis mechanisms have been postulated. Furthermore, the possibility exists that several mechanisms occur simultaneously. We present here an investigation of the cell membrane dynamics during exocytosis in anterior pituitary cells, especially gonadotropes, which secrete LH, a hormone central to ovulation. Gonadotrope enrichment was achieved using immunolabeled magnetic nanobeads. Three complementary imaging techniques were used to realize a fine structure study of the dynamics of the exocytosis-like sites occurring during secretion. Living pituitary and gonadotrope-enriched cells were imaged with atomic force microscopy, as well as cells that had been fixed to obtain better resolution. Atomic force microscopy, along with scanning and transmission electron microscopy, studies of these cells revealed that there are at least two different site configurations: simple single fusion pores and a complex association of pores consisting of a simple primary site combined with secondary attachments.

  2. Corrugated Membrane Fuel Cell Structures

    Energy Technology Data Exchange (ETDEWEB)

    Grot, Stephen [President, Ion Power Inc.

    2013-09-30

    One of the most challenging aspects of traditional PEM fuel cell stacks is the difficulty achieving the platinum catalyst utilization target of 0.2 gPt/kWe set forth by the DOE. Good catalyst utilization can be achieved with state-of-the-art catalyst coated membranes (CCM) when low catalyst loadings (<0.3 mg/cm2) are used at a low current. However, when low platinum loadings are used, the peak power density is lower than conventional loadings, requiring a larger total active area and a larger bipolar plate. This results in a lower overall stack power density not meeting the DOE target. By corrugating the fuel cell membrane electrode structure, Ion Power?s goal is to realize both the Pt utilization targets as well as the power density targets of the DOE. This will be achieved by demonstrating a fuel cell single cell (50 cm2) with a twofold increase in the membrane active area over the geometric area of the cell by corrugating the MEA structure. The corrugating structure must be able to demonstrate the target properties of < 10 mOhm-cm2 electrical resistance at > 20 psi compressive strength over the active area, in combination with offering at least 80% of power density that can be achieved by using the same MEA in a flat plate structure. Corrugated membrane fuel cell structures also have the potential to meet DOE power density targets by essentially packaging more membrane area into the same fuel cell volume as compared to conventional stack constructions.

  3. Increased activity of cell membrane-associated prothrombinase, fibrinogen-like protein 2, in peripheral blood mononuclear cells of B-cell lymphoma patients.

    Directory of Open Access Journals (Sweden)

    Esther Rabizadeh

    Full Text Available Fibrinogen-like protein 2, FGL-2, was reported to be overexpressed in various cancer tissues, where it acts as a transmembrane prothrombinase. This study aims to determine the prothrombinase activity of FGL-2 in peripheral blood mononuclear cells (PBMC of patients with B-cell lymphoma. FGL-2 activity was determined in patients with B-cell lymphoma (n = 53, and healthy controls (n = 145. FGL-2 activity in patients at diagnosis increased 3 ± 0.3 fold (p < 0.001. Sensitivity and specificity of the test was established at 73.6% and 80.7%, respectively, using a cutoff of 150% activity over control. Moreover, FGL-2 activity in 10 of 11 patients in remission decreased by 76%. In contrast, no significant difference was observed in expression levels of fgl-2 gene in patients and controls. Taken together, our study indicates that FGL-2 prothrombinase activity in PBMC of lymphoma patients is increased in active disease and normalizes during remission, thus being a potential marker for follow up of lymphoma patients.

  4. Major Intrinsic Proteins in Biomimetic Membranes

    DEFF Research Database (Denmark)

    Helix Nielsen, Claus

    2010-01-01

    this challenge by developing membranes in the form of lipid bilayers in which specialized transport proteins are incorporated. This raises the question: is it possible to mimic biological membranes and create a membrane based sensor and/or separation device? In the development of a biomimetic sensor...... or as sensor devices based on e.g., the selective permeation of metalloids. In principle a MIP based membrane sensor/separation device requires the supporting biomimetic matrix to be virtually impermeable to anything but water or the solute in question. In practice, however, a biomimetic support matrix...... will generally have finite permeabilities to both electrolytes and non-electrolytes. The feasibility of a biomimetic MIP device thus depends on the relative transport contribution from both protein and biomimetic support matrix. Also the biomimetic matrix must be encapsulated in order to protect it and make...

  5. Visualization of the African swine fever virus infection in living cells by incorporation into the virus particle of green fluorescent protein-p54 membrane protein chimera

    International Nuclear Information System (INIS)

    Hernaez, Bruno; Escribano, Jose M.; Alonso, Covadonga

    2006-01-01

    Many stages of African swine fever virus infection have not yet been studied in detail. To track the behavior of African swine fever virus (ASFV) in the infected cells in real time, we produced an infectious recombinant ASFV (B54GFP-2) that expresses and incorporates into the virus particle a chimera of the p54 envelope protein fused to the enhanced green fluorescent protein (EGFP). The incorporation of the fusion protein into the virus particle was confirmed immunologically and it was determined that p54-EGFP was fully functional by confirmation that the recombinant virus made normal-sized plaques and presented similar growth curves to the wild-type virus. The tagged virus was visualized as individual fluorescent particles during the first stages of infection and allowed to visualize the infection progression in living cells through the viral life cycle by confocal microscopy. In this work, diverse potential applications of B54GFP-2 to study different aspects of ASFV infection are shown. By using this recombinant virus it was possible to determine the trajectory and speed of intracellular virus movement. Additionally, we have been able to visualize for first time the ASFV factory formation dynamics and the cytophatic effect of the virus in live infected cells. Finally, we have analyzed virus progression along the infection cycle and infected cell death as time-lapse animations

  6. Structural adaptations of proteins to different biological membranes

    Science.gov (United States)

    Pogozheva, Irina D.; Tristram-Nagle, Stephanie; Mosberg, Henry I.; Lomize, Andrei L.

    2013-01-01

    To gain insight into adaptations of proteins to their membranes, intrinsic hydrophobic thicknesses, distributions of different chemical groups and profiles of hydrogen-bonding capacities (α and β) and the dipolarity/polarizability parameter (π*) were calculated for lipid-facing surfaces of 460 integral α-helical, β-barrel and peripheral proteins from eight types of biomembranes. For comparison, polarity profiles were also calculated for ten artificial lipid bilayers that have been previously studied by neutron and X-ray scattering. Estimated hydrophobic thicknesses are 30-31 Å for proteins from endoplasmic reticulum, thylakoid, and various bacterial plasma membranes, but differ for proteins from outer bacterial, inner mitochondrial and eukaryotic plasma membranes (23.9, 28.6 and 33.5 Å, respectively). Protein and lipid polarity parameters abruptly change in the lipid carbonyl zone that matches the calculated hydrophobic boundaries. Maxima of positively charged protein groups correspond to the location of lipid phosphates at 20-22 Å distances from the membrane center. Locations of Tyr atoms coincide with hydrophobic boundaries, while distributions maxima of Trp rings are shifted by 3-4 Å toward the membrane center. Distributions of Trp atoms indicate the presence of two 5-8 Å-wide midpolar regions with intermediate π* values within the hydrocarbon core, whose size and symmetry depend on the lipid composition of membrane leaflets. Midpolar regions are especially asymmetric in outer bacterial membranes and cell membranes of mesophilic but not hyperthermophilic archaebacteria, indicating the larger width of the central nonpolar region in the later case. In artificial lipid bilayers, midpolar regions are observed up to the level of acyl chain double bonds. PMID:23811361

  7. Facilitation of yeast-lethal membrane protein production by detoxifying with GFP tagging.

    Science.gov (United States)

    Oshikane, Hiroyuki; Watabe, Masahiko; Nakaki, Toshio

    2018-03-27

    Recombinant techniques for target protein production have been rapidly established and widely utilised in today's biological research. Nevertheless, methods for membrane protein production have yet to be developed, since membrane proteins generally tend to be expressed at low levels, easily aggregated, and/or even toxic to their host cells. Here we report that a GFP-tagging technique can be applied for the stable production of membrane proteins that are toxic to their host cells when overexpressed, paving the way for future advances in membrane protein biochemistry and drug development. Copyright © 2018 Elsevier Inc. All rights reserved.

  8. Structural investigation of membrane proteins by electron microscopy

    NARCIS (Netherlands)

    Moscicka, Katarzyna Beata

    2009-01-01

    Biological membranes are vital components of all living systems, forming the boundaries of cells and their organelles. They consist of a lipid bilayer and embedded proteins, which are nanomachines that fulfill key functions such as energy conversion, solute transport, secretion, and signal

  9. Topology of membrane proteins-predictions, limitations and variations.

    Science.gov (United States)

    Tsirigos, Konstantinos D; Govindarajan, Sudha; Bassot, Claudio; Västermark, Åke; Lamb, John; Shu, Nanjiang; Elofsson, Arne

    2017-10-26

    Transmembrane proteins perform a variety of important biological functions necessary for the survival and growth of the cells. Membrane proteins are built up by transmembrane segments that span the lipid bilayer. The segments can either be in the form of hydrophobic alpha-helices or beta-sheets which create a barrel. A fundamental aspect of the structure of transmembrane proteins is the membrane topology, that is, the number of transmembrane segments, their position in the protein sequence and their orientation in the membrane. Along these lines, many predictive algorithms for the prediction of the topology of alpha-helical and beta-barrel transmembrane proteins exist. The newest algorithms obtain an accuracy close to 80% both for alpha-helical and beta-barrel transmembrane proteins. However, lately it has been shown that the simplified picture presented when describing a protein family by its topology is limited. To demonstrate this, we highlight examples where the topology is either not conserved in a protein superfamily or where the structure cannot be described solely by the topology of a protein. The prediction of these non-standard features from sequence alone was not successful until the recent revolutionary progress in 3D-structure prediction of proteins. Copyright © 2017 Elsevier Ltd. All rights reserved.

  10. Effect of therapeutic concentration of lithium on live HEK293 cells; increase of Na+/K+-ATPase, change of overall protein composition and alteration of surface layer of plasma membrane.

    Science.gov (United States)

    Vosahlikova, Miroslava; Ujcikova, Hana; Chernyavskiy, Oleksandr; Brejchova, Jana; Roubalova, Lenka; Alda, Martin; Svoboda, Petr

    2017-05-01

    The effect of long-term exposure of live cells to lithium cations (Li) was studied in HEK293 cells cultivated in the presence of 1mM LiCl for 7 or 21days. The alteration of Na + /K + -ATPase level, protein composition and biophysical state of plasma membrane was determined with the aim to characterize the physiological state of Li-treated cells. Na + /K + -ATPase level was determined by [ 3 H]ouabain binding and immunoblot assays. Overall protein composition was determined by 2D electrophoresis followed by proteomic analysis by MALDI-TOF MS/MS and LFQ. Li interaction with plasma membrane was characterized by fluorescent probes DPH, TMA-DPH and Laurdan. Na + /K + -ATPase was increased in plasma membranes isolated from cells exposed to Li. Identification of Li-altered proteins by 2D electrophoresis, MALDI-TOF MS/MS and LFQ suggests a change of energy metabolism in mitochondria and cytosol and alteration of cell homeostasis of calcium. Measurement of Laurdan generalized polarization indicated a significant alteration of surface layer of isolated plasma membranes prepared from both types of Li-treated cells. Prolonged exposure of HEK293 cells to 1mM LiCl results in up-regulation of Na + /K + -ATPase expression, reorganization of overall cellular metabolism and alteration of the surface layer/polar head-group region of isolated plasma membranes. Our findings demonstrate adaptation of live HEK293 cell metabolism to prolonged exposure to therapeutic concentration of Li manifested as up-regulation of Na + /K + -ATPase expression, alteration of protein composition and change of the surface layer of plasma membrane. Copyright © 2017 Elsevier B.V. All rights reserved.

  11. Epstein-Barr virus-encoded latent membrane protein 1 impairs G2 checkpoint in human nasopharyngeal epithelial cells through defective Chk1 activation.

    Directory of Open Access Journals (Sweden)

    Wen Deng

    Full Text Available Nasopharyngeal carcinoma (NPC is a common cancer in Southeast Asia, particularly in southern regions of China. EBV infection is closely associated with NPC and has long been postulated to play an etiological role in the development of NPC. However, the role of EBV in malignant transformation of nasopharyngeal epithelial cells remains enigmatic. The current hypothesis of NPC development is that premalignant nasopharyngeal epithelial cells harboring genetic alterations support EBV infection and expression of EBV genes induces further genomic instability to facilitate the development of NPC. The latent membrane protein 1 (LMP1 is a well-documented EBV-encoded oncogene. The involvement of LMP1 in human epithelial malignancies has been implicated, but the mechanisms of oncogenic actions of LMP1, particularly in nasopharyngeal cells, are unclear. Here we observed that LMP1 expression in nasopharyngeal epithelial cells impaired G2 checkpoint, leading to formation of unrepaired chromatid breaks in metaphases after γ-ray irradiation. We further found that defective Chk1 activation was involved in the induction of G2 checkpoint defect in LMP1-expressing nasopharyngeal epithelial cells. Impairment of G2 checkpoint could result in loss of the acentrically broken chromatids and propagation of broken centric chromatids in daughter cells exiting mitosis, which facilitates chromosome instability. Our findings suggest that LMP1 expression facilitates genomic instability in cells under genotoxic stress. Elucidation of the mechanisms involved in LMP1-induced genomic instability in nasopharyngeal epithelial cells will shed lights on the understanding of role of EBV infection in NPC development.

  12. Comparison of membrane electroporation and protein denature in response to pulsed electric field with different durations.

    Science.gov (United States)

    Huang, Feiran; Fang, Zhihui; Mast, Jason; Chen, Wei

    2013-05-01

    In this paper, we compared the minimum potential differences in the electroporation of membrane lipid bilayers and the denaturation of membrane proteins in response to an intensive pulsed electric field with various pulse durations. Single skeletal muscle fibers were exposed to a pulsed external electric field. The field-induced changes in the membrane integrity (leakage current) and the Na channel currents were monitored to identify the minimum electric field needed to damage the membrane lipid bilayer and the membrane proteins, respectively. We found that in response to a relatively long pulsed electric shock (longer than the membrane intrinsic time constant), a lower membrane potential was needed to electroporate the cell membrane than for denaturing the membrane proteins, while for a short pulse a higher membrane potential was needed. In other words, phospholipid bilayers are more sensitive to the electric field than the membrane proteins for a long pulsed shock, while for a short pulse the proteins become more vulnerable. We can predict that for a short or ultrashort pulsed electric shock, the minimum membrane potential required to start to denature the protein functions in the cell plasma membrane is lower than that which starts to reduce the membrane integrity. Copyright © 2013 Wiley Periodicals, Inc.

  13. Molecular machines open cell membranes.

    Science.gov (United States)

    García-López, Víctor; Chen, Fang; Nilewski, Lizanne G; Duret, Guillaume; Aliyan, Amir; Kolomeisky, Anatoly B; Robinson, Jacob T; Wang, Gufeng; Pal, Robert; Tour, James M

    2017-08-30

    Beyond the more common chemical delivery strategies, several physical techniques are used to open the lipid bilayers of cellular membranes. These include using electric and magnetic fields, temperature, ultrasound or light to introduce compounds into cells, to release molecular species from cells or to selectively induce programmed cell death (apoptosis) or uncontrolled cell death (necrosis). More recently, molecular motors and switches that can change their conformation in a controlled manner in response to external stimuli have been used to produce mechanical actions on tissue for biomedical applications. Here we show that molecular machines can drill through cellular bilayers using their molecular-scale actuation, specifically nanomechanical action. Upon physical adsorption of the molecular motors onto lipid bilayers and subsequent activation of the motors using ultraviolet light, holes are drilled in the cell membranes. We designed molecular motors and complementary experimental protocols that use nanomechanical action to induce the diffusion of chemical species out of synthetic vesicles, to enhance the diffusion of traceable molecular machines into and within live cells, to induce necrosis and to introduce chemical species into live cells. We also show that, by using molecular machines that bear short peptide addends, nanomechanical action can selectively target specific cell-surface recognition sites. Beyond the in vitro applications demonstrated here, we expect that molecular machines could also be used in vivo, especially as their design progresses to allow two-photon, near-infrared and radio-frequency activation.

  14. Molecular machines open cell membranes

    Science.gov (United States)

    García-López, Víctor; Chen, Fang; Nilewski, Lizanne G.; Duret, Guillaume; Aliyan, Amir; Kolomeisky, Anatoly B.; Robinson, Jacob T.; Wang, Gufeng; Pal, Robert; Tour, James M.

    2017-08-01

    Beyond the more common chemical delivery strategies, several physical techniques are used to open the lipid bilayers of cellular membranes. These include using electric and magnetic fields, temperature, ultrasound or light to introduce compounds into cells, to release molecular species from cells or to selectively induce programmed cell death (apoptosis) or uncontrolled cell death (necrosis). More recently, molecular motors and switches that can change their conformation in a controlled manner in response to external stimuli have been used to produce mechanical actions on tissue for biomedical applications. Here we show that molecular machines can drill through cellular bilayers using their molecular-scale actuation, specifically nanomechanical action. Upon physical adsorption of the molecular motors onto lipid bilayers and subsequent activation of the motors using ultraviolet light, holes are drilled in the cell membranes. We designed molecular motors and complementary experimental protocols that use nanomechanical action to induce the diffusion of chemical species out of synthetic vesicles, to enhance the diffusion of traceable molecular machines into and within live cells, to induce necrosis and to introduce chemical species into live cells. We also show that, by using molecular machines that bear short peptide addends, nanomechanical action can selectively target specific cell-surface recognition sites. Beyond the in vitro applications demonstrated here, we expect that molecular machines could also be used in vivo, especially as their design progresses to allow two-photon, near-infrared and radio-frequency activation.

  15. Detergent-Mediated Reconstitution of Membrane Proteins

    NARCIS (Netherlands)

    Knol, J; Sjollema, K.A; Poolman, B.

    1998-01-01

    The efficiency of reconstitution of the lactose transport protein (LacS) of Streptococcus thermophilus is markedly higher with Triton X-100 than with other detergents commonly employed to mediate the membrane insertion. To rationalize these differences, the lipid/detergent structures that are formed

  16. A Chitin-binding Protein Purified from Moringa oleifera Seeds Presents Anticandidal Activity by Increasing Cell Membrane Permeability and Reactive Oxygen Species Production

    Directory of Open Access Journals (Sweden)

    João X.S. Neto

    2017-06-01

    Full Text Available Candida species are opportunistic pathogens that infect immunocompromised and/or immunosuppressed patients, particularly in hospital facilities, that besides representing a significant threat to health increase the risk of mortality. Apart from echinocandins and triazoles, which are well tolerated, most of the antifungal drugs used for candidiasis treatment can cause side effects and lead to the development of resistant strains. A promising alternative to the conventional treatments is the use of plant proteins. M. oleifera Lam. is a plant with valuable medicinal properties, including antimicrobial activity. This work aimed to purify a chitin-binding protein from M. oleifera seeds and to evaluate its antifungal properties against Candida species. The purified protein, named Mo-CBP2, represented about 0.2% of the total seed protein and appeared as a single band on native PAGE. By mass spectrometry, Mo-CBP2 presented 13,309 Da. However, by SDS-PAGE, Mo-CBP2 migrated as a single band with an apparent molecular mass of 23,400 Da. Tricine-SDS-PAGE of Mo-CBP2 under reduced conditions revealed two protein bands with apparent molecular masses of 7,900 and 4,600 Da. Altogether, these results suggest that Mo-CBP2 exists in different oligomeric forms. Moreover, Mo-CBP2 is a basic glycoprotein (pI 10.9 with 4.1% (m/m sugar and it did not display hemagglutinating and hemolytic activities upon rabbit and human erythrocytes. A comparative analysis of the sequence of triptic peptides from Mo-CBP2 in solution, after LC-ESI-MS/MS, revealed similarity with other M. oleifera proteins, as the 2S albumin Mo-CBP3 and flocculating proteins, and 2S albumins from different species. Mo-CBP2 possesses in vitro antifungal activity against Candida albicans, C. parapsilosis, C. krusei, and C. tropicalis, with MIC50 and MIC90 values ranging between 9.45–37.90 and 155.84–260.29 μM, respectively. In addition, Mo-CBP2 (18.90 μM increased the cell membrane permeabilization

  17. Identification of novel autophagic Radix Polygalae fraction by cell membrane chromatography and UHPLC-(Q)TOF-MS for degradation of neurodegenerative disease proteins.

    Science.gov (United States)

    Wu, An-Guo; Wong, Vincent Kam-Wai; Zeng, Wu; Liu, Liang; Law, Betty Yuen-Kwan

    2015-11-24

    With its traditional use in relieving insomnia and anxiety, our previous study has identified onjisaponin B from Radix Polygalae (RP), as a novel autophagic enhancer with potential neuroprotective effects. In current study, we have further identified a novel active fraction from RP, contains 17 major triterpenoid saponins including the onjisaponin B, by the combinational use of cell membrane chromatography (CMC) and ultra-performance liquid chromatography coupled to (quadrupole) time-of-flight mass spectrometry {UHPLC-(Q)TOF-MS}. By exhibiting more potent autophagic effect in cells, the active fraction enhances the clearance of mutant huntingtin, and reduces protein level and aggregation of α-synuclein in a higher extent when compared with onjisaponin B. Here, we have reported for the first time the new application of cell-based CMC and UHPLC-(Q)TOF-MS analysis in identifying new autophagy inducers with neuroprotective effects from Chinese medicinal herb. This result has provided novel insights into the possible pharmacological actions of the active components present in the newly identified active fraction of RP, which may help to improve the efficacy of the traditional way of prescribing RP, and also provide new standard for the quality control of decoction of RP or its medicinal products in the future.

  18. Towards understanding of Nipah virus attachment protein assembly and the role of protein affinity and crowding for membrane curvature events.

    Energy Technology Data Exchange (ETDEWEB)

    Stachowiak, Jeanne C.; Hayden, Carl C.; Negrete, Oscar.; Davis, Ryan Wesley; Sasaki, Darryl Y

    2013-10-01

    Pathogenic viruses are a primary threat to our national security and to the health and economy of our world. Effective defense strategies to combat viral infection and spread require the development of understanding of the mechanisms that these pathogens use to invade the host cell. We present in this report results of our research into viral particle recognition and fusion to cell membranes and the role that protein affinity and confinement in lipid domains plays in membrane curvature in cellular fusion and fission events. Herein, we describe 1) the assembly of the G attachment protein of Nipah virus using point mutation studies to define its role in viral particle fusion to the cell membrane, 2) how lateral pressure of membrane bound proteins induce curvature in model membrane systems, and 3) the role of membrane curvature in the selective partitioning of molecular receptors and specific affinity of associated proteins.

  19. The outer membrane protein TolC of Vibrio cholerae serves as a second cell-surface receptor for the VP3 phage.

    Science.gov (United States)

    Fan, Fenxia; Li, Xu; Pang, Bo; Zhang, Cheng; Li, Zhe; Zhang, Lijuan; Li, Jie; Zhang, Jingyun; Yan, Meiying; Liang, Weili; Kan, Biao

    2017-12-19

    Receptor recognition is a key step in the initiation of phage infection. Previously, we found that VP3, the T7 family phage of the Vibrio cholerae serogroup O1 biotype El Tor, can adsorb to the core oligosaccharide (OS) of lipopolysaccharides of V. cholerae. However, some wild-type strains of V. cholerae possessing the intact OS gene cluster still have VP3 binding but are resistant to VP3 infection. Moreover, an OS gene deletion mutant still exhibits weak VP3 binding, suggesting multiple factors are possibly involved in VP3 binding to V. cholerae. Here, we report that the outer-membrane protein TolC of V. cholerae is involved in the host adsorption of VP3. We observed that TolC directly interacts with the VP3 tail fiber protein gp44 and its C-terminal domains, and also found that three amino acid residues in the outside loops of TolC, at positions 78, 290 and 291, are critical for binding to gp44. Among VP3-resistant wild type V. cholerae strains, frequent amino acid residue mutations were observed in the loops around the sites 78, 290 and 291 which were predicted to be exposed to the cell surface. These findings reveal a co-receptor-binding mechanism for VP3 infection of V. cholerae and that both outer membrane TolC and OS are necessary for successful VP3 infection of V. cholerae. We conclude that mutations on the outside loops of the receptor may confer V. cholerae strains with VP3 phage-resistance, enabling these strains to survive in environments containing VP3 or related phages. Copyright © 2017, The American Society for Biochemistry and Molecular Biology.

  20. Combinatorial Method for Overexpression of Membrane Proteins in Escherichia coli*

    Science.gov (United States)

    Leviatan, Shani; Sawada, Keisuke; Moriyama, Yoshinori; Nelson, Nathan

    2010-01-01

    Membrane proteins constitute 20–30% of all proteins encoded by the genome of various organisms. Large amounts of purified proteins are required for activity and crystallization attempts. Thus, there is an unmet need for a heterologous membrane protein overexpression system for purification, crystallization, and activity determination. We developed a combinatorial method for overexpressing and purifying membrane proteins using Escherichia coli. This method utilizes short hydrophilic bacterial proteins, YaiN and YbeL, fused to the ends of the membrane proteins to serve as facilitating factors for expression and purification. Fourteen prokaryotic and mammalian membrane proteins were expressed using this system. Moderate to high expression was obtained for most proteins, and detergent solubilization combined with a short purification process produced stable, monodispersed membrane proteins. Five of the mammalian membrane proteins, overexpressed using our system, were reconstituted into liposomes and exhibited transport activity comparable with the native transporters. PMID:20525689

  1. Combinatorial method for overexpression of membrane proteins in Escherichia coli.

    Science.gov (United States)

    Leviatan, Shani; Sawada, Keisuke; Moriyama, Yoshinori; Nelson, Nathan

    2010-07-30

    Membrane proteins constitute 20-30% of all proteins encoded by the genome of various organisms. Large amounts of purified proteins are required for activity and crystallization attempts. Thus, there is an unmet need for a heterologous membrane protein overexpression system for purification, crystallization, and activity determination. We developed a combinatorial method for overexpressing and purifying membrane proteins using Escherichia coli. This method utilizes short hydrophilic bacterial proteins, YaiN and YbeL, fused to the ends of the membrane proteins to serve as facilitating factors for expression and purification. Fourteen prokaryotic and mammalian membrane proteins were expressed using this system. Moderate to high expression was obtained for most proteins, and detergent solubilization combined with a short purification process produced stable, monodispersed membrane proteins. Five of the mammalian membrane proteins, overexpressed using our system, were reconstituted into liposomes and exhibited transport activity comparable with the native transporters.

  2. Correlation between membrane fluidity cellular development and stem cell differentiation

    KAUST Repository

    Noutsi, Pakiza

    2016-12-01

    Cell membranes are made up of a complex structure of lipids and proteins that diffuse laterally giving rise to what we call membrane fluidity. During cellular development, such as neuronal differentiation, cell membranes undergo dramatic structural changes induced by proteins such as ARC and Cofilin among others in the case of synaptic modification. In this study we used the generalized polarization (GP) property of fluorescent probe Laurdan using two-photon microscopy to determine membrane fluidity as a function of time and for various cell lines. A low GP value corresponds to a higher fluidity and a higher GP value is associated with a more rigid membrane. Four different cell lines were monitored such as hN2, NIH3T3, HEK293 and L6 cells. As expected, NIH3T3 cells have more rigid membrane at earlier stages of their development. On the other hand neurons tend to have the highest membrane fluidity early in their development emphasizing its correlation with plasticity and the need for this malleability during differentiation. This study sheds light on the involvement of membrane fluidity during neuronal differentiation and development of other cell lines.

  3. Chemically Stable Lipids for Membrane Protein Crystallization

    Energy Technology Data Exchange (ETDEWEB)

    Ishchenko, Andrii; Peng, Lingling; Zinovev, Egor; Vlasov, Alexey; Lee, Sung Chang; Kuklin, Alexander; Mishin, Alexey; Borshchevskiy, Valentin; Zhang, Qinghai; Cherezov, Vadim (MIPT); (USC); (Scripps)

    2017-05-01

    The lipidic cubic phase (LCP) has been widely recognized as a promising membrane-mimicking matrix for biophysical studies of membrane proteins and their crystallization in a lipidic environment. Application of this material to a wide variety of membrane proteins, however, is hindered due to a limited number of available host lipids, mostly monoacylglycerols (MAGs). Here, we designed, synthesized, and characterized a series of chemically stable lipids resistant to hydrolysis, with properties complementary to the widely used MAGs. In order to assess their potential to serve as host lipids for crystallization, we characterized the phase properties and lattice parameters of mesophases made of two most promising lipids at a variety of different conditions by polarized light microscopy and small-angle X-ray scattering. Both lipids showed remarkable chemical stability and an extended LCP region in the phase diagram covering a wide range of temperatures down to 4 °C. One of these lipids has been used for crystallization and structure determination of a prototypical membrane protein bacteriorhodopsin at 4 and 20 °C.

  4. Computational Approaches for Designing Protein/Inhibitor Complexes and Membrane Protein Variants

    Science.gov (United States)

    Vijayendran, Krishna Gajan

    Drug discovery of small-molecule protein inhibitors is a vast enterprise that involves several scientific disciplines (i.e. genomics, cell biology, x-ray crystallography, chemistry, computer science, statistics), with each discipline focusing on a particular aspect of the process. In this thesis, I use computational and experimental approaches to explore the most fundamental aspect of drug discovery: the molecular interactions of small-molecules inhibitors with proteins. In Part I (Chapters I and II), I describe how computational docking approaches can be used to identify structurally diverse molecules that can inhibit multiple protein targets in the brain. I illustrate this approach using the examples of microtubule-stabilizing agents and inhibitors of cyclooxygenase(COX)-I and 5-lipoxygenase (5-LOX). In Part II (Chapters III and IV), I focus on membrane proteins, which are notoriously difficult to work with due to their low natural abundances, low yields for heterologous over expression, and propensities toward aggregation. I describe a general approach for designing water-soluble variants of membrane proteins, for the purpose of developing cell-free, label-free, detergent-free, solution-phase studies of protein structure and small-molecule binding. I illustrate this approach through the design of a water-soluble variant of the membrane protein Smoothened, wsSMO. This wsSMO stands to serve as a first-step towards developing membrane protein analogs of this important signaling protein and drug target.

  5. Bcl-2 apoptosis proteins, mitochondrial membrane curvature, and cancer

    Science.gov (United States)

    Hwee Lai, Ghee; Schmidt, Nathan; Sanders, Lori; Mishra, Abhijit; Wong, Gerard; Ivashyna, Olena; Christenson, Eric; Schlesinger, Paul; Akabori, Kiyotaka; Santangelo, Christian

    2012-02-01

    Critical interactions between Bcl-2 family proteins permeabilize the outer mitochondrial membrane, a common decision point early in the intrinsic apoptotic pathway that irreversibly commits the cell to death. However, a unified picture integrating the essential non-passive role of lipid membranes with the contested dynamics of Bcl-2 regulation remains unresolved. Correlating results between synchrotron x-ray diffraction and microscopy in cell-free assays, we report activation of pro-apoptotic Bax induces strong pure negative Gaussian membrane curvature topologically necessary for pore formation and membrane remodeling events. Strikingly, Bcl-xL suppresses not only Bax-induced pore formation, but also membrane remodeling by disparate systems including cell penetrating, antimicrobial or viral fusion peptides, and bacterial toxin, none of which have BH3 allosteric domains to mediate direct binding. We propose a parallel mode of Bcl-2 pore regulation in which Bax and Bcl-xL induce antagonistic and mutually interacting Gaussian membrane curvatures. The universal nature of curvature-mediated interactions allows synergy with direct binding mechanisms, and potentially accounts for the Bcl-2 family modulation of mitochondrial fission/fusion dynamics.

  6. Mycoplasma hyopneumoniae-derived lipid-associated membrane proteins induce inflammation and apoptosis in porcine peripheral blood mononuclear cells in vitro.

    Science.gov (United States)

    Bai, Fangfang; Ni, Bo; Liu, Maojun; Feng, Zhixin; Xiong, Qiyan; Shao, Guoqing

    2015-01-30

    Mycoplasma hyopneumoniae is the causative agent of swine enzootic pneumonia (EP), a disease that causes considerable economic losss in swine industry. Lipid-associated membrane proteins (LAMPs) of mycoplasma play important roles in causing mycoplasma diseases. The present study explores the pathogenic mechanisms of M. hyopneumoniae LAMPs by elucidating their role in modulating the inflammation, apoptosis, and relevant signaling pathways of peripheral blood mononuclear cells (PBMCs) of pig. LAMP treatment inhibited the growth of PBMCs. Up-regulation of cytokines, such as IL-6 and IL-1β, as well as increased production of nitric oxide (NO) and superoxide anion were all detected in the supernatant of LAMPs-treated PBMCs. Furthermore, flow cytometric analysis using dual staining with annexin-V-FITC and propidium iodide (PI) showed that LAMPs of M. hyopneumoniae induced a time-dependent apoptosis in lymphocyts and monocytes from PBMCs, which was blocked by NOS inhibitor or antioxidant. In addition, LAMPs induced the phosphorylation of p38, the ratio of pro-apoptotic Bax protein to anti-apoptotic Bcl-2, activation of caspase-3 and caspase-8, and poly ADP-ribose polymerase (PARP) cleavage in PBMCs. These findings demonstrated that M. hyopneumoniae LAMPs induced the production of proinflammatory cytokines, NO and reactive oxygen species (ROS), and apoptosis of PBMCs in vitro through p38 MAPK and Bax/Bcl-2 signaling pathways, as well as caspase activation. Copyright © 2014 Elsevier B.V. All rights reserved.

  7. [Construction of epithelial membrane protein 1 eukaryotic expression vector and its influence on migration and invasion of human oral tongue squamous carcinoma cells].

    Science.gov (United States)

    Xiaohua, Dai; Jun, Zhang; Huiru, Zou; Xiaoli, Lian; Yanni, Li; Guanhua, Wang; Yan, Yan

    2016-08-01

    This study aimed to construct a eukaryotic expression vector pEGFP-N1-EMP1 of epithelial mem-brane protein 1 (EMP1) and investigate its influence on migration and invasion of human oral tongue squamous carcinoma cells. The human EMP1 gene was amplified by reverse transcription polymerase chain reaction and then ligated into the pEGFP-N1 vector by double restriction endonuclease digestion to construct pEGFP-N1-EMP1 recombinant plasmid. After sequencing identification, pEGFP-N1-EMP1 recombinant plasmid and pEGFP-N1 plasmid were transfected into human oral tongue squamous carcinoma Tb3.1 cell line. The expression of green fluorescent protein in cells was observed after transfection using an inverted fluorescence microscope. The overexpression of EMP1 mRNA was identified at 24, 48, and 72 h after transfection by real-time fluorescence quantitative polymerase chain reaction. The effect of EMP1 overexpression on migration and invasion of Tb3.1 cells was detected by Transwell assay. The full-length EMP1 gene sequence was successfully obtained. Sequence analysis showed that the EMP1 gene was inserted into the pEGFP-N1 vector correctly. Green fluorescence was observed in the transfected cells under fluorescence microscopy. The results of real-time fluorescence quantitative polymerase chain reaction indicated that the expression of EMP1 at 24 h after pEGFP-N1-EMP1 transfection was significantly higher than the other groups. Transwell assays indicated that overexpression of the EMP1 gene could significantly inhibit the migration and invasion ability of Tb3.1 cells. The eukaryotic expression vector of EMP1 was successfully constructed, and EMP1 overexpression was confirmed to inhibit the migration and inva-sion of oral tongue squamous carcinoma cells in vitro. This study laid a foundation for further investigation on the influence of the EMP1 gene on the metastasis of oral tongue squamous carcinoma and its molecular mechanism.
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  8. An auxin-binding protein is localized to the plasma membrane of maize coleoptile cells: Identification by photoaffinity labeling and purification of a 23-kDa polypeptide

    Energy Technology Data Exchange (ETDEWEB)

    Feldwisch, J.; Zettl, R.; Hesse, F.; Schell, J.; Palme, K. (Max-Planck-Inst. fuer Zuechtungsforschung, Koeln (West Germany))

    1992-01-15

    Plasma membrane vesicles were isolated from maize (Zea mays L.) coleoptile tissue by aqueous two-phase partitioning and assayed for homogeneity by the use of membrane-specific enzymatic assays. Using 5-azido-(7-{sup 3}H)indole-3-acetic acid (({sup 3}H)N{sub 3}IAA), the authors identified several IAA-binding proteins with the molecular masses of 60 kDa (pm60), 58 kDa (pm58), and 23 kDa (pm23). Using Triton X-114, they were able to selectively extract pm23 from the plasma membrane. They show that auxins and functional analogues compete with ({sup 3}H)N{sub 3}IAA for binding to pm23. They found that PAB130, a polyclonal antibody raised against auxin-binding protein 1 (ABP-1), recognized ABP-1 as well as pm23. This suggests that pm23 shares common epitopes with ABP-1. In addition, they identified an auxin-binding protein with a molecular mass of 24 kDa (pm24), which was detected in microsomal but not in plasma membrane vesicle preparations. Like pm23 this protein was extracted from membrane vesicles with Triton X-114. They designed a purification scheme allowing simultaneous purification of pm23 and pm24. Homogeneous pm23 and pm24 were obtained from coleoptile extracts after 7,000-fold purification.

  9. High-resolution atomic force microscopy and spectroscopy of native membrane proteins

    Science.gov (United States)

    Bippes, Christian A.; Muller, Daniel J.

    2011-08-01

    Membranes confining cells and cellular compartments are essential for life. Membrane proteins are molecular machines that equip cell membranes with highly sophisticated functionality. Examples of such functions are signaling, ion pumping, energy conversion, molecular transport, specific ligand binding, cell adhesion and protein trafficking. However, it is not well understood how most membrane proteins work and how the living cell regulates their function. We review how atomic force microscopy (AFM) can be applied for structural and functional investigations of native membrane proteins. High-resolution time-lapse AFM imaging records membrane proteins at work, their oligomeric state and their dynamic assembly. The AFM stylus resembles a multifunctional toolbox that allows the measurement of several chemical and physical parameters at the nanoscale. In the single-molecule force spectroscopy (SMFS) mode, AFM quantifies and localizes interactions in membrane proteins that stabilize their folding and modulate their functional state. Dynamic SMFS discloses fascinating insights into the free energy landscape of membrane proteins. Single-cell force spectroscopy quantifies the interactions of live cells with their environment to single-receptor resolution. In the future, technological progress in AFM-based approaches will enable us to study the physical nature of biological interactions in more detail and decipher how cells control basic processes.

  10. BAR domains, amphipathic helices and membrane-anchored proteins use the same mechanism to sense membrane curvature

    DEFF Research Database (Denmark)

    Madsen, Kenneth Lindegaard; Bhatia, V K; Gether, U

    2010-01-01

    The internal membranes of eukaryotic cells are all twists and bends characterized by high curvature. During recent years it has become clear that specific proteins sustain these curvatures while others simply recognize membrane shape and use it as "molecular information" to organize cellular proc...... on curved membranes instead of higher affinity as assumed so far. Finally, we integrate these new insights into the debate about which motifs are involved in sensing versus induction of membrane curvature and what role MCS proteins may play in biology....

  11. Green fluorescent protein-based expression screening of membrane proteins in Escherichia coli.

    Science.gov (United States)

    Bird, Louise E; Rada, Heather; Verma, Anil; Gasper, Raphael; Birch, James; Jennions, Matthew; Lӧwe, Jan; Moraes, Isabel; Owens, Raymond J

    2015-01-06

    The production of recombinant membrane proteins for structural and functional studies remains technically challenging due to low levels of expression and the inherent instability of many membrane proteins once solubilized in detergents. A protocol is described that combines ligation independent cloning of membrane proteins as GFP fusions with expression in Escherichia coli detected by GFP fluorescence. This enables the construction and expression screening of multiple membrane protein/variants to identify candidates suitable for further investment of time and effort. The GFP reporter is used in a primary screen of expression by visualizing GFP fluorescence following SDS polyacrylamide gel electrophoresis (SDS-PAGE). Membrane proteins that show both a high expression level with minimum degradation as indicated by the absence of free GFP, are selected for a secondary screen. These constructs are scaled and a total membrane fraction prepared and solubilized in four different detergents. Following ultracentrifugation to remove detergent-insoluble material, lysates are analyzed by fluorescence detection size exclusion chromatography (FSEC). Monitoring the size exclusion profile by GFP fluorescence provides information about the mono-dispersity and integrity of the membrane proteins in different detergents. Protein: detergent combinations that elute with a symmetrical peak with little or no free GFP and minimum aggregation are candidates for subsequent purification. Using the above methodology, the heterologous expression in E. coli of SED (shape, elongation, division, and sporulation) proteins from 47 different species of bacteria was analyzed. These proteins typically have ten transmembrane domains and are essential for cell division. The results show that the production of the SEDs orthologues in E. coli was highly variable with respect to the expression levels and integrity of the GFP fusion proteins. The experiment identified a subset for further investigation.

  12. MODIFICATION OF ERYTHROCYTE MEMBRANE PROTEINS WITH POLYETHYLENE GLYCOL 1500

    Directory of Open Access Journals (Sweden)

    N. G. Zemlianskykh

    2016-10-01

    Full Text Available The aim of the work was to study the effect of polyethylene glycol PEG-1500 on the Ca2+-ATPase activity and changes in CD44 surface marker expression in human erythrocyte membranes. Determination of the Ca2+-ATPase activity was carried out in sealed erythrocyte ghosts by the level of accumulation of inorganic phosphorus. Changes in the expression of CD44 and amount of CD44+-erythrocytes were evaluated by flow cytometry. The inhibition of Ca2+-ATPase activity and a reduction in the level of CD44 expression and also the decrease in the amount CD44+-cells were found, reflecting a fairly complex restructuring in the membrane-cytoskeleton complex of erythrocytes under the influence of PEG-1500. Effect of PEG-1500 on the surface CD44 marker could be mediated by modification of proteins of membrane-cytoskeleton complex, as indicated by accelerated loss of CD44 in erythrocyte membranes after application of protein cross-linking reagent diamide. Reduced activity of Ca2+-ATPase activity may contribute to the increase in intracellular Ca2+ level and thus leads to a modification of interactions of integral proteins with cytoskeletal components that eventually could result in membrane vesiculation and decreasing in expression of the CD44 marker, which is dynamically linked to the cytoskeleton.

  13. Dendronic trimaltoside amphiphiles (DTMs) for membrane protein study

    DEFF Research Database (Denmark)

    Sadaf, Aiman; Du, Yang; Santillan, Claudia

    2017-01-01

    The critical contribution of membrane proteins in normal cellular function makes their detailed structure and functional analysis essential. Detergents, amphipathic agents with the ability to maintain membrane proteins in a soluble state in aqueous solution, have key roles in membrane protein...... alkyl chains by introducing dendronic hydrophobic groups connected to a trimaltoside head group, designated dendronic trimaltosides (DTMs). Representative DTMs conferred enhanced stabilization to multiple membrane proteins compared to the benchmark conventional detergent, DDM. One DTM (i.e., DTM-A6...

  14. Outer membrane proteins of pathogenic spirochetes.

    Science.gov (United States)

    Cullen, Paul A; Haake, David A; Adler, Ben

    2004-06-01

    Pathogenic spirochetes are the causative agents of several important diseases including syphilis, Lyme disease, leptospirosis, swine dysentery, periodontal disease and some forms of relapsing fever. Spirochetal bacteria possess two membranes and the proteins present in the outer membrane are at the site of interaction with host tissue and the immune system. This review describes the current knowledge in the field of spirochetal outer membrane protein (OMP) biology. What is known concerning biogenesis and structure of OMPs, with particular regard to the atypical signal peptide cleavage sites observed amongst the spirochetes, is discussed. We examine the functions that have been determined for several spirochetal OMPs including those that have been demonstrated to function as adhesins, porins or to have roles in complement resistance. A detailed description of the role of spirochetal OMPs in immunity, including those that stimulate protective immunity or that are involved in antigenic variation, is given. A final section is included which covers experimental considerations in spirochetal outer membrane biology. This section covers contentious issues concerning cellular localization of putative OMPs, including determination of surface exposure. A more detailed knowledge of spirochetal OMP biology will hopefully lead to the design of new vaccines and a better understanding of spirochetal pathogenesis.

  15. An approach to membrane protein structure without crystals

    Science.gov (United States)

    Sorgen, Paul L.; Hu, Yonglin; Guan, Lan; Kaback, H. Ronald; Girvin, Mark E.

    2002-01-01

    The lactose permease of Escherichia coli catalyzes coupled translocation of galactosides and H+ across the cell membrane. It is the best-characterized member of the Major Facilitator Superfamily, a related group of membrane proteins with 12 transmembrane domains that mediate transport of various substrates across cell membranes. Despite decades of effort and their functional importance in all kingdoms of life, no high-resolution structures have been solved for any member of this family. However, extensive biochemical, genetic, and biophysical studies on lactose permease have established its transmembrane topology, secondary structure, and numerous interhelical contacts. Here we demonstrate that this information is sufficient to calculate a structural model at the level of helix packing or better. PMID:12391320

  16. Fuel-Cell Structure Prevents Membrane Drying

    Science.gov (United States)

    Mcelroy, J.

    1986-01-01

    Embossed plates direct flows of reactants and coolant. Membrane-type fuel-cell battery has improved reactant flow and heat removal. Compact, lightweight battery produces high current and power without drying of membranes.

  17. Serial Millisecond Crystallography of Membrane Proteins.

    Science.gov (United States)

    Jaeger, Kathrin; Dworkowski, Florian; Nogly, Przemyslaw; Milne, Christopher; Wang, Meitian; Standfuss, Joerg

    2016-01-01

    Serial femtosecond crystallography (SFX) at X-ray free-electron lasers (XFELs) is a powerful method to determine high-resolution structures of pharmaceutically relevant membrane proteins. Recently, the technology has been adapted to carry out serial millisecond crystallography (SMX) at synchrotron sources, where beamtime is more abundant. In an injector-based approach, crystals grown in lipidic cubic phase (LCP) or embedded in viscous medium are delivered directly into the unattenuated beam of a microfocus beamline. Pilot experiments show the application of microjet-based SMX for solving the structure of a membrane protein and compatibility of the method with de novo phasing. Planned synchrotron upgrades, faster detectors and software developments will go hand-in-hand with developments at free-electron lasers to provide a powerful methodology for solving structures from microcrystals at room temperature, ligand screening or crystal optimization for time-resolved studies with minimal or no radiation damage.

  18. The fusion-related hydrophobic domain of Sendai F protein can be moved through the cytoplasmic membrane of Escherichia coli.

    OpenAIRE

    Davis, N G; Hsu, M C

    1986-01-01

    Recent work on a prokaryotic membrane protein, gene III protein (pIII) of coliphage f1, showed that polypeptide segments of sufficient hydrophobicity functioned to stop transfer of the polypeptide across the cell membrane: strings of 16 or more hydrophobic amino acids sufficed. A fusion-related hydrophobic domain (FRHD) of Sendai F protein, a sequence of 26 consecutive uncharged residues, has been implicated in the fusion of the viral membrane envelope and the target-cell membrane through a h...

  19. Vesicle-associated membrane protein 2 mediates trafficking of {alpha}5{beta}1 integrin to the plasma membrane

    Energy Technology Data Exchange (ETDEWEB)

    Hasan, Nazarul [Department of Biochemistry and Molecular Biology, University of Louisville School of Medicine, 319 Abraham Flexner Way, Room 515, Louisville, KY 40202 (United States); Hu, Chuan, E-mail: chuan.hu@louisville.edu [Department of Biochemistry and Molecular Biology, University of Louisville School of Medicine, 319 Abraham Flexner Way, Room 515, Louisville, KY 40202 (United States)

    2010-01-01

    Integrins are major receptors for cell adhesion to the extracellular matrix (ECM). As transmembrane proteins, the levels of integrins at the plasma membrane or the cell surface are ultimately determined by the balance between two vesicle trafficking events: endocytosis of integrins at the plasma membrane and exocytosis of the vesicles that transport integrins. Here, we report that vesicle-associated membrane protein 2 (VAMP2), a SNARE protein that mediates vesicle fusion with the plasma membrane, is involved in the trafficking of {alpha}5{beta}1 integrin. VAMP2 was present on vesicles containing endocytosed {beta}1 integrin. Small interfering RNA (siRNA) silencing of VAMP2 markedly reduced cell surface {alpha}5{beta}1 and inhibited cell adhesion and chemotactic migration to fibronectin, the ECM ligand of {alpha}5{beta}1, without altering cell surface expression of {alpha}2{beta}1 integrin or {alpha}3{beta}1 integrin. By contrast, silencing of VAMP8, another SNARE protein, had no effect on cell surface expression of the integrins or cell adhesion to fibronectin. In addition, VAMP2-mediated trafficking is involved in cell adhesion to collagen but not to laminin. Consistent with disruption of integrin functions in cell proliferation and survival, VAMP2 silencing diminished proliferation and triggered apoptosis. Collectively, these data indicate that VAMP2 mediates the trafficking of {alpha}5{beta}1 integrin to the plasma membrane and VAMP2-dependent integrin trafficking is critical in cell adhesion, migration and survival.

  20. Outer membrane protein A (OmpA of Shigella flexneri 2a induces TLR2-mediated activation of B cells: involvement of protein tyrosine kinase, ERK and NF-κB.

    Directory of Open Access Journals (Sweden)

    Rajsekhar Bhowmick

    Full Text Available B cells are critically important in combating bacterial infections and their differentiation into plasma cells and memory cells aids bacterial clearance and long-lasting immunity conferred by essentially all vaccines. Outer membrane protein A (OmpA of Shigella flexneri 2a has been demonstrated to induce the production of IgG and IgA in vivo following immunization of mice through intranasal route, but the direct involvement of B cells in OmpA-mediated immune regulation was not determined. Consequently, we investigated whether OmpA can modulate B cell functions and identified the molecular events involved in OmpA-induced B cell immune response in vitro. We show that OmpA of S. flexneri 2a activates B cells to produce protective cytokines, IL-6 and IL-10 as well as facilitates their differentiation into antibody secreting cells (ASCs. The immunostimulatory properties of OmpA are attributed to the increased surface expression of MHCII and CD86 on B cells. We also report here that B cell activation by OmpA is mediated strictly through recognition by TLR2, resulting in initiation of cascades of signal transduction events, involving increased phosphorylation of protein tyrosine kinases (PTKs, ERK and IκBα, leading to nuclear translocation of NF-κB. Importantly, a TLR2 antibody diminishes OmpA-induced upregulation of MHCII and CD86 on B cell surface as well as significantly inhibits B cell differentiation and cytokine secretion. Furthermore, we illustrate that B cell differentiation into ASCs and induction of cytokine secretion by OmpA are dependent on PTKs activity. Moreover, we identify that OmpA-induced B cell differentiation is entirely dependent on ERK pathway, whereas both NF-κB and ERK are essential for cytokine secretion by B cells. Overall, our data demonstrate that OmpA of S. flexneri 2a amplifies TLR signaling in B cells and triggers B cell immune response, which is critical for the development of an effective adaptive immunity to an

  1. Membrane re-modelling by BAR domain superfamily proteins via molecular and non-molecular factors.

    Science.gov (United States)

    Nishimura, Tamako; Morone, Nobuhiro; Suetsugu, Shiro

    2018-03-14

    Lipid membranes are structural components of cell surfaces and intracellular organelles. Alterations in lipid membrane shape are accompanied by numerous cellular functions, including endocytosis, intracellular transport, and cell migration. Proteins containing Bin-Amphiphysin-Rvs (BAR) domains (BAR proteins) are unique, because their structures correspond to the membrane curvature, that is, the shape of the lipid membrane. BAR proteins present at high concentration determine the shape of the membrane, because BAR domain oligomers function as scaffolds that mould the membrane. BAR proteins co-operate with various molecular and non-molecular factors. The molecular factors include cytoskeletal proteins such as the regulators of actin filaments and the membrane scission protein dynamin. Lipid composition, including saturated or unsaturated fatty acid tails of phospholipids, also affects the ability of BAR proteins to mould the membrane. Non-molecular factors include the external physical forces applied to the membrane, such as tension and friction. In this mini-review, we will discuss how the BAR proteins orchestrate membrane dynamics together with various molecular and non-molecular factors. © 2018 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.

  2. Formation of Cell Membrane Component Domains in Artificial Lipid Bilayer.

    Science.gov (United States)

    Tero, Ryugo; Fukumoto, Kohei; Motegi, Toshinori; Yoshida, Miyu; Niwano, Michio; Hirano-Iwata, Ayumi

    2017-12-20

    The lipid bilayer environment around membrane proteins strongly affects their structure and functions. Here, we aimed to study the fusion of proteoliposomes (PLs) derived from cultured cells with an artificial lipid bilayer membrane and the distribution of the PL components after the fusion. PLs, which were extracted as a crude membrane fraction from Chinese hamster ovary (CHO) cells, formed isolated domains in a supported lipid bilayer (SLB), comprising phosphatidylcholine (PC), phosphatidylethanolamine (PE), and cholesterol (Chol), after the fusion. Observation with a fluorescence microscope and an atomic force microscope showed that the membrane fusion occurred selectively at microdomains in the PC + PE + Chol-SLB, and that almost all the components of the PL were retained in the domain. PLs derived from human embryonic kidney 293 (HEK) cells also formed isolated domains in the PC + PE + Chol-SLB, but their fusion kinetics was different from that of the CHO-PLs. We attempted to explain the mechanism of the PL-SLB fusion and the difference between CHO- and HEK-PLs, based on a kinetic model. The domains that contained the whole cell membrane components provided environments similar to that of natural cell membranes, and were thus effective for studying membrane proteins using artificial lipid bilayer membranes.

  3. Heterologous expression of membrane proteins: choosing the appropriate host.

    Directory of Open Access Journals (Sweden)

    Florent Bernaudat

    Full Text Available BACKGROUND: Membrane proteins are the targets of 50% of drugs, although they only represent 1% of total cellular proteins. The first major bottleneck on the route to their functional and structural characterisation is their overexpression; and simply choosing the right system can involve many months of trial and error. This work is intended as a guide to where to start when faced with heterologous expression of a membrane protein. METHODOLOGY/PRINCIPAL FINDINGS: The expression of 20 membrane proteins, both peripheral and integral, in three prokaryotic (E. coli, L. lactis, R. sphaeroides and three eukaryotic (A. thaliana, N. benthamiana, Sf9 insect cells hosts was tested. The proteins tested were of various origins (bacteria, plants and mammals, functions (transporters, receptors, enzymes and topologies (between 0 and 13 transmembrane segments. The Gateway system was used to clone all 20 genes into appropriate vectors for the hosts to be tested. Culture conditions were optimised for each host, and specific strategies were tested, such as the use of Mistic fusions in E. coli. 17 of the 20 proteins were produced at adequate yields for functional and, in some cases, structural studies. We have formulated general recommendations to assist with choosing an appropriate system based on our observations of protein behaviour in the different hosts. CONCLUSIONS/SIGNIFICANCE: Most of the methods presented here can be quite easily implemented in other laboratories. The results highlight certain factors that should be considered when selecting an expression host. The decision aide provided should help both newcomers and old-hands to select the best system for their favourite membrane protein.

  4. A Peptidomimetic Antibiotic Targets Outer Membrane Proteins and Disrupts Selectively the Outer Membrane in Escherichia coli.

    Science.gov (United States)

    Urfer, Matthias; Bogdanovic, Jasmina; Lo Monte, Fabio; Moehle, Kerstin; Zerbe, Katja; Omasits, Ulrich; Ahrens, Christian H; Pessi, Gabriella; Eberl, Leo; Robinson, John A

    2016-01-22

    Increasing antibacterial resistance presents a major challenge in antibiotic discovery. One attractive target in Gram-negative bacteria is the unique asymmetric outer membrane (OM), which acts as a permeability barrier that protects the cell from external stresses, such as the presence of antibiotics. We describe a novel β-hairpin macrocyclic peptide JB-95 with potent antimicrobial activity against Escherichia coli. This peptide exhibits no cellular lytic activity, but electron microscopy and fluorescence studies reveal an ability to selectively disrupt the OM but not the inner membrane of E. coli. The selective targeting of the OM probably occurs through interactions of JB-95 with selected β-barrel OM proteins, including BamA and LptD as shown by photolabeling experiments. Membrane proteomic studies reveal rapid depletion of many β-barrel OM proteins from JB-95-treated E. coli, consistent with induction of a membrane stress response and/or direct inhibition of the Bam folding machine. The results suggest that lethal disruption of the OM by JB-95 occurs through a novel mechanism of action at key interaction sites within clusters of β-barrel proteins in the OM. These findings open new avenues for developing antibiotics that specifically target β-barrel proteins and the integrity of the Gram-negative OM. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  5. In-membrane micro fuel cell

    Energy Technology Data Exchange (ETDEWEB)

    Omosebi, Ayokunle; Besser, Ronald

    2016-09-06

    An in-membrane micro fuel cell comprises an electrically-insulating membrane that is permissive to the flow of cations, such as protons, and a pair of electrodes deposited on channels formed in the membrane. The channels are arranged as conduits for fluids, and define a membrane ridge between the channels. The electrodes are porous and include catalysts for promoting the liberation of a proton and an electron from a chemical species and/or or the recombination of a proton and an electron with a chemical specie. The fuel cell may be provided a biosensor, an electrochemical sensor, a microfluidic device, or other microscale devices fabricated in the fuel cell membrane.

  6. Silica nanoparticles for the oriented encapsulation of membrane proteins into artificial bilayer lipid membranes.

    Science.gov (United States)

    Schadauer, Florian; Geiss, Andreas F; Srajer, Johannes; Siebenhofer, Bernhard; Frank, Pinar; Reiner-Rozman, Ciril; Ludwig, Bernd; Richter, Oliver-M H; Nowak, Christoph; Naumann, Renate L C

    2015-03-03

    An artificial bilayer lipid membrane system is presented, featuring the oriented encapsulation of membrane proteins in a functionally active form. Nickel nitrilo-triacetic acid-functionalized silica nanoparticles, of a diameter of around 25 nm, are used to attach the proteins via a genetically engineered histidine tag in a uniform orientation. Subsequently, the proteins are reconstituted within a phospholipid bilayer, formed around the particles by in situ dialysis to form so-called proteo-lipobeads (PLBs). With a final size of about 50 nm, the PLBs can be employed for UV/vis spectroscopy studies, particularly of multiredox center proteins, because the effects of light scattering are negligible. As a proof of concept, we use cytochrome c oxidase (CcO) from P. denitrificans with the his tag genetically engineered to subunit I. In this orientation, the P side of CcO is directed to the outside and hence electron transfer can be initiated by reduced cytochrome c (cc). UV/vis measurements are used in order to determine the occupancy by CcO molecules encapsulated in the lipid bilayer as well as the kinetics of electron transfer between CcO and cc. The kinetic data are analyzed in terms of the Michaelis-Menten kinetics showing that the turnover rate of CcO is significantly decreased compared to that of solubilized protein, whereas the binding characteristics are improved. The data demonstrate the suitability of PLBs for functional cell-free bioassays of membrane proteins.

  7. High efficiency penetration of antibody-immobilized nanoneedle thorough plasma membrane for in situ detection of cytoskeletal proteins in living cells.

    Science.gov (United States)

    Kawamura, R; Shimizu, K; Matsumoto, Y; Yamagishi, A; Silberberg, Y R; Iijima, M; Kuroda, S; Fukazawa, K; Ishihara, K; Nakamura, C

    2016-11-03

    The field of structural dynamics of cytoskeletons in living cells is gathering wide interest, since better understanding of cytoskeleton intracellular organization will provide us with not only insights into basic cell biology but may also enable development of new strategies in regenerative medicine and cancer therapy, fields in which cytoskeleton-dependent dynamics play a pivotal role. The nanoneedle technology is a powerful tool allowing for intracellular investigations, as it can be directly inserted into live cells by penetrating through the plasma membrane causing minimal damage to cells, under the precise manipulation using atomic force microscope. Modifications of the nanoneedles using antibodies have allowed for accurate mechanical detection of various cytoskeletal components, including actin, microtubules and intermediate filaments. However, successful penetration of the nanoneedle through the plasma membrane has been shown to vary greatly between different cell types and conditions. In an effort to overcome this problem and improve the success rate of nanoneedle insertion into the live cells, we have focused here on the fluidity of the membrane lipid bilayer, which may hinder nanoneedle penetration into the cytosolic environment. We aimed to reduce apparent fluidity of the membrane by either increasing the approach velocity or reducing experimental temperatures. Although changes in approach velocity did not have much effect, lowering the temperature was found to greatly improve the detection of unbinding forces, suggesting that alteration in the plasma membrane fluidity led to increase in nanoneedle penetration. Operation at a lower temperature of 4 °C greatly improved the success rate of nanoneedle insertion to live cells at an optimized approach velocity, while it did not affect the binding of antibodies immobilized on the nanoneedle to vimentins for mechanical detection. As these experimental parameters can be applied to various cell types, these

  8. Quantification of detergent using colorimetric methods in membrane protein crystallography.

    Science.gov (United States)

    Prince, Chelsy; Jia, Zongchao

    2015-01-01

    Membrane protein crystallography has the potential to greatly aid our understanding of membrane protein biology. Yet, membrane protein crystals remain challenging to produce. Although robust methods for the expression and purification of membrane proteins continue to be developed, the detergent component of membrane protein samples is equally important to crystallization efforts. This chapter describes the development of three colorimetric assays for the quantitation of detergent in membrane protein samples and provides detailed protocols. All of these techniques use small sample volumes and have potential applications in crystallography. The application of these techniques in crystallization prescreening, detergent concentration modification, and detergent exchange experiments is demonstrated. It has been observed that the concentration of detergent in a membrane protein sample can be just as important as the protein concentration when attempting to reproduce crystallization lead conditions. © 2015 Elsevier Inc. All rights reserved.

  9. Magic-Angle-Spinning Solid-State NMR of Membrane Proteins

    NARCIS (Netherlands)

    Baker, Lindsay A.; Folkers, Gert E.; Sinnige, Tessa; Houben, Klaartje; Kaplan, M.; van der Cruijsen, Elwin A W; Baldus, Marc

    2015-01-01

    Solid-state NMR spectroscopy (ssNMR) provides increasing possibilities to examine membrane proteins in different molecular settings, ranging from synthetic bilayers to whole cells. This flexibility often enables ssNMR experiments to be directly correlated with membrane protein function. In this

  10. Chorioamnionitis and diagnostic value of C-reactive protein, erythrocyte sedimentation rate and white blood cell count in its diagnosis among pregnant women with premature rupture of membranes.

    Science.gov (United States)

    Amirabi, Afsaneh; Naji, Siamak; Yekta, Zahra; Sadeghi, Yeganeh

    2012-05-01

    Several laboratory parameters have been used in these studies to diagnose chorioamnionitis leading to controversies to some extent. The aim of this study was to assess the diagnostic value of C-reactive protein (CRP), erythrocyte sedimentation rate (ESR) and white blood cell (WBC) count in chorioamnionitis among pregnant women with premature rupture of membranes (PROM). In a cross-sectional diagnostic test research, 71 patients presented with PROM before the 37th week of gestation were enrolled. A blood sample was taken from all the patients. Hematologic automatic blood cell counter was used to count the blood cells and their differentiation. ESR and CRP were also measured using the same blood sample at the laboratory. Sensitivity, specificity, correct classification rate and likelihood ratios were calculated. Receiver operating curves were plotted and area under curve was estimated along with its 95% confidence interval. A total of 71 patients were studied. None of the patients had a positive drug history or a history of hypertension before the 20th week of gestation or during her previous pregnancy. Contrary to ESR, WBC count and CRP results didn't provide minimum acceptable diagnostic accuracy measures for diagnosis of chorioamnionitis. The sensitivity and specificity of a positive ESR test at a cutoff value of 52 were 66.7 and 60%, respectively. The area under curve was calculated to be 0.62. The findings of the present study were not supportive of using CRP, WBC as a reliable diagnostic test to identify chorioamnionitis in women with PROM. The results of CRP and WBC were not acceptable but ESR diagnostic value was minimally acceptable.

  11. Atomic-level Analysis of Membrane Protein Structure

    OpenAIRE

    Hendrickson, Wayne A.

    2016-01-01

    Membrane proteins are substantially more challenging than natively soluble proteins as subjects for structural analysis. Thus, membrane proteins are greatly under-represented in structural databases. Recently, as a consequence of focused attention by consortium efforts and advances in methodology, the pace has accelerated for atomic-level structure determination of membrane proteins. Enabling advances have come in methods for protein production, for crystallographic analysis, and for cryo-EM ...

  12. Amphipathic agents for membrane protein study.

    Science.gov (United States)

    Sadaf, Aiman; Cho, Kyung Ho; Byrne, Bernadette; Chae, Pil Seok

    2015-01-01

    Membrane proteins (MPs) are insoluble in aqueous media as a result of incompatibility between the hydrophilic property of the solvent molecules and the hydrophobic nature of MP surfaces, normally associated with lipid membranes. Amphipathic compounds are necessary for extraction of these macromolecules from the native membranes and their maintenance in solution. The amphipathic agents surround the hydrophobic segments of MPs, thus serving as a membrane mimetic system. Of the available amphipathic agents, detergents are most widely used for MP manipulation. However, MPs encapsulated by conventional detergent micelles have a tendency to undergo structural degradation, hampering MP advance, and necessitating the development of novel detergents with enhanced efficacy for MP study. In this chapter, we will introduce both conventional and novel classes of detergents and discuss about the chemical structures, design principles, and efficacies of these compounds for MP solubilization and stabilization. The behaviors of those agents toward MP crystallization will be a primary topic in our discussion. This discussion highlights the common features of popular conventional/novel detergents essential for successful MP structural study. The conclusions reached by this discussion would not only enable MP scientists to rationally select a set of detergent candidates among a large number of detergents but also provide detergent inventors with useful guidelines in designing novel amphipathic systems. © 2015 Elsevier Inc. All rights reserved.

  13. The impact of hemodialysis on erythrocyte membrane cytoskeleton proteins

    Directory of Open Access Journals (Sweden)

    Maria Olszewska

    2015-02-01

    Full Text Available Background: Hemodialysis (HD is one of the methods of renal replacement therapy, but it also contributes to an increase in oxidative stress. Hemodialysis leads to changes in the erythrocyte cytoskeleton structure, whilst the presence of glucose in the dialysis fluid which activates the pentose phosphate pathway contributes to the intensification of oxidative stress. Available literature lacks reports on the effect of glucose in the dialytic fluid on the composition of proteins of the cell membrane cytoskeleton.Material/Methods: Red blood cells for this analysis were collected from patients with chronic renal failure treated with hemodialysis using both glucose-containing and glucose-free dialysis fluid. Following the preparation of membranes, the electrophoretic separation of proteins was performed in denaturing conditions according to Laemmli. The level of tryptophan in membranes was determined by spectrofluorimetry, whilst the activity of glucose-6-phosphate dehydrogenase was determined by measuring the reduction of oxidated NADP.Results: Hemodialysis in both groups of patients resulted in a statistically significant reduction of tryptophan as an oxidative stress indicator when compared to the control group. Moreover, the activity of glucose-6-phosphate dehydrogenase in the group of patients was higher than in the control group, and following the HD procedure it decreased, which may have been caused by a reduced concentration of dialyzed glucose. The HD procedure affects the structure of the erythrocyte membrane cytoskeleton, which is reflected in the concentration changes in individual proteins and in their mutual relationships corresponding to vertical and horizontal interactions stabilizing the structure of the erythrocyte membrane cytoskeleton. These changes may contribute to the shortening of cell lifespan.

  14. Stochastic lattice model of synaptic membrane protein domains

    Science.gov (United States)

    Li, Yiwei; Kahraman, Osman; Haselwandter, Christoph A.

    2017-05-01

    Neurotransmitter receptor molecules, concentrated in synaptic membrane domains along with scaffolds and other kinds of proteins, are crucial for signal transmission across chemical synapses. In common with other membrane protein domains, synaptic domains are characterized by low protein copy numbers and protein crowding, with rapid stochastic turnover of individual molecules. We study here in detail a stochastic lattice model of the receptor-scaffold reaction-diffusion dynamics at synaptic domains that was found previously to capture, at the mean-field level, the self-assembly, stability, and characteristic size of synaptic domains observed in experiments. We show that our stochastic lattice model yields quantitative agreement with mean-field models of nonlinear diffusion in crowded membranes. Through a combination of analytic and numerical solutions of the master equation governing the reaction dynamics at synaptic domains, together with kinetic Monte Carlo simulations, we find substantial discrepancies between mean-field and stochastic models for the reaction dynamics at synaptic domains. Based on the reaction and diffusion properties of synaptic receptors and scaffolds suggested by previous experiments and mean-field calculations, we show that the stochastic reaction-diffusion dynamics of synaptic receptors and scaffolds provide a simple physical mechanism for collective fluctuations in synaptic domains, the molecular turnover observed at synaptic domains, key features of the observed single-molecule trajectories, and spatial heterogeneity in the effective rates at which receptors and scaffolds are recycled at the cell membrane. Our work sheds light on the physical mechanisms and principles linking the collective properties of membrane protein domains to the stochastic dynamics that rule their molecular components.

  15. Stochastic lattice model of synaptic membrane protein domains.

    Science.gov (United States)

    Li, Yiwei; Kahraman, Osman; Haselwandter, Christoph A

    2017-05-01

    Neurotransmitter receptor molecules, concentrated in synaptic membrane domains along with scaffolds and other kinds of proteins, are crucial for signal transmission across chemical synapses. In common with other membrane protein domains, synaptic domains are characterized by low protein copy numbers and protein crowding, with rapid stochastic turnover of individual molecules. We study here in detail a stochastic lattice model of the receptor-scaffold reaction-diffusion dynamics at synaptic domains that was found previously to capture, at the mean-field level, the self-assembly, stability, and characteristic size of synaptic domains observed in experiments. We show that our stochastic lattice model yields quantitative agreement with mean-field models of nonlinear diffusion in crowded membranes. Through a combination of analytic and numerical solutions of the master equation governing the reaction dynamics at synaptic domains, together with kinetic Monte Carlo simulations, we find substantial discrepancies between mean-field and stochastic models for the reaction dynamics at synaptic domains. Based on the reaction and diffusion properties of synaptic receptors and scaffolds suggested by previous experiments and mean-field calculations, we show that the stochastic reaction-diffusion dynamics of synaptic receptors and scaffolds provide a simple physical mechanism for collective fluctuations in synaptic domains, the molecular turnover observed at synaptic domains, key features of the observed single-molecule trajectories, and spatial heterogeneity in the effective rates at which receptors and scaffolds are recycled at the cell membrane. Our work sheds light on the physical mechanisms and principles linking the collective properties of membrane protein domains to the stochastic dynamics that rule their molecular components.

  16. Translocation of microfilament-associated inhibitory guanine-nucleotide-binding proteins to the plasma membrane in myeloid differentiated human leukemia (HL-60) cells

    NARCIS (Netherlands)

    Meyer zu Heringdorf, D.; Liedel, K.; Kaldenberg-Stasch, S.; Michel, M. C.; Jakobs, K. H.; Wieland, T.

    1996-01-01

    The cytoskeletal localization of inhibitory guanine-nucleotide-binding (Gi) proteins and the coupling of these proteins to formyl peptide receptors were studied in myeloid differentiated human leukemia (HL-60) cells. Treatment of HL-60 cells with cytochalasin B or botulinum C2 toxin, which leads to

  17. Membrane alterations induced by nonstructural proteins of human norovirus.

    Directory of Open Access Journals (Sweden)

    Sylvie Y Doerflinger

    2017-10-01

    Full Text Available Human noroviruses (huNoV are the most frequent cause of non-bacterial acute gastroenteritis worldwide, particularly genogroup II genotype 4 (GII.4 variants. The viral nonstructural (NS proteins encoded by the ORF1 polyprotein induce vesical clusters harboring the viral replication sites. Little is known so far about the ultrastructure of these replication organelles or the contribution of individual NS proteins to their biogenesis. We compared the ultrastructural changes induced by expression of norovirus ORF1 polyproteins with those induced upon infection with murine norovirus (MNV. Characteristic membrane alterations induced by ORF1 expression resembled those found in MNV infected cells, consisting of vesicle accumulations likely built from the endoplasmic reticulum (ER which included single membrane vesicles (SMVs, double membrane vesicles (DMVs and multi membrane vesicles (MMVs. In-depth analysis using electron tomography suggested that MMVs originate through the enwrapping of SMVs with tubular structures similar to mechanisms reported for picornaviruses. Expression of GII.4 NS1-2, NS3 and NS4 fused to GFP revealed distinct membrane alterations when analyzed by correlative light and electron microscopy. Expression of NS1-2 induced proliferation of smooth ER membranes forming long tubular structures that were affected by mutations in the active center of the putative NS1-2 hydrolase domain. NS3 was associated with ER membranes around lipid droplets (LDs and induced the formation of convoluted membranes, which were even more pronounced in case of NS4. Interestingly, NS4 was the only GII.4 protein capable of inducing SMV and DMV formation when expressed individually. Our work provides the first ultrastructural analysis of norovirus GII.4 induced vesicle clusters and suggests that their morphology and biogenesis is most similar to picornaviruses. We further identified NS4 as a key factor in the formation of membrane alterations of huNoV and

  18. The synthesis of recombinant membrane proteins in yeast for structural studies.

    Science.gov (United States)

    Routledge, Sarah J; Mikaliunaite, Lina; Patel, Anjana; Clare, Michelle; Cartwright, Stephanie P; Bawa, Zharain; Wilks, Martin D B; Low, Floren; Hardy, David; Rothnie, Alice J; Bill, Roslyn M

    2016-02-15

    Historically, recombinant membrane protein production has been a major challenge meaning that many fewer membrane protein structures have been published than those of soluble proteins. However, there has been a recent, almost exponential increase in the number of membrane protein structures being deposited in the Protein Data Bank. This suggests that empirical methods are now available that can ensure the required protein supply for these difficult targets. This review focuses on methods that are available for protein production in yeast, which is an important source of recombinant eukaryotic membrane proteins. We provide an overview of approaches to optimize the expression plasmid, host cell and culture conditions, as well as the extraction and purification of functional protein for crystallization trials in preparation for structural studies. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.

  19. Flippase activity in proteoliposomes reconstituted with Spinacea oleracea endoplasmic reticulum membrane proteins: evidence of biogenic membrane flippase in plants.

    Science.gov (United States)

    Sahu, Santosh Kumar; Gummadi, Sathyanarayana N

    2008-09-30

    Phospholipid translocation (flip-flop) in biogenic (self-synthesizing) membranes such as the endoplasmic reticulum of eukaryotic cells (rat liver) and bacterial cytoplasmic membranes is a fundamental step in membrane biogenesis. It is known that flip-flop in these membranes occurs without a metabolic energy requirement, bidirectionally with no specificity for phospholipid headgroup. In this study, we demonstrate for the first time ATP-independent flippase activity in endoplasmic reticulum membranes of plants using spinach as a model system. For this, we generated proteoliposomes from a Triton X-100 extract of endoplasmic reticulum membranes of spinach and assayed them for flippase activity using fluorescently labeled phospholipids. The half-time for flipping was found to be 0.7-1.0 min. We also show that (a) proteoliposomes can flip fluorescently labeled analogues of phosphatidylcholine and phosphatidylethanolamine, (b) flipping activity is protein-mediated, (c) more than one class of lipid translocator (flippase) is present in spinach membranes, based on the sensitivity to protease and protein-modifying reagents, and (d) translocation of PC and PE is affected differently upon treatment with protease and protein-modifying reagents. Ca (2+)-dependent scrambling activity was not observed in the vesicles reconstituted from plant ER membranes, ruling out the possibility of the involvement of scramblase in translocation of phospholipids. These results suggest the existence of biogenic membrane flippases in plants and that the mechanism of membrane biogenesis is similar to that found in animals.

  20. Membrane Targeting of P-type ATPases in Plant Cells

    International Nuclear Information System (INIS)

    Harper, Jeffrey F.

    2004-01-01

    How membrane proteins are targeted to specific subcellular locations is a very complex and poorly understood area of research. Our long-term goal is to use P-type ATPases (ion pumps), in a model plant system Arabidopsis, as a paradigm to understand how members of a family of closely related membrane proteins can be targeted to different subcellular locations. The research is divided into two specific aims. The first aim is focused on determining the targeting destination of all 10 ACA-type calcium pumps (Arabidopsis Calcium ATPase) in Arabidopsis. ACAs represent a plant specific-subfamily of plasma membrane-type calcium pumps. In contrast to animals, the plant homologs have been found in multiple membrane systems, including the ER (ACA2), tonoplast (ACA4) and plasma membrane (ACA8). Their high degree of similarity provides a unique opportunity to use a comparative approach to delineate the membrane specific targeting information for each pump. One hypothesis to be tested is that an endomembrane located ACA can be re-directed to the plasma membrane by including targeting information from a plasma membrane isoform, ACA8. Our approach is to engineer domain swaps between pumps and monitor the targeting of chimeric proteins in plant cells using a Green Fluorescence Protein (GFP) as a tag. The second aim is to test the hypothesis that heterologous transporters can be engineered into plants and targeted to the plasma membrane by fusing them to a plasma membrane proton pump. As a test case we are evaluating the targeting properties of fusions made between a yeast sodium/proton exchanger (Sod2) and a proton pump (AHA2). This fusion may potentially lead to a new strategy for engineering salt resistant plants. Together these aims are designed to provide fundamental insights into the biogenesis and function of plant cell membrane systems

  1. Membrane Targeting of P-type ATPases in Plant Cells

    Energy Technology Data Exchange (ETDEWEB)

    Jeffrey F. Harper, Ph.D.

    2004-06-30

    How membrane proteins are targeted to specific subcellular locations is a very complex and poorly understood area of research. Our long-term goal is to use P-type ATPases (ion pumps), in a model plant system Arabidopsis, as a paradigm to understand how members of a family of closely related membrane proteins can be targeted to different subcellular locations. The research is divided into two specific aims. The first aim is focused on determining the targeting destination of all 10 ACA-type calcium pumps (Arabidopsis Calcium ATPase) in Arabidopsis. ACAs represent a plant specific-subfamily of plasma membrane-type calcium pumps. In contrast to animals, the plant homologs have been found in multiple membrane systems, including the ER (ACA2), tonoplast (ACA4) and plasma membrane (ACA8). Their high degree of similarity provides a unique opportunity to use a comparative approach to delineate the membrane specific targeting information for each pump. One hypothesis to be tested is that an endomembrane located ACA can be re-directed to the plasma membrane by including targeting information from a plasma membrane isoform, ACA8. Our approach is to engineer domain swaps between pumps and monitor the targeting of chimeric proteins in plant cells using a Green Fluorescence Protein (GFP) as a tag. The second aim is to test the hypothesis that heterologous transporters can be engineered into plants and targeted to the plasma membrane by fusing them to a plasma membrane proton pump. As a test case we are evaluating the targeting properties of fusions made between a yeast sodium/proton exchanger (Sod2) and a proton pump (AHA2). This fusion may potentially lead to a new strategy for engineering salt resistant plants. Together these aims are designed to provide fundamental insights into the biogenesis and function of plant cell membrane systems.

  2. Efficient activation of T cells by human monocyte-derived dendritic cells (HMDCs pulsed with Coxiella burnetii outer membrane protein Com1 but not by HspB-pulsed HMDCs

    Directory of Open Access Journals (Sweden)

    Wang Xile

    2011-09-01

    Full Text Available Abstract Background Coxiella burnetii is an obligate intracellular bacterium and the etiologic agent of Q fever; both coxiella outer membrane protein 1 (Com1 and heat shock protein B (HspB are its major immunodominant antigens. It is not clear whether Com1 and HspB have the ability to mount immune responses against C. burnetii infection. Results The recombinant proteins Com1 and HspB were applied to pulse human monocyte-derived dendritic cells (HMDCs, and the pulsed HMDCs were used to stimulate isogenic T cells. Com1-pulsed HMDCs expressed substantially higher levels of surface molecules (CD83, CD40, CD80, CD86, CD54, and CD58 and a higher level of interleukin-12 than HspB-pulsed HMDCs. Moreover, Com1-pulsed HMDCs induced high-level proliferation and activation of CD4+ and CD8+ cells, which expressed high levels of T-cell activation marker CD69 and inflammatory cytokines IFN-γ and TNF-α. In contrast, HspB-pulsed HMDCs were unable to induce efficient T-cell proliferation and activation. Conclusions Our results demonstrate that Com1-pulsed HMDCs are able to induce efficient T-cell proliferation and drive T cells toward Th1 and Tc1 polarization; however, HspB-pulsed HMDCs are unable to do so. Unlike HspB, Com1 is a protective antigen, which was demonstrated by the adoptive transfer of Com1-pulsed bone marrow dendritic cells into naive BALB/c mice.

  3. Selectivity of biopolymer membranes using HepG2 cells.

    Science.gov (United States)

    Lü, Dongyuan; Gao, Yuxin; Luo, Chunhua; Lü, Shouqian; Wang, Qian; Xu, Xianghong; Sun, Shujin; Wang, Chengzhi; Long, Mian

    2015-03-01

    Bioartificial liver (BAL) system has emerged as an alternative treatment to bridge acute liver failure to either liver transplantation or liver regeneration. One of the main reasons that the efficacy of the current BAL systems was not convincing in clinical trials is attributed to the lack of friendly interface between the membrane and the hepatocytes in liver bioreactor, the core unit of BAL system. Here, we systematically compared the biological responses of hepatosarcoma HepG2 cells seeded on eight, commercially available biocompatible membranes made of acetyl cellulose-nitrocellulose mixed cellulose (CA-NC), acetyl cellulose (CA), nylon (JN), polypropylene (PP), nitrocellulose (NC), polyvinylidene fluoride (PVDF), polycarbonate (PC) and polytetrafluoroethylene (PTFE). Physicochemical analysis and mechanical tests indicated that CA, JN and PP membranes yield high adhesivity and reasonable compressive and/or tensile features with friendly surface topography for cell seeding. Cells prefer to adhere on CA, JN, PP or PTFE membranes with high proliferation rate in spheriod-like shape. Actin, albumin and cytokeratin 18 expressions are favorable for cells on CA or PP membrane, whereas protein filtration is consistent among all the eight membranes. These results further the understandings of cell growth, morphology and spreading, as well as protein filtration on distinct membranes in designing a liver bioreactor.

  4. Self-assembling peptides form nanodiscs that stabilize membrane proteins

    DEFF Research Database (Denmark)

    Midtgaard, Søren Roi; Pedersen, Martin Cramer; Kirkensgaard, Jacob Judas Kain

    2014-01-01

    New methods to handle membrane bound proteins, e.g. G-protein coupled receptors (GPCRs), are highly desirable. Recently, apoliprotein A1 (ApoA1) based lipoprotein particles have emerged as a new platform for studying membrane proteins, and it has been shown that they can self-assemble in combinat......New methods to handle membrane bound proteins, e.g. G-protein coupled receptors (GPCRs), are highly desirable. Recently, apoliprotein A1 (ApoA1) based lipoprotein particles have emerged as a new platform for studying membrane proteins, and it has been shown that they can self...

  5. Monoclonal antibody against membrane protein of transmissible gastroenteritis virus.

    Science.gov (United States)

    Sun, Xuejiao; Ren, Yudong; Li, Yu; Zhu, Jiayi; Zhu, Weijuan; Ding, Fan; Li, Guangxing; Wang, Chunfeng; Gao, Ming; Gao, Yunhang; Cao, Liyan; Ren, Xiaofeng

    2013-02-01

    Transmissible gastroenteritis virus (TGEV) is a porcine coronavirus that can cause piglet diarrhea with high mortality rates. TGEV membrane (M) protein not only plays a vital role in the process of virus assembly and budding, but also induces the production of interferon-α during infection. In this study, a monoclonal antibody (MAb) designated 7G7, against the TGEV M protein was generated by inoculating BALB/c mice with TGEV followed by hybridoma technique. Immunofluorescence assays indicated that MAb 7G7 was capable of detecting cell infection by TGEV. Virus-based ELISA demonstrated that MAb 7G7 can be used as a highly specific diagnostic reagent for TGEV.

  6. Membrane progesterone receptor beta (mPR?/Paqr8) promotes progesterone-dependent neurite outgrowth in PC12 neuronal cells via non-G protein-coupled receptor (GPCR) signaling

    OpenAIRE

    Kasubuchi, Mayu; Watanabe, Keita; Hirano, Kanako; Inoue, Daisuke; Li, Xuan; Terasawa, Kazuya; Konishi, Morichika; Itoh, Nobuyuki; Kimura, Ikuo

    2017-01-01

    Recently, sex steroid membrane receptors garnered world-wide attention because they may be related to sex hormone-mediated unknown rapid non-genomic action that cannot be currently explained by their genomic action via nuclear receptors. Progesterone affects cell proliferation and survival via non-genomic effects. In this process, membrane progesterone receptors (mPRα, mPRβ, mPRγ, mPRδ, and mPRε) were identified as putative G protein-coupled receptors (GPCRs) for progesterone. However, the st...

  7. Ion transport across the biological membrane by computational protein design

    Science.gov (United States)

    Grigoryan, Gevorg

    The cellular membrane is impermeable to most of the chemicals the cell needs to take in or discard to survive. Therefore, transporters-a class of transmembrane proteins tasked with shuttling cargo chemicals in and out of the cell-are essential to all cellular life. From existing crystal structures, we know transporters to be complex machines, exquisitely tuned for specificity and controllability. But how could membrane-bound life have evolved if it needed such complex machines to exist first? To shed light onto this question, we considered the task of designing a transporter de novo. As our guiding principle, we took the ``alternating-access model''-a conceptual mechanism stating that transporters work by rocking between two conformations, each exposing the cargo-binding site to either the intra- or the extra-cellular environment. A computational design framework was developed to encode an anti-parallel four-helix bundle that rocked between two alternative states to orchestrate the movement of Zn(II) ions across the membrane. The ensemble nature of both states was accounted for using a free energy-based approach, and sequences were chosen based on predicted formation of the targeted topology in the membrane and bi-stability. A single sequence was prepared experimentally and shown to function as a Zn(II) transporter in lipid vesicles. Further, transport was specific to Zn(II) ions and several control peptides supported the underlying design principles. This included a mutant designed to retain all properties but with reduced rocking, which showed greatly depressed transport ability. These results suggest that early transporters could have evolved in the context of simple topologies, to be later tuned by evolution for improved properties and controllability. Our study also serves as an important advance in computational protein design, showing the feasibility of designing functional membrane proteins and of tuning conformational landscapes for desired function

  8. Fuel cell and membrane therefore

    Energy Technology Data Exchange (ETDEWEB)

    Aindow, Tai-Tsui

    2016-08-09

    A fuel cell includes first and second flow field plates, and an anode electrode and a cathode electrode between the flow field plates. A polymer electrolyte membrane (PEM) is arranged between the electrodes. At least one of the flow field plates influences, at least in part, an in-plane anisotropic physical condition of the PEM that varies in magnitude between a high value direction and a low value direction. The PEM has an in-plane physical property that varies in magnitude between a high value direction and a low value direction. The PEM is oriented with its high value direction substantially aligned with the high value direction of the flow field plate.

  9. Quantification of green fluorescent protein-(GFP-) tagged membrane proteins by capillary gel electrophoresis.

    Science.gov (United States)

    Danish, Azeem; Lee, Sang-Yong; Müller, Christa E

    2017-10-07

    A fast and robust procedure for the quantification of GFP-tagged membrane proteins in cell homogenates was developed employing capillary gel electrophoresis coupled to laser-induced fluorescence detection (CGE-LIF). The new method was found to be highly sensitive and applicable to structurally diverse membrane proteins including synaptic vesicle protein 2A (SV2A), adenosine A 2A receptor (A 2A AR), and connexin 43 (Cx43). Quantification of SV2A and A 2A AR using radioligand binding assays confirmed the results obtained with CGE-LIF. The CGE-LIF method showed significantly higher sensitivity as compared to fluorimetric measurement in a microplate. Importantly, CGE-LIF involves separation of the target proteins and their degradation products prior to quantification and thereby ensures specificity. We anticipate broad applicability of the method for any fluorophore-tagged protein.

  10. G protein-membrane interactions II: Effect of G protein-linked lipids on membrane structure and G protein-membrane interactions.

    Science.gov (United States)

    Casas, Jesús; Ibarguren, Maitane; Álvarez, Rafael; Terés, Silvia; Lladó, Victoria; Piotto, Stefano P; Concilio, Simona; Busquets, Xavier; López, David J; Escribá, Pablo V

    2017-09-01

    G proteins often bear myristoyl, palmitoyl and isoprenyl moieties, which favor their association with the membrane and their accumulation in G Protein Coupled Receptor-rich microdomains. These lipids influence the biophysical properties of membranes and thereby modulate G protein binding to bilayers. In this context, we showed here that geranylgeraniol, but neither myristate nor palmitate, increased the inverted hexagonal (H II ) phase propensity of phosphatidylethanolamine-containing membranes. While myristate and palmitate preferentially associated with phosphatidylcholine membranes, geranylgeraniol favored nonlamellar-prone membranes. In addition, Gαi 1 monomers had a higher affinity for lamellar phases, while Gβγ and Gαβγ showed a marked preference for nonlamellar prone membranes. Moreover, geranylgeraniol enhanced the binding of G protein dimers and trimers to phosphatidylethanolamine-containing membranes, yet it decreased that of monomers. By contrast, both myristate and palmitate increased the Gαi 1 preference for lamellar membranes. Palmitoylation reinforced the binding of the monomer to PC membranes and myristoylation decreased its binding to PE-enriched bilayer. Finally, binding of dimers and trimers to lamellar-prone membranes was decreased by palmitate and myristate, but it was increased in nonlamellar-prone bilayers. These results demonstrate that co/post-translational G protein lipid modifications regulate the membrane lipid structure and that they influence the physico-chemical properties of membranes, which in part explains why G protein subunits sort to different plasma membrane domains. This article is part of a Special Issue entitled: Membrane Lipid Therapy: Drugs Targeting Biomembranes edited by Pablo V. Escribá. Copyright © 2017 Elsevier B.V. All rights reserved.

  11. Channels in cell membranes and synchrotron radiation

    International Nuclear Information System (INIS)

    Yan Xiaohui; Tian Liang; Zhang Xinyi

    2004-01-01

    For long time a lot of scientists have devoted to study how matter, such as water molecules and K + , Na + , Ca 2+ , Cl - ions, move through cell membranes and complete the matter exchange between the inside and outside of cells. Peter Agre discovered and characterized the first water channel protein in 1988 and Roderick MacKinnon elucidated the structural and mechanistic basis for ion channel function in 1998. These achievements have made it possible for us to 'see' these exquisitely designed molecular machines in action at the atomic level. The Nobel Prize in Chemistry for 2003 is shared between these two scientists. In determining the high resolution 3D structure of these channels, the synchrotron X-ray diffraction plays an important role

  12. Plasma membranes from insect midgut cells

    Directory of Open Access Journals (Sweden)

    Walter R. Terra

    2006-06-01

    Full Text Available Plasma membranes from insect midgut cells are separated into apical and basolateral domains. The apical domain is usually modified into microvilli with a molecular structure similar to other animals. Nevertheless, the microvillar structure should differ in some insects to permit the traffic inside them of secretory vesicles that may budd laterally or pinch-off from the tips of microvilli. Other microvillar modifications are associated with proton-pumping or with the interplay with an ensheathing lipid membrane (the perimicrovilllar membrane observed in the midgut cells of hemipterans (aphids and bugs. The perimicrovillar membranes are thought to be involved in amino acid absorption from diluted diets. The microvillar and perimicrovillar membranes have densities (and protein content that depend on the insect taxon. The role played by the microvillar and perimicrovillar proteins in insect midgut physiology is reviewed here trying to provide a coherent picture of data and highlighting further research areas.As membranas plasmáticas das células intestinais dos insetos apresentam um domínio apical e outro basal. O domínio apical é geralmente modificado em microvilosidades com organização molecular similar a de outros animais, embora possam diferir naqueles insetos que apresentam vesículas secretoras em trânsito que brotam lateralmente ou destacam-se das extremidades das microvilosidades. Outras modificações microvilares estão associadas a bombeamento de prótons ou a interrelações com uma membrana lipídica (a membrana perimicrovilar que reveste as microvilosidades de células intestinais de hemípteros (pulgões e percevejos. Admite-se que as membranas perimicrovilares estejam envolvidas na absorção de aminoácidos a partir de dietas diluídas. As membranas microvilares e perimicrovilares tem densidades distintas (e conteúdo protéico que dependem do táxon do inseto. O papel desempenhado pelas proteínas microvilares e

  13. Membrane orientation of droplets prepared from Chara corallina internodal cells.

    Science.gov (United States)

    Berecki, G; Eijken, M; Van Iren, F; Van Duijn, B

    2001-01-01

    It is generally accepted that the membrane surrounding droplets from characean cells originates from the tonoplast, but there is some uncertainty regarding droplet membrane sidedness. This issue was addressed directly by combining two different droplet isolation methods and the patch clamp technique. Neutral red accumulation was used to demonstrate the presence of H(+)-transport over the membrane and to predict membrane orientation. Two types of droplet populations with differently oriented membranes could be formed in an iso-osmotic bath solution. Cytoplasmic droplets (cytosolic side of the tonoplast inside) contained cytoplasm, while the second type of droplet population contained vacuolar sap (vacuolar droplets, vacuolar side of the tonoplast inside). Smaller vesicels also appeared inside the droplets, with an apparently inversely oriented membrane. Confocal laser scanning microscopy indirectly demonstrated that, at least with one of the droplet isolation methods, the plasma membrane entirely remains in the internodal cell after intracellular perfusion. Both types of droplet populations allowed the formation of excised patches and single-channel measurements by the patch clamp technique. Properties of anion channels in the tonoplast could be used to prove the predicted membrane orientation, knowing that Ca2+ can only activate these channels from the cytosolic side. These results provide useful data for studies addressing ligand-binding, block and modulation, organization and interaction of proteins within the membrane or with other regulatory factors, where it is important to control membrane orientation.

  14. Extracellular vesicles as a platform for membrane-associated therapeutic protein delivery.

    Science.gov (United States)

    Yang, Yoosoo; Hong, Yeonsun; Cho, Eunji; Kim, Gi Beom; Kim, In-San

    2018-01-01

    Membrane proteins are of great research interest, particularly because they are rich in targets for therapeutic application. The suitability of various membrane proteins as targets for therapeutic formulations, such as drugs or antibodies, has been studied in preclinical and clinical studies. For therapeutic application, however, a protein must be expressed and purified in as close to its native conformation as possible. This has proven difficult for membrane proteins, as their native conformation requires the association with an appropriate cellular membrane. One solution to this problem is to use extracellular vesicles as a display platform. Exosomes and microvesicles are membranous extracellular vesicles that are released from most cells. Their membranes may provide a favourable microenvironment for membrane proteins to take on their proper conformation, activity, and membrane distribution; moreover, membrane proteins can cluster into microdomains on the surface of extracellular vesicles following their biogenesis. In this review, we survey the state-of-the-art of extracellular vesicle (exosome and small-sized microvesicle)-based therapeutics, evaluate the current biological understanding of these formulations, and forecast the technical advances that will be needed to continue driving the development of membrane protein therapeutics.

  15. Cubic membranes: a legend beyond the Flatland* of cell membrane organization.

    Science.gov (United States)

    Almsherqi, Zakaria A; Kohlwein, Sepp D; Deng, Yuru

    2006-06-19

    Cubic membranes represent highly curved, three-dimensional nanoperiodic structures that correspond to mathematically well defined triply periodic minimal surfaces. Although they have been observed in numerous cell types and under different conditions, particularly in stressed, diseased, or virally infected cells, knowledge about the formation and function of nonlamellar, cubic structures in biological systems is scarce, and research so far is restricted to the descriptive level. We show that the "organized smooth endoplasmic reticulum" (OSER; Snapp, E.L., R.S. Hegde, M. Francolini, F. Lombardo, S. Colombo, E. Pedrazzini, N. Borgese, and J. Lippincott-Schwartz. 2003. J. Cell Biol. 163:257-269), which is formed in response to elevated levels of specific membrane-resident proteins, is actually the two-dimensional representation of two subtypes of cubic membrane morphology. Controlled OSER induction may thus provide, for the first time, a valuable tool to study cubic membrane formation and function at the molecular level.

  16. A new approach to the modification of cell membrane glycosphingolipids: Ganglioside composition of JTC-12 P3 cells altered by feeding with galactose as a sole carbohydrate source in protein- and lipid-free synthetic medium

    International Nuclear Information System (INIS)

    Kawaguchi, Tatsuya; Takaoka, Toshiko; Yoshida, Eiko; Iwamori, Masao; Nagai, Yoshitaka; Takatsuki, Kiyoshi

    1988-01-01

    A significant difference in the glycosphingolipid composition of JTC-12 P3 cells established from monkey kidney tissue was observed when cells cultured in a protein- and lipid-free synthetic medium containing glucose (DM-160) as a sole carbohydrate source were transferred and cultured in the same medium containing galactose and pyruvic acid (DM-170) in place of glucose. In particular, the amounts of gangliosides GM3, GM2, and GD3 in the cells cultured in DM-170 were 5.3-, 17.8-, and more than 8-fold those in the cells cultured in DM-160, respectively, indicating that anabolism of gangliosides is greatly enhanced in cells cultured in the presence of galactose and pyruvic acid, as compared with cells cultured in the presence of glucose. In fact, after cultivation of cells in the medium with N-acetyl-D-[ 14 C]mannosamine for 96 h, the radioactivity incorporated into the gangliosides of the cells in DM-170 was 10-fold that of the cells in DM-160. Among the gangliosides of the cells in DM-170, highly sialylated molecules such as GD3, GD1a, GD1b, and GT1b were preferentially labeled, indicating that the sialytransferases responsible for the synthesis of gangliosides are significantly more activated in cells cultured in DM-170 than in DM-160. These observations reveal that the glycosphingolipid composition of the plasma membrane can be modified epigenetically under well-defined conditions and provide important clues for clarifying the roles of glycosphingolipids associated with particular cell functions

  17. Membrane complement regulatory protein reduces the damage of transplanting autologous bone marrow mesenchymal stem cells by suppressing the activation of complement.

    Science.gov (United States)

    Xiao, Kai; Fang, Zhenhua; Gao, Xinfeng; Zhao, Jingjing; Huang, Ruokun; Xie, Ming

    2017-10-01

    There are few studies on the interaction of transplanting autologous bone marrow mesenchymal stem cells (BMSCs) and complement. In order to further explore the effect of complement on BMSCs, BMSCs were obtained from bone marrow of 20 cases clinical patients, and then experimented in vitro. The cytotoxicity of complement on the mesenchymal stem cells in autologous human serum (AHS) was measured by Europium cytotoxicity assay. The complement membrane attack complex (MAC) deposited on the membrane surface was detected by flow cytometry. Finally, the cytotoxicity on BMSCs was measured after mCRPs overexpression or knockdown. We found that more than 90% of cells derived from bone marrow were identified to be mesenchymal stem cells through detection of cell membrane surface markers by flow cytometry. BMSCs harvested from the 20 patients all had cytotoxicity after incubated with AHS, and the cytotoxicity was significant higher than that incubated with complement inactivated autologous human serum (iAHS). Complement attack complex (MAC) could be detected on the BMSCs incubated with AHS, which implied the complement activation. We also found that mCRPs CD55 and CD59 overexpressions can resist the cytotoxicity induced by complement activation, while mCRPs CD55 and CD59 knockdown can enhance the cytotoxicity. Thus, the results indicated that mCRPs could effectively protect BMSCs from attacking by complement by suppressing the activation of complement. Copyright © 2017 Elsevier Ltd. All rights reserved.

  18. Proteomic analysis reveals the diversity and complexity of membrane proteins in chickpea (Cicer arietinum L.

    Directory of Open Access Journals (Sweden)

    Jaiswal Dinesh Kumar

    2012-10-01

    Full Text Available Abstract Background Compartmentalization is a unique feature of eukaryotes that helps in maintaining cellular homeostasis not only in intra- and inter-organellar context, but also between the cells and the external environment. Plant cells are highly compartmentalized with a complex metabolic network governing various cellular events. The membranes are the most important constituents in such compartmentalization, and membrane-associated proteins play diverse roles in many cellular processes besides being part of integral component of many signaling cascades. Results To obtain valuable insight into the dynamic repertoire of membrane proteins, we have developed a proteome reference map of a grain legume, chickpea, using two-dimensional gel electrophoresis. MALDI-TOF/TOF and LC-ESI-MS/MS analysis led to the identification of 91 proteins involved in a variety of cellular functions viz., bioenergy, stress-responsive and signal transduction, metabolism, protein synthesis and degradation, among others. Significantly, 70% of the identified proteins are putative integral membrane proteins, possessing transmembrane domains. Conclusions The proteomic analysis revealed many resident integral membrane proteins as well as membrane-associated proteins including those not reported earlier. To our knowledge, this is the first report of membrane proteome from aerial tissues of a crop plant. The findings may provide a better understanding of the biochemical machinery of the plant membranes at the molecular level that might help in functional genomics studies of different developmental pathways and stress-responses.

  19. Pinkbar is an epithelial-specific BAR domain protein that generates planar membrane structures

    Energy Technology Data Exchange (ETDEWEB)

    Pykäläinen, Anette; Boczkowska, Malgorzata; Zhao, Hongxia; Saarikangas, Juha; Rebowski, Grzegorz; Jansen, Maurice; Hakanen, Janne; Koskela, Essi V.; Peränen, Johan; Vihinen, Helena; Jokitalo, Eija; Salminen, Marjo; Ikonen, Elina; Dominguez, Roberto; Lappalainen, Pekka (Helsinki); (Penn)

    2013-05-29

    Bin/amphipysin/Rvs (BAR)-domain proteins sculpt cellular membranes and have key roles in processes such as endocytosis, cell motility and morphogenesis. BAR domains are divided into three subfamilies: BAR- and F-BAR-domain proteins generate positive membrane curvature and stabilize cellular invaginations, whereas I-BAR-domain proteins induce negative curvature and stabilize protrusions. We show that a previously uncharacterized member of the I-BAR subfamily, Pinkbar, is specifically expressed in intestinal epithelial cells, where it localizes to Rab13-positive vesicles and to the plasma membrane at intercellular junctions. Notably, the BAR domain of Pinkbar does not induce membrane tubulation but promotes the formation of planar membrane sheets. Structural and mutagenesis analyses reveal that the BAR domain of Pinkbar has a relatively flat lipid-binding interface and that it assembles into sheet-like oligomers in crystals and in solution, which may explain its unique membrane-deforming activity.

  20. Anion permselective membrane. [For redox fuel cells

    Energy Technology Data Exchange (ETDEWEB)

    Alexander, S.S.; Hodgdon, R.B.

    1978-01-01

    Experimental anion permeselective membranes were improved and characterized for use as separators in a chemical redox, power storage cell being developed at the NASA Lewis Research Center. The goal of minimal Fe/sup +3/ ion transfer was achieved for each candidate membrane system. Minimal membrane resistivity was demonstrated by reduction of film thickness using synthetic backing materials but usefulness of thin membranes was limited by the scarcity of compatible fabrics. The most durable and useful backing fabrics were modacrylics. One membrane, a copolymer of 4 vinylpyridine and vinyl benzylchloride was outstanding in overall electrochemical and physical properties. Long term (1000 hrs) membrane chemical and thermal durability in redox environment was shown by three candidate polymers and two membranes. The remainder had good durability at ambient temperature. Manufacturing capability was demonstrated for large scale production of membrane sheets 5.5 ft/sup 2/ in area for two candidate systems.

  1. Roles of the outer membrane protein AsmA of Salmonella enterica in the control of marRAB expression and invasion of epithelial cells.

    Science.gov (United States)

    Prieto, Ana I; Hernández, Sara B; Cota, Ignacio; Pucciarelli, M Graciela; Orlov, Yuri; Ramos-Morales, Francisco; García-del Portillo, Francisco; Casadesús, Josep

    2009-06-01

    A genetic screen for suppressors of bile sensitivity in DNA adenine methylase (dam) mutants of Salmonella enterica serovar Typhimurium yielded insertions in an uncharacterized locus homologous to the Escherichia coli asmA gene. Disruption of asmA suppressed bile sensitivity also in phoP and wec mutants of S. enterica and increased the MIC of sodium deoxycholate for the parental strain ATCC 14028. Increased levels of marA mRNA were found in asmA, asmA dam, asmA phoP, and asmA wec strains of S. enterica, suggesting that lack of AsmA activates expression of the marRAB operon. Hence, asmA mutations may enhance bile resistance by inducing gene expression changes in the marRAB-controlled Mar regulon. In silico analysis of AsmA structure predicted the existence of one transmembrane domain. Biochemical analysis of subcellular fractions revealed that the asmA gene of S. enterica encodes a protein of approximately 70 kDa located in the outer membrane. Because AsmA is unrelated to known transport and/or efflux systems, we propose that activation of marRAB in asmA mutants may be a consequence of envelope reorganization. Competitive infection of BALB/c mice with asmA(+) and asmA isogenic strains indicated that lack of AsmA attenuates Salmonella virulence by the oral route but not by the intraperitoneal route. Furthermore, asmA mutants showed a reduced ability to invade epithelial cells in vitro.

  2. Major proteins of the goat milk fat globule membrane.

    Science.gov (United States)

    Cebo, C; Caillat, H; Bouvier, F; Martin, P

    2010-03-01

    Fat is present in milk as droplets of triglycerides surrounded by a complex membrane derived from the mammary epithelial cell called milk fat globule membrane (MFGM). Although numerous studies have been published on human or bovine MFGM proteins, to date few studies exist on MFGM proteins from goat milk. The objective of this study was thus to investigate the protein composition of the goat MFGM. Milk fat globule membrane proteins from goat milk were separated by 6% and 10% sodium dodecyl sulfate-PAGE and were Coomassie or periodic acid-Schiff stained. Most of MFGM proteins [mucin-1, fatty acid synthase, xanthine oxidase, butyrophilin, lactadherin (MFG EGF-8, MFG-E8), and adipophilin] already described in cow milk were identified in goat milk using peptide mass fingerprinting. In addition, lectin staining provided a preliminary characterization of carbohydrate structures occurring on MFGM proteins from goat milk depending on alpha(S1)-casein genotype and lactation stage. We provide here first evidence of the presence of O-glycans on fatty acid synthase and xanthine oxidase from goat milk. A prominent difference between the cow and the goat species was demonstrated for lactadherin. Indeed, whereas 2 polypeptide chains were easily identified by peptide mass fingerprinting matrix-assisted laser desorption/ionization-time of flight analysis within bovine MFGM proteins, lactadherin from goat milk consisted of a single polypeptide chain. Another striking observation was the presence of caseins associated with MFGM preparations from goat milk, whereas virtually no caseins were found in MFGM extracts from bovine milk. Taken together, these observations strongly support the existence of a singular secretion mode previously hypothesized in the goat.

  3. Hematopoietic protein-1 regulates the actin membrane skeleton and membrane stability in murine erythrocytes.

    Directory of Open Access Journals (Sweden)

    Maia M Chan

    Full Text Available Hematopoietic protein-1 (Hem-1 is a hematopoietic cell specific member of the WAVE (Wiskott-Aldrich syndrome verprolin-homologous protein complex, which regulates filamentous actin (F-actin polymerization in many cell types including immune cells. However, the roles of Hem-1 and the WAVE complex in erythrocyte biology are not known. In this study, we utilized mice lacking Hem-1 expression due to a non-coding point mutation in the Hem1 gene to show that absence of Hem-1 results in microcytic, hypochromic anemia characterized by abnormally shaped erythrocytes with aberrant F-actin foci and decreased lifespan. We find that Hem-1 and members of the associated WAVE complex are normally expressed in wildtype erythrocyte progenitors and mature erythrocytes. Using mass spectrometry and global proteomics, Coomassie staining, and immunoblotting, we find that the absence of Hem-1 results in decreased representation of essential erythrocyte membrane skeletal proteins including α- and β- spectrin, dematin, p55, adducin, ankyrin, tropomodulin 1, band 3, and band 4.1. Hem1⁻/⁻ erythrocytes exhibit increased protein kinase C-dependent phosphorylation of adducin at Ser724, which targets adducin family members for dissociation from spectrin and actin, and subsequent proteolysis. Increased adducin Ser724 phosphorylation in Hem1⁻/⁻ erythrocytes correlates with decreased protein expression of the regulatory subunit of protein phosphatase 2A (PP2A, which is required for PP2A-dependent dephosphorylation of PKC targets. These results reveal a novel, critical role for Hem-1 in the homeostasis of structural proteins required for formation and stability of the actin membrane skeleton in erythrocytes.

  4. Plasma membrane changes during programmed cell deaths.

    Science.gov (United States)

    Zhang, Yingying; Chen, Xin; Gueydan, Cyril; Han, Jiahuai

    2018-01-01

    Ruptured and intact plasma membranes are classically considered as hallmarks of necrotic and apoptotic cell death, respectively. As such, apoptosis is usually considered a non-inflammatory process while necrosis triggers inflammation. Recent studies on necroptosis and pyroptosis, two types of programmed necrosis, revealed that plasma membrane rupture is mediated by MLKL channels during necroptosis but depends on non-selective gasdermin D (GSDMD) pores during pyroptosis. Importantly, the morphology of dying cells executed by MLKL channels can be distinguished from that executed by GSDMD pores. Interestingly, it was found recently that secondary necrosis of apoptotic cells, a previously believed non-regulated form of cell lysis that occurs after apoptosis, can be programmed and executed by plasma membrane pore formation like that of pyroptosis. In addition, pyroptosis is associated with pyroptotic bodies, which have some similarities to apoptotic bodies. Therefore, different cell death programs induce distinctive reshuffling processes of the plasma membrane. Given the fact that the nature of released intracellular contents plays a crucial role in dying/dead cell-induced immunogenicity, not only membrane rupture or integrity but also the nature of plasma membrane breakdown would determine the fate of a cell as well as its ability to elicit an immune response. In this review, we will discuss recent advances in the field of apoptosis, necroptosis and pyroptosis, with an emphasis on the mechanisms underlying plasma membrane changes observed on dying cells and their implication in cell death-elicited immunogenicity.

  5. NMR-based screening of membrane protein ligands

    NARCIS (Netherlands)

    Yanamala, Naveena; Dutta, Arpana; Beck, Barbara; Van Fleet, Bart; Hay, Kelly; Yazbak, Ahmad; Ishima, Rieko; Doemling, Alexander; Klein-Seetharaman, Judith

    2010-01-01

    Membrane proteins pose problems for the application of NMR-based ligand-screening methods because of the need to maintain the proteins in a membrane mimetic environment such as detergent micelles: they add to the molecular weight of the protein, increase the viscosity of the solution, interact with

  6. Highly Branched Pentasaccharide-Bearing Amphiphiles for Membrane Protein Studies

    DEFF Research Database (Denmark)

    Ehsan, Muhammad; Du, Yang; Scull, Nicola J

    2016-01-01

    Detergents are essential tools for membrane protein manipulation. Micelles formed by detergent molecules have the ability to encapsulate the hydrophobic domains of membrane proteins. The resulting protein-detergent complexes (PDCs) are compatible with the polar environments of aqueous media, maki...

  7. Proteomic Analysis of Intracellular and Membrane Proteins From Voriconazole-Resistant Candida glabrata

    OpenAIRE

    Yoo, Jae Il; Kim, Hwa Su; Choi, Chi Won; Yoo, Jung Sik; Yu, Jae Yon; Lee, Yeong Seon

    2013-01-01

    Objectives The proteomic analysis of voriconazole resistant Candida glabrata strain has not yet been investigated. In this study, differentially expressed proteins of intracellular and membrane fraction from voriconazole-susceptible, susceptible dose-dependent (S-DD), resistant C. glabrata strains were compared with each other and several proteins were identified. Methods The proteins of intracellular and membrane were isolated by disrupting cells with glass bead and centrifugation from voric...

  8. Extraction and identification of membrane proteins from black widow spider eggs.

    Science.gov (United States)

    Fu, Si-Ling; Li, Jiang-Lin; Chen, Jia; Wang, Qiu-Ting; Li, Jian-Jun; Wang, Xian-Chun

    2015-07-18

    The eggs of oviparous animals are storehouses of maternal proteins required for embryonic development. Identification and molecular characterization of such proteins will provide much insight into the regulation of embryonic development. We previously analyzed soluble proteins in the eggs of the black widow spider (Latrodectus tredecimguttatus), and report here on the extraction and mass spectrometric identification of the egg membrane proteins. Comparison of different lysis solutions indicated that the highest extraction of the membrane proteins was achieved with 3%-4% sodium laurate in 40 mmol/L Tris-HCl buffer containing 4% CHAPS and 2% DTT (pH 7.4). SDS-PAGE combined with nLC-MS/MS identified 39 proteins with membrane-localization annotation, including those with structural, catalytic, and regulatory activities. Nearly half of the identified membrane proteins were metabolic enzymes involved in various cellular processes, particularly energy metabolism and biosynthesis, suggesting that relevant metabolic processes were active during the embryonic development of the eggs. Several identified cell membrane proteins were involved in the special structure formation and function of the egg cell membranes. The present proteomic analysis of the egg membrane proteins provides new insight into the molecular mechanisms of spider embryonic development.

  9. Membrane-mediated interaction between strongly anisotropic protein scaffolds.

    Directory of Open Access Journals (Sweden)

    Yonatan Schweitzer

    2015-02-01

    Full Text Available Specialized proteins serve as scaffolds sculpting strongly curved membranes of intracellular organelles. Effective membrane shaping requires segregation of these proteins into domains and is, therefore, critically dependent on the protein-protein interaction. Interactions mediated by membrane elastic deformations have been extensively analyzed within approximations of large inter-protein distances, small extents of the protein-mediated membrane bending and small deviations of the protein shapes from isotropic spherical segments. At the same time, important classes of the realistic membrane-shaping proteins have strongly elongated shapes with large and highly anisotropic curvature. Here we investigated, computationally, the membrane mediated interaction between proteins or protein oligomers representing membrane scaffolds with strongly anisotropic curvature, and addressed, quantitatively, a specific case of the scaffold geometrical parameters characterizing BAR domains, which are crucial for membrane shaping in endocytosis. In addition to the previously analyzed contributions to the interaction, we considered a repulsive force stemming from the entropy of the scaffold orientation. We computed this interaction to be of the same order of magnitude as the well-known attractive force related to the entropy of membrane undulations. We demonstrated the scaffold shape anisotropy to cause a mutual aligning of the scaffolds and to generate a strong attractive interaction bringing the scaffolds close to each other to equilibrium distances much smaller than the scaffold size. We computed the energy of interaction between scaffolds of a realistic geometry to constitute tens of kBT, which guarantees a robust segregation of the scaffolds into domains.

  10. Proteomic Analysis of Intracellular and Membrane Proteins From Voriconazole-Resistant Candida glabrata.

    Science.gov (United States)

    Yoo, Jae Il; Kim, Hwa Su; Choi, Chi Won; Yoo, Jung Sik; Yu, Jae Yon; Lee, Yeong Seon

    2013-12-01

    The proteomic analysis of voriconazole resistant Candida glabrata strain has not yet been investigated. In this study, differentially expressed proteins of intracellular and membrane fraction from voriconazole-susceptible, susceptible dose-dependent (S-DD), resistant C. glabrata strains were compared with each other and several proteins were identified. The proteins of intracellular and membrane were isolated by disrupting cells with glass bead and centrifugation from voriconazole susceptible, S-DD, and resistant C. glabrata strains. The abundance of expressed proteins was compared using two-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis and proteins showing continuous twofold or more increase or reduction of expression in resistant strains compared to susceptible and S-DD strain were analyzed by liquid chromatography/mass spectrometry-mass spectrometry method. Of 34 intracellular proteins, 15 proteins showed expression increase or reduction (twofold or more). The identified proteins included regulation, energy production, carbohydrate transport, amino acid transport, and various metabolism related proteins. The increase of expression of heat shock protein 70 was found. Among membrane proteins, 12, 31 proteins showed expression increase or decrease in the order of susceptible, S-DD, and resistant strains. This expression included carbohydrate metabolism, amino acid synthesis, and response to stress-related proteins. In membrane fractions, the change of expression of 10 heat shock proteins was observed, and 9 heat shock protein 70 (Hsp70) showed the reduction of expression. The expression of Hsp70 protein in membrane fraction is related to voriconazole resistant C. glabrata strains.

  11. In Plant and Animal Cells, Detergent-Resistant Membranes Do Not Define Functional Membrane Rafts

    Czech Academy of Sciences Publication Activity Database

    Tanner, W.; Malínský, Jan; Opekarová, Miroslava

    2011-01-01

    Roč. 23, č. 4 (2011), s. 1191-1193 ISSN 1040-4651 Institutional research plan: CEZ:AV0Z50390512; CEZ:AV0Z50200510 Keywords : plasma-membrane * lipod rafts * proteins Subject RIV: EA - Cell Biology Impact factor: 8.987, year: 2011

  12. Membrane protein expression triggers chromosomal locus repositioning in bacteria

    OpenAIRE

    Libby, Elizabeth A.; Roggiani, Manuela; Goulian, Mark

    2012-01-01

    It has long been hypothesized that subcellular positioning of chromosomal loci in bacteria may be influenced by gene function and expression state. Here we provide direct evidence that membrane protein expression affects the position of chromosomal loci in Escherichia coli. For two different membrane proteins, we observed a dramatic shift of their genetic loci toward the membrane upon induction. In related systems in which a cytoplasmic protein was produced, or translation was eliminated by m...

  13. Membrane Protein Production in Lactococcus lactis for Functional Studies.

    Science.gov (United States)

    Seigneurin-Berny, Daphne; King, Martin S; Sautron, Emiline; Moyet, Lucas; Catty, Patrice; André, François; Rolland, Norbert; Kunji, Edmund R S; Frelet-Barrand, Annie

    2016-01-01

    Due to their unique properties, expression and study of membrane proteins in heterologous systems remains difficult. Among the bacterial systems available, the Gram-positive lactic bacterium, Lactococcus lactis, traditionally used in food fermentations, is nowadays widely used for large-scale production and functional characterization of bacterial and eukaryotic membrane proteins. The aim of this chapter is to describe the different possibilities for the functional characterization of peripheral or intrinsic membrane proteins expressed in Lactococcus lactis.

  14. Immunohistochemical expression of latent membrane protein 1 ...

    African Journals Online (AJOL)

    Methods: Archival formalin-fixed, paraffin-embedded NPC biopsies were evaluated in 23 Moroccan patients for the presence of LMP1 and p53 using immunohistochemistry (IHC). Results: No LMP1 expression was observed whereas 8 of 23 cases (34. 7%) had detectable p53 protein in the nuclei of tumor cells.

  15. Identification of novel autophagic Radix Polygalae fraction by cell membrane chromatography and UHPLC-(Q)TOF-MS for degradation of neurodegenerative disease proteins

    OpenAIRE

    An-Guo Wu; Vincent Kam-Wai Wong; Wu Zeng; Liang Liu; Betty Yuen-Kwan Law

    2015-01-01

    With its traditional use in relieving insomnia and anxiety, our previous study has identified onjisaponin B from Radix Polygalae (RP), as a novel autophagic enhancer with potential neuroprotective effects. In current study, we have further identified a novel active fraction from RP, contains 17 major triterpenoid saponins including the onjisaponin B, by the combinational use of cell membrane chromatography (CMC) and ultra-performance liquid chromatography coupled to (quadrupole) time-of-fligh...

  16. An Integrated Framework Advancing Membrane Protein Modeling and Design.

    Directory of Open Access Journals (Sweden)

    Rebecca F Alford

    2015-09-01

    Full Text Available Membrane proteins are critical functional molecules in the human body, constituting more than 30% of open reading frames in the human genome. Unfortunately, a myriad of difficulties in overexpression and reconstitution into membrane mimetics severely limit our ability to determine their structures. Computational tools are therefore instrumental to membrane protein structure prediction, consequently increasing our understanding of membrane protein function and their role in disease. Here, we describe a general framework facilitating membrane protein modeling and design that combines the scientific principles for membrane protein modeling with the flexible software architecture of Rosetta3. This new framework, called RosettaMP, provides a general membrane representation that interfaces with scoring, conformational sampling, and mutation routines that can be easily combined to create new protocols. To demonstrate the capabilities of this implementation, we developed four proof-of-concept applications for (1 prediction of free energy changes upon mutation; (2 high-resolution structural refinement; (3 protein-protein docking; and (4 assembly of symmetric protein complexes, all in the membrane environment. Preliminary data show that these algorithms can produce meaningful scores and structures. The data also suggest needed improvements to both sampling routines and score functions. Importantly, the applications collectively demonstrate the potential of combining the flexible nature of RosettaMP with the power of Rosetta algorithms to facilitate membrane protein modeling and design.

  17. A cDNA Immunization Strategy to Generate Nanobodies against Membrane Proteins in Native Conformation

    Directory of Open Access Journals (Sweden)

    Thomas Eden

    2018-01-01

    Full Text Available Nanobodies (Nbs are soluble, versatile, single-domain binding modules derived from the VHH variable domain of heavy-chain antibodies naturally occurring in camelids. Nbs hold huge promise as novel therapeutic biologics. Membrane proteins are among the most interesting targets for therapeutic Nbs because they are accessible to systemically injected biologics. In order to be effective, therapeutic Nbs must recognize their target membrane protein in native conformation. However, raising Nbs against membrane proteins in native conformation can pose a formidable challenge since membrane proteins typically contain one or more hydrophobic transmembrane regions and, therefore, are difficult to purify in native conformation. Here, we describe a highly efficient genetic immunization strategy that circumvents these difficulties by driving expression of the target membrane protein in native conformation by cells of the immunized camelid. The strategy encompasses ballistic transfection of skin cells with cDNA expression plasmids encoding one or more orthologs of the membrane protein of interest and, optionally, other costimulatory proteins. The plasmid is coated onto 1 µm gold particles that are then injected into the shaved and depilated skin of the camelid. A gene gun delivers a helium pulse that accelerates the DNA-coated particles to a velocity sufficient to penetrate through multiple layers of cells in the skin. This results in the exposure of the extracellular domains of the membrane protein on the cell surface of transfected cells. Repeated immunization drives somatic hypermutation and affinity maturation of target-specific heavy-chain antibodies. The VHH/Nb coding region is PCR-amplified from B cells obtained from peripheral blood or a lymph node biopsy. Specific Nbs are selected by phage display or by screening of Nb-based heavy-chain antibodies expressed as secretory proteins in transfected HEK cells. Using this strategy, we have successfully

  18. A cDNA Immunization Strategy to Generate Nanobodies against Membrane Proteins in Native Conformation

    Science.gov (United States)

    Eden, Thomas; Menzel, Stephan; Wesolowski, Janusz; Bergmann, Philine; Nissen, Marion; Dubberke, Gudrun; Seyfried, Fabienne; Albrecht, Birte; Haag, Friedrich; Koch-Nolte, Friedrich

    2018-01-01

    Nanobodies (Nbs) are soluble, versatile, single-domain binding modules derived from the VHH variable domain of heavy-chain antibodies naturally occurring in camelids. Nbs hold huge promise as novel therapeutic biologics. Membrane proteins are among the most interesting targets for therapeutic Nbs because they are accessible to systemically injected biologics. In order to be effective, therapeutic Nbs must recognize their target membrane protein in native conformation. However, raising Nbs against membrane proteins in native conformation can pose a formidable challenge since membrane proteins typically contain one or more hydrophobic transmembrane regions and, therefore, are difficult to purify in native conformation. Here, we describe a highly efficient genetic immunization strategy that circumvents these difficulties by driving expression of the target membrane protein in native conformation by cells of the immunized camelid. The strategy encompasses ballistic transfection of skin cells with cDNA expression plasmids encoding one or more orthologs of the membrane protein of interest and, optionally, other costimulatory proteins. The plasmid is coated onto 1 µm gold particles that are then injected into the shaved and depilated skin of the camelid. A gene gun delivers a helium pulse that accelerates the DNA-coated particles to a velocity sufficient to penetrate through multiple layers of cells in the skin. This results in the exposure of the extracellular domains of the membrane protein on the cell surface of transfected cells. Repeated immunization drives somatic hypermutation and affinity maturation of target-specific heavy-chain antibodies. The VHH/Nb coding region is PCR-amplified from B cells obtained from peripheral blood or a lymph node biopsy. Specific Nbs are selected by phage display or by screening of Nb-based heavy-chain antibodies expressed as secretory proteins in transfected HEK cells. Using this strategy, we have successfully generated agonistic

  19. A simple detection method for low-affinity membrane protein interactions by baculoviral display.

    Directory of Open Access Journals (Sweden)

    Toshiko Sakihama

    Full Text Available BACKGROUND: Membrane protein interactions play an important role in cell-to-cell recognition in various biological activities such as in the immune or neural system. Nevertheless, there has remained the major obstacle of expression of the membrane proteins in their active form. Recently, we and other investigators found that functional membrane proteins express on baculovirus particles (budded virus, BV. In this study, we applied this BV display system to detect interaction between membrane proteins important for cell-to-cell interaction in immune system. METHODOLOGY/PRINCIPAL FINDINGS: We infected Sf9 cells with recombinant baculovirus encoding the T cell membrane protein CD2 or its ligand CD58 and recovered the BV. We detected specific interaction between CD2-displaying BV and CD58-displaying BV by an enzyme-linked immunosorbent assay (ELISA. Using this system, we also detected specific interaction between two other membrane receptor-ligand pairs, CD40-CD40 ligand (CD40L, and glucocorticoid-induced TNFR family-related protein (GITR-GITR ligand (GITRL. Furthermore, we observed specific binding of BV displaying CD58, CD40L, or GITRL to cells naturally expressing their respective receptors by flowcytometric analysis using anti-baculoviral gp64 antibody. Finally we isolated CD2 cDNA from a cDNA expression library by magnetic separation using CD58-displaying BV and anti-gp64 antibody. CONCLUSIONS: We found the BV display system worked effectively in the detection of the interaction of membrane proteins. Since various membrane proteins and their oligomeric complexes can be displayed on BV in the native form, this BV display system should prove highly useful in the search for natural ligands or to develop screening systems for therapeutic antibodies and/or compounds.

  20. Adamantane-based amphiphiles (ADAs) for membrane protein study: importance of a detergent hydrophobic group in membrane protein solubilisation.

    Science.gov (United States)

    Chae, Pil Seok; Bae, Hyoung Eun; Das, Manabendra

    2014-10-21

    We prepared adamantane-containing amphiphiles and evaluated them using a large membrane protein complex in terms of protein solubilisation and stabilization efficacy. These agents were superior to conventional detergents, especially in terms of the membrane protein solubilisation efficiency, implying a new detergent structure-property relationship.

  1. INTERACTION OF ALDEHYDES DERIVED FROM LIPID PEROXIDATION AND MEMBRANE PROTEINS.

    Directory of Open Access Journals (Sweden)

    Stefania ePizzimenti

    2013-09-01

    Full Text Available A great variety of compounds are formed during lipid peroxidation of polyunsaturated fatty acids of membrane phospholipids. Among them, bioactive aldehydes, such as 4-hydroxyalkenals, malondialdehyde (MDA and acrolein, have received particular attention since they have been considered as toxic messengers that can propagate and amplify oxidative injury. In the 4-hydroxyalkenal class, 4-hydroxy-2-nonenal (HNE is the most intensively studied aldehyde, in relation not only to its toxic function, but also to its physiological role. Indeed, HNE can be found at low concentrations in human tissues and plasma and participates in the control of biological processes, such as signal transduction, cell proliferation and differentiation. Moreover, at low doses, HNE exerts an anti-cancer effect, by inhibiting cell proliferation, angiogenesis, cell adhesion and by inducing differentiation and/or apoptosis in various tumor cell lines. It is very likely that a substantial fraction of the effects observed in cellular responses, induced by HNE and related aldehydes, be mediated by their interaction with proteins, resulting in the formation of covalent adducts or in the modulation of their expression and/or activity. In this review we focus on membrane proteins affected by lipid peroxidation-derived aldehydes, under physiological and pathological conditions.

  2. The human multidrug resistance-associated protein MRP is a plasma membrane drug-efflux pump

    NARCIS (Netherlands)

    Zaman, G. J.; Flens, M. J.; van Leusden, M. R.; de Haas, M.; Mülder, H. S.; Lankelma, J.; Pinedo, H. M.; Scheper, R. J.; Baas, F.; Broxterman, H. J.

    1994-01-01

    The multidrug-resistance associated protein MRP is a 180- to 195-kDa membrane protein associated with resistance of human tumor cells to cytotoxic drugs. We have investigated how MRP confers drug resistance in SW-1573 human lung carcinoma cells by generating a subline stably transfected with an

  3. Layer-by-layer cell membrane assembly

    Science.gov (United States)

    Matosevic, Sandro; Paegel, Brian M.

    2013-11-01

    Eukaryotic subcellular membrane systems, such as the nuclear envelope or endoplasmic reticulum, present a rich array of architecturally and compositionally complex supramolecular targets that are as yet inaccessible. Here we describe layer-by-layer phospholipid membrane assembly on microfluidic droplets, a route to structures with defined compositional asymmetry and lamellarity. Starting with phospholipid-stabilized water-in-oil droplets trapped in a static droplet array, lipid monolayer deposition proceeds as oil/water-phase boundaries pass over the droplets. Unilamellar vesicles assembled layer-by-layer support functional insertion both of purified and of in situ expressed membrane proteins. Synthesis and chemical probing of asymmetric unilamellar and double-bilayer vesicles demonstrate the programmability of both membrane lamellarity and lipid-leaflet composition during assembly. The immobilized vesicle arrays are a pragmatic experimental platform for biophysical studies of membranes and their associated proteins, particularly complexes that assemble and function in multilamellar contexts in vivo.

  4. Membrane protein expression triggers chromosomal locus repositioning in bacteria

    Science.gov (United States)

    Libby, Elizabeth A.; Roggiani, Manuela; Goulian, Mark

    2012-01-01

    It has long been hypothesized that subcellular positioning of chromosomal loci in bacteria may be influenced by gene function and expression state. Here we provide direct evidence that membrane protein expression affects the position of chromosomal loci in Escherichia coli. For two different membrane proteins, we observed a dramatic shift of their genetic loci toward the membrane upon induction. In related systems in which a cytoplasmic protein was produced, or translation was eliminated by mutating the start codon, a shift was not observed. Antibiotics that block transcription and translation similarly prevented locus repositioning toward the membrane. We also found that repositioning is relatively rapid and can be detected at positions that are a considerable distance on the chromosome from the gene encoding the membrane protein (>90 kb). Given that membrane protein-encoding genes are distributed throughout the chromosome, their expression may be an important mechanism for maintaining the bacterial chromosome in an expanded and dynamic state. PMID:22529375

  5. Discriminating lysosomal membrane protein types using dynamic neural network.

    Science.gov (United States)

    Tripathi, Vijay; Gupta, Dwijendra Kumar

    2014-01-01

    This work presents a dynamic artificial neural network methodology, which classifies the proteins into their classes from their sequences alone: the lysosomal membrane protein classes and the various other membranes protein classes. In this paper, neural networks-based lysosomal-associated membrane protein type prediction system is proposed. Different protein sequence representations are fused to extract the features of a protein sequence, which includes seven feature sets; amino acid (AA) composition, sequence length, hydrophobic group, electronic group, sum of hydrophobicity, R-group, and dipeptide composition. To reduce the dimensionality of the large feature vector, we applied the principal component analysis. The probabilistic neural network, generalized regression neural network, and Elman regression neural network (RNN) are used as classifiers and compared with layer recurrent network (LRN), a dynamic network. The dynamic networks have memory, i.e. its output depends not only on the input but the previous outputs also. Thus, the accuracy of LRN classifier among all other artificial neural networks comes out to be the highest. The overall accuracy of jackknife cross-validation is 93.2% for the data-set. These predicted results suggest that the method can be effectively applied to discriminate lysosomal associated membrane proteins from other membrane proteins (Type-I, Outer membrane proteins, GPI-Anchored) and Globular proteins, and it also indicates that the protein sequence representation can better reflect the core feature of membrane proteins than the classical AA composition.

  6. Identification of Salt-Tolerant Sinorhizobium sp Strain BL3 Membrane Proteins Based on Proteomics

    DEFF Research Database (Denmark)

    Tanthanuch, Waraporn; Mohammed, Shabaz; Matthiesen, Rune

    2010-01-01

    Sinorhizobium sp. BL3 is a salt-tolerant strain that can fix atmospheric nitrogen in symbiosis with leguminous host plants under salt-stress conditions. Since cell membranes are the first barrier to environmental change, it is interesting to explore the membrane proteins within this protective...

  7. One-step isolation of plasma membrane proteins using magnetic beads with immobilized concanavalin A

    DEFF Research Database (Denmark)

    Lee, Yu-Chen; Block, Gregory; Chen, Huiwen

    2008-01-01

    We have developed a simple method for isolating and purifying plasma membrane proteins from various cell types. This one-step affinity-chromatography method uses the property of the lectin concanavalin A (ConA) and the technique of magnetic bead separation to obtain highly purified plasma membran...

  8. Engineering Escherichia coli for Functional Expression of Membrane Proteins

    NARCIS (Netherlands)

    Ho, Franz Y; Poolman, Bert

    2015-01-01

    A major bottleneck in the characterization of membrane proteins is low yield of functional protein in recombinant expression. Microorganisms are widely used for recombinant protein production, because of ease of cultivation and high protein yield. However, the target proteins do not always obtain

  9. Intermolecular detergent-membrane protein noes for the characterization of the dynamics of membrane protein-detergent complexes.

    Science.gov (United States)

    Eichmann, Cédric; Orts, Julien; Tzitzilonis, Christos; Vögeli, Beat; Smrt, Sean; Lorieau, Justin; Riek, Roland

    2014-12-11

    The interaction between membrane proteins and lipids or lipid mimetics such as detergents is key for the three-dimensional structure and dynamics of membrane proteins. In NMR-based structural studies of membrane proteins, qualitative analysis of intermolecular nuclear Overhauser enhancements (NOEs) or paramagnetic resonance enhancement are used in general to identify the transmembrane segments of a membrane protein. Here, we employed a quantitative characterization of intermolecular NOEs between (1)H of the detergent and (1)H(N) of (2)H-perdeuterated, (15)N-labeled α-helical membrane protein-detergent complexes following the exact NOE (eNOE) approach. Structural considerations suggest that these intermolecular NOEs should show a helical-wheel-type behavior along a transmembrane helix or a membrane-attached helix within a membrane protein as experimentally demonstrated for the complete influenza hemagglutinin fusion domain HAfp23. The partial absence of such a NOE pattern along the amino acid sequence as shown for a truncated variant of HAfp23 and for the Escherichia coli inner membrane protein YidH indicates the presence of large tertiary structure fluctuations such as an opening between helices or the presence of large rotational dynamics of the helices. Detergent-protein NOEs thus appear to be a straightforward probe for a qualitative characterization of structural and dynamical properties of membrane proteins embedded in detergent micelles.

  10. Diffuse Charge Effects in Fuel Cell Membranes

    NARCIS (Netherlands)

    Biesheuvel, P.M.; Franco, A.A.; Bazant, M.Z.

    2009-01-01

    It is commonly assumed that electrolyte membranes in fuel cells are electrically neutral, except in unsteady situations, when the double-layer capacitance is heuristically included in equivalent circuit calculations. Indeed, the standard model for electron transfer kinetics at the membrane/electrode

  11. Studying Membrane Protein Structure and Function Using Nanodiscs

    DEFF Research Database (Denmark)

    Huda, Pie

    The structure and dynamic of membrane proteins can provide valuable information about general functions, diseases and effects of various drugs. Studying membrane proteins are a challenge as an amphiphilic environment is necessary to stabilise the protein in a functionally and structurally relevant...... form. This is most typically achieved through the use of detergent based reconstitution systems. However, time and again such systems fail to provide a suitable environment causing aggregation and inactivation. Nanodiscs are self-assembled lipoproteins containing two membrane scaffold proteins...... and a lipid bilayer in defined nanometer size, which can act as a stabiliser for membrane proteins. This enables both functional and structural investigation of membrane proteins in a detergent free environment which is closer to the native situation. Understanding the self-assembly of nanodiscs is important...

  12. Plasma membrane microdomains regulate turnover of transport proteins in yeast

    Czech Academy of Sciences Publication Activity Database

    Grossmann, G.; Malínský, Jan; Stahlschmidt, W.; Loibl, M.; Weig-Meckl, I.; Frommer, W.B.; Opekarová, Miroslava; Tanner, W.

    2008-01-01

    Roč. 183, č. 6 (2008), s. 1075-1088 ISSN 0021-9525 R&D Projects: GA ČR GA204/06/0009; GA ČR GA204/07/0133; GA ČR GC204/08/J024 Institutional research plan: CEZ:AV0Z50390703; CEZ:AV0Z50200510 Keywords : Lithium acetate * Membrane compartment of Can1 * Monomeric red fluorescent protein Subject RIV: EA - Cell Biology Impact factor: 9.120, year: 2008

  13. Cell biology symposium: Membrane trafficking and signal transduction

    Science.gov (United States)

    In general, membrane trafficking is a broad group of processes where proteins and other large molecules are distributed throughout the cell as well as adjacent extracellular spaces. Whereas signal transduction is a process where signals are transmitted through a series of chemical or molecular event...

  14. Membrane interaction of retroviral Gag proteins

    Directory of Open Access Journals (Sweden)

    Robert Alfred Dick

    2014-04-01

    Full Text Available Assembly of an infectious retroviral particle relies on multimerization of the Gag polyprotein at the inner leaflet of the plasma membrane. The three domains of Gag common to all retroviruses-- MA, CA, and NC-- provide the signals for membrane binding, assembly, and viral RNA packaging, respectively. These signals do not function independently of one another. For example, Gag multimerization enhances membrane binding and is more efficient when NC is interacting with RNA. MA binding to the plasma membrane is governed by several principles, including electrostatics, recognition of specific lipid head groups, hydrophobic interactions, and membrane order. HIV-1 uses many of these principles while Rous sarcoma virus (RSV appears to use fewer. This review describes the principles that govern Gag interactions with membranes, focusing on RSV and HIV-1 Gag. The review also defines lipid and membrane behavior, and discusses the complexities in determining how lipid and membrane behavior impact Gag membrane binding.

  15. Static light scattering to characterize membrane proteins in detergent solution

    NARCIS (Netherlands)

    Slotboom, Dirk Jan; Duurkens, Ria H.; Olieman, Kees; Erkens, Guus B.

    2008-01-01

    Determination of the oligomeric state or the subunit stoichiometry of integral membrane proteins in detergent solution is notoriously difficult, because the amount of detergent (and lipid) associated with the proteins is usually not known. Only two classical methods (sedimentation equilibrium

  16. Consequences of membrane protein overexpression in Escherichia coli.

    Science.gov (United States)

    Wagner, Samuel; Baars, Louise; Ytterberg, A Jimmy; Klussmeier, Anja; Wagner, Claudia S; Nord, Olof; Nygren, Per-Ake; van Wijk, Klaas J; de Gier, Jan-Willem

    2007-09-01

    Overexpression of membrane proteins is often essential for structural and functional studies, but yields are frequently too low. An understanding of the physiological response to overexpression is needed to improve such yields. Therefore, we analyzed the consequences of overexpression of three different membrane proteins (YidC, YedZ, and LepI) fused to green fluorescent protein (GFP) in the bacterium Escherichia coli and compared this with overexpression of a soluble protein, GST-GFP. Proteomes of total lysates, purified aggregates, and cytoplasmic membranes were analyzed by one- and two-dimensional gel electrophoresis and mass spectrometry complemented with flow cytometry, microscopy, Western blotting, and pulse labeling experiments. Composition and accumulation levels of protein complexes in the cytoplasmic membrane were analyzed with improved two-dimensional blue native PAGE. Overexpression of the three membrane proteins, but not soluble GST-GFP, resulted in accumulation of cytoplasmic aggregates containing the overexpressed proteins, chaperones (DnaK/J and GroEL/S), and soluble proteases (HslUV and ClpXP) as well as many precursors of periplasmic and outer membrane proteins. This was consistent with lowered accumulation levels of secreted proteins in the three membrane protein overexpressors and is likely to be a direct consequence of saturation of the cytoplasmic membrane protein translocation machinery. Importantly accumulation levels of respiratory chain complexes in the cytoplasmic membrane were strongly reduced. Induction of the acetate-phosphotransacetylase pathway for ATP production and a down-regulated tricarboxylic acid cycle indicated the activation of the Arc two-component system, which mediates adaptive responses to changing respiratory states. This study provides a basis for designing rational strategies to improve yields of membrane protein overexpression in E. coli.

  17. Role of membrane contact sites in protein import into mitochondria.

    Science.gov (United States)

    Horvath, Susanne E; Rampelt, Heike; Oeljeklaus, Silke; Warscheid, Bettina; van der Laan, Martin; Pfanner, Nikolaus

    2015-03-01

    Mitochondria import more than 1,000 different proteins from the cytosol. The proteins are synthesized as precursors on cytosolic ribosomes and are translocated by protein transport machineries of the mitochondrial membranes. Five main pathways for protein import into mitochondria have been identified. Most pathways use the translocase of the outer mitochondrial membrane (TOM) as the entry gate into mitochondria. Depending on specific signals contained in the precursors, the proteins are subsequently transferred to different intramitochondrial translocases. In this article, we discuss the connection between protein import and mitochondrial membrane architecture. Mitochondria possess two membranes. It is a long-standing question how contact sites between outer and inner membranes are formed and which role the contact sites play in the translocation of precursor proteins. A major translocation contact site is formed between the TOM complex and the presequence translocase of the inner membrane (TIM23 complex), promoting transfer of presequence-carrying preproteins to the mitochondrial inner membrane and matrix. Recent findings led to the identification of contact sites that involve the mitochondrial contact site and cristae organizing system (MICOS) of the inner membrane. MICOS plays a dual role. It is crucial for maintaining the inner membrane cristae architecture and forms contacts sites to the outer membrane that promote translocation of precursor proteins into the intermembrane space and outer membrane of mitochondria. The view is emerging that the mitochondrial protein translocases do not function as independent units, but are embedded in a network of interactions with machineries that control mitochondrial activity and architecture. © 2014 The Protein Society.

  18. A 39-kD plasma membrane protein (IP39) is an anchor for the unusual membrane skeleton of Euglena gracilis

    International Nuclear Information System (INIS)

    Rosiere, T.K.; Marrs, J.A.; Bouck, G.B.

    1990-01-01

    The major integral plasma membrane protein (IP39) of Euglena gracilis was radiolabeled, peptide mapped, and dissected with proteases to identify cytoplasmic domains that bind and anchor proteins of the cell surface. When plasma membranes were radioiodinated and extracted with octyl glucoside, 98% of the extracted label was found in IP39 or the 68- and 110-kD oligomers of IP39. The octyl glucoside extracts were incubated with unlabeled cell surface proteins immobilized on nitrocellulose (overlays). Radiolabel from the membrane extract bound one (80 kD) of the two (80 and 86 kD) major membrane skeletal protein bands. Resolubilization of the bound label yielded a radiolabeled polypeptide identical in Mr to IP39. Intact plasma membranes were also digested with papain before or after radioiodination, thereby producing a cytoplasmically truncated IP39. The octyl glucoside extract of truncated IP39 no longer bound to the 80-kD membrane skeletal protein in the nitrocellulose overlays. EM of intact or trypsin digested plasma membranes incubated with membrane skeletal proteins under stringent conditions similar to those used in the nitrocellulose overlays revealed a partially reformed membrane skeletal layer. Little evidence of a membrane skeletal layer was found, however, when plasma membranes were predigested with papain before reassociation. A candidate 80-kD binding domain of IP39 has been tentatively identified as a peptide fragment that was present after trypsin digestion of plasma membranes, but was absent after papain digestion in two-dimensional peptide maps of IP39. Together, these data suggest that the unique peripheral membrane skeleton of Euglena binds to the plasma membrane through noncovalent interactions between the major 80-kD membrane skeletal protein and a small, papain sensitive cytoplasmic domain of IP39

  19. Randomly organized lipids and marginally stable proteins: a coupling of weak interactions to optimize membrane signaling.

    Science.gov (United States)

    Rice, Anne M; Mahling, Ryan; Fealey, Michael E; Rannikko, Anika; Dunleavy, Katie; Hendrickson, Troy; Lohese, K Jean; Kruggel, Spencer; Heiling, Hillary; Harren, Daniel; Sutton, R Bryan; Pastor, John; Hinderliter, Anne

    2014-09-01

    Eukaryotic lipids in a bilayer are dominated by weak cooperative interactions. These interactions impart highly dynamic and pliable properties to the membrane. C2 domain-containing proteins in the membrane also interact weakly and cooperatively giving rise to a high degree of conformational plasticity. We propose that this feature of weak energetics and plasticity shared by lipids and C2 domain-containing proteins enhance a cell's ability to transduce information across the membrane. We explored this hypothesis using information theory to assess the information storage capacity of model and mast cell membranes, as well as differential scanning calorimetry, carboxyfluorescein release assays, and tryptophan fluorescence to assess protein and membrane stability. The distribution of lipids in mast cell membranes encoded 5.6-5.8bits of information. More information resided in the acyl chains than the head groups and in the inner leaflet of the plasma membrane than the outer leaflet. When the lipid composition and information content of model membranes were varied, the associated C2 domains underwent large changes in stability and denaturation profile. The C2 domain-containing proteins are therefore acutely sensitive to the composition and information content of their associated lipids. Together, these findings suggest that the maximum flow of signaling information through the membrane and into the cell is optimized by the cooperation of near-random distributions of membrane lipids and proteins. This article is part of a Special Issue entitled: Interfacially Active Peptides and Proteins. Guest Editors: William C. Wimley and Kalina Hristova. Copyright © 2014 Elsevier B.V. All rights reserved.

  20. Cell-penetrating peptides for drug delivery across membrane barriers

    DEFF Research Database (Denmark)

    Foged, Camilla; Nielsen, Hanne Moerck

    2008-01-01

    During the last decade, cell-penetrating peptides have been investigated for their ability to overcome the plasma membrane barrier of mammalian cells for the intracellular or transcellular delivery of cargoes as diverse as low molecular weight drugs, imaging agents, oligonucleotides, peptides......-penetrating peptides as transmembrane drug delivery agents, according to the recent literature, and discusses critical issues and future challenges in relation to fully understanding the fundamental principles of the cell-penetrating peptide-mediated membrane translocation of cargoes and the exploitation......, proteins and colloidal carriers such as liposomes and polymeric nanoparticles. Their ability to cross biological membranes in a non-disruptive way without apparent toxicity is highly desired for increasing drug bioavailability. This review provides an overview of the application of cell...

  1. Scaffolding proteins in membrane trafficking : the role of ELKS

    NARCIS (Netherlands)

    Yu, K.L.

    2015-01-01

    Intracellular membrane trafficking is an essential cellular process that involves cooperation of many factors such as scaffolding proteins, GTPases and SNAREs. These proteins work together to ensure proper delivery of different membrane-enclosed cargoes to specific cellular destinations. In this

  2. Advanced membrane electrode assemblies for fuel cells

    Science.gov (United States)

    Kim, Yu Seung; Pivovar, Bryan S.

    2012-07-24

    A method of preparing advanced membrane electrode assemblies (MEA) for use in fuel cells. A base polymer is selected for a base membrane. An electrode composition is selected to optimize properties exhibited by the membrane electrode assembly based on the selection of the base polymer. A property-tuning coating layer composition is selected based on compatibility with the base polymer and the electrode composition. A solvent is selected based on the interaction of the solvent with the base polymer and the property-tuning coating layer composition. The MEA is assembled by preparing the base membrane and then applying the property-tuning coating layer to form a composite membrane. Finally, a catalyst is applied to the composite membrane.

  3. Molecular dynamics simulations of large integral membrane proteins with an implicit membrane model.

    Science.gov (United States)

    Tanizaki, Seiichiro; Feig, Michael

    2006-01-12

    The heterogeneous dielectric generalized Born (HDGB) methodology is an the extension of the GBMV model for the simulation of integral membrane proteins with an implicit membrane environment. Three large integral membrane proteins, the bacteriorhodopsin monomer and trimer and the BtuCD protein, were simulated with the HDGB model in order to evaluate how well thermodynamic and dynamic properties are reproduced. Effects of the truncation of electrostatic interactions were examined. For all proteins, the HDGB model was able to generate stable trajectories that remained close to the starting experimental structures, in excellent agreement with explicit membrane simulations. Dynamic properties evaluated through a comparison of B-factors are also in good agreement with experiment and explicit membrane simulations. However, overall flexibility was slightly underestimated with the HDGB model unless a very large electrostatic cutoff is employed. Results with the HDGB model are further compared with equivalent simulations in implicit aqueous solvent, demonstrating that the membrane environment leads to more realistic simulations.

  4. The role of antioxidant-protein interactions in biological membrane

    International Nuclear Information System (INIS)

    McGillivray, Duncan J; Singh, Rachna; Melton, Laurence D.; Worcester, David L.; Gilbert, Elliot P.

    2009-01-01

    Full text: Oxidative damage of cellular membranes has been linked to a variety of disease pathologies, including cardiac disease, Alzheimer's and complications due to diabetes. The oxidation of unsaturated and polyunsaturated fatty acid chains found in cellular membranes leads to significant alteration in membrane physical properties, including lipid orientation and membrane permeability, which ultimately affect biological function. Polyphenols are naturally occurring phytochemicals present in a number of fruit and vegetables that are of interest for their anti-oxidative powers. These polyphenols inhibit lipid oxidation in cellular membrane surfaces, although the mechanism of this inhibition is not entirely clear. Moreover, the polyphenols have significant binding affinity for proteins, which can lead to the formation of soluble and insoluble protein-polyphenol complexes Significantly, in the presence of casein proteins the oxidation inhibition the polyphenols in the membrane is significantly enhanced (as assessed by Lipid Peroxidation Inhibition Capacity assays). Thus the antioxidant pathway appears to involve these protein/polyphenol complexes, as well as direct antioxidant action by the polyphenol. Here we discuss neutron and x-ray scattering results from phospholipid membranes, looking at the positioning of two examples of polyphenolic antioxidants in phospholipid membranes, quercetin and phloretin, the antioxidants' impact on the membrane organisation, and the interaction between antioxidant and extra-membranous protein. This information sheds light on the mechanism of antioxidant protection in these systems, which may be used to understand biological responses to oxidative stress.

  5. A novel bioactive membrane by cell electrospinning.

    Science.gov (United States)

    Chen, Haiping; Liu, Yuanyuan; Hu, Qingxi

    2015-11-01

    Electrospinning permits fabrication of biodegradable matrices that can resemble the both scale and mechanical behavior of the native extracellular matrix. However, achieving high-cellular density and infiltration of cells within matrices with traditional technique remain challenging and time consuming. The cell electrospinning technique presented in this paper can mitigate the problems associated with these limitations. Cells encapsulated by the material in the cell electrospinning technique survived well and distributed homogenously within the nanofibrous membrane, and their vitality was improved to 133% after being cultured for 28 days. The electrospun nanofibrous membrane has a certain degradation property and favorable cell-membrane interaction that supports the active biocompatibility of the membrane. Its properties are helpful for supporting cell attachment and growth, maintaining phenotypic shape, and secreting an ample amount of extracellular matrix (ECM). This novel membrane may be a potential application within the field of tissue engineering. The ability of cell electrospinning to microintegrate cells into a biodegradable fibrous matrix embodies a novel tissue engineering approach that could be applied to fabricate a high cell density elastic tissue mimetic. Copyright © 2015 Elsevier Inc. All rights reserved.

  6. Integral and peripheral association of proteins and protein complexes with Yersinia pestis inner and outer membranes

    Directory of Open Access Journals (Sweden)

    Bunai Christine L

    2009-02-01

    Full Text Available Abstract Yersinia pestis proteins were sequentially extracted from crude membranes with a high salt buffer (2.5 M NaBr, an alkaline solution (180 mM Na2CO3, pH 11.3 and membrane denaturants (8 M urea, 2 M thiourea and 1% amidosulfobetaine-14. Separation of proteins by 2D gel electrophoresis was followed by identification of more than 600 gene products by MS. Data from differential 2D gel display experiments, comparing protein abundances in cytoplasmic, periplasmic and all three membrane fractions, were used to assign proteins found in the membrane fractions to three protein categories: (i integral membrane proteins and peripheral membrane proteins with low solubility in aqueous solutions (220 entries; (ii peripheral membrane proteins with moderate to high solubility in aqueous solutions (127 entries; (iii cytoplasmic or ribosomal membrane-contaminating proteins (80 entries. Thirty-one proteins were experimentally associated with the outer membrane (OM. Circa 50 proteins thought to be part of membrane-localized, multi-subunit complexes were identified in high Mr fractions of membrane extracts via size exclusion chromatography. This data supported biologically meaningful assignments of many proteins to the membrane periphery. Since only 32 inner membrane (IM proteins with two or more predicted transmembrane domains (TMDs were profiled in 2D gels, we resorted to a proteomic analysis by 2D-LC-MS/MS. Ninety-four additional IM proteins with two or more TMDs were identified. The total number of proteins associated with Y. pestis membranes increased to 456 and included representatives of all six β-barrel OM protein families and 25 distinct IM transporter families.

  7. Membrane's Eleven: heavy-atom derivatives of membrane-protein crystals

    DEFF Research Database (Denmark)

    Morth, Jens Preben; Sørensen, Thomas Lykke-Møller; Nissen, Poul

    2006-01-01

    A database has been assembled of heavy-atom derivatives used in the structure determination of membrane proteins. The database can serve as a guide to the design of experiments in the search for heavy-atom derivatives of new membrane-protein crystals. The database pinpoints organomercurials, plat...

  8. Rational design of a fusion partner for membrane protein expression in E. coli.

    Science.gov (United States)

    Luo, Jianying; Choulet, Julie; Samuelson, James C

    2009-08-01

    We have designed a novel protein fusion partner (P8CBD) to utilize the co-translational SRP pathway in order to target heterologous proteins to the E. coli inner membrane. SRP-dependence was demonstrated by analyzing the membrane translocation of P8CBD-PhoA fusion proteins in wt and SRP-ffh77 mutant cells. We also demonstrate that the P8CBD N-terminal fusion partner promotes over-expression of a Thermotoga maritima polytopic membrane protein by replacement of the native signal anchor sequence. Furthermore, the yeast mitochondrial inner membrane protein Oxa1p was expressed as a P8CBD fusion and shown to function within the E. coli inner membrane. In this example, the mitochondrial targeting peptide was replaced by P8CBD. Several practical features were incorporated into the P8CBD expression system to aid in protein detection, purification, and optional in vitro processing by enterokinase. The basis of membrane protein over-expression toxicity is discussed and solutions to this problem are presented. We anticipate that this optimized expression system will aid in the isolation and study of various recombinant forms of membrane-associated protein.

  9. Membrane androgen receptor characteristics of human ZIP9 (SLC39A) zinc transporter in prostate cancer cells: Androgen-specific activation and involvement of an inhibitory G protein in zinc and MAP kinase signaling.

    Science.gov (United States)

    Thomas, Peter; Pang, Yefei; Dong, Jing

    2017-05-15

    Characteristics of novel human membrane androgen receptor (mAR), ZIP9 (SLC39A9), were investigated in ZIP9-transfected PC-3 cells (PC3-ZIP9). Ligand blot analysis showed plasma membrane [ 3 H]-T binding corresponds to the position of ZIP9 on Western blots which suggests ZIP9 can bind [ 3 H]-T alone, without a protein partner. Progesterone antagonized testosterone actions, blocking increases in zinc, Erk phosphorylation and apoptosis, further evidence that ZIP9 is specifically activated by androgens. Pre-treatment with GTPγS and pertussis toxin decreased plasma membrane [ 3 H]-T binding and blocked testosterone-induced increases in Erk phosphorylation and intracellular zinc, indicating ZIP9 is coupled to an inhibitory G protein (Gi) that mediates both MAP kinase and zinc signaling. Testosterone treatment of nuclei and mitochondria which express ZIP9 decreased their zinc contents, suggesting ZIP9 also regulates free zinc through releasing it from these intracellular organelles. The results show ZIP9 is a specific Gi coupled-mAR mediating testosterone-induced MAP kinase and zinc signaling in PC3-ZIP9 cells. Copyright © 2017 Elsevier B.V. All rights reserved.

  10. Effects of cholesterol depletion on membrane nanostructure in MCF-7 cells by atomic force microscopy

    Science.gov (United States)

    Wang, Yuhua; Jiang, Ningcheng; Shi, Aisi; Zheng, Liqin; Yang, Hongqin; Xie, Shusen

    2017-02-01

    The cell membrane is composed of phospholipids, glycolipids, cholesterol and proteins that are dynamic and heterogeneous distributed in the bilayer structure and many researches have showed that the plasma membrane in eukaryotic cells contains microdomains termed "lipid raft" in which cholesterol, sphingolipids and specific membrane proteins are enriched. Cholesterol extraction induced lipid raft disruption is one of the most widely used methods for lipid raft research and MβCD is a type of solvent to extract the cholesterol from cell membranes. In this study, the effect of MβCD treatment on the membrane nanostructure in MCF-7 living cells was investigated by atomic force microscopy. Different concentrations of MβCD were selected to deplete cholesterol for 30 min and the viability of cells was tested by MTT assay to obtain the optimal concentration. Then the nanostructure of the cell membrane was detected. The results show that an appropriate concentration of MβCD can induce the alteration of cell membranes nanostructure and the roughness of membrane surface decreases significantly. This may indicate that microdomains of the cell membrane disappear and the cell membrane appears more smoothly. Cholesterol can affect nanostructure and inhomogeneity of the plasma membrane in living cells.

  11. Red Blood Cell Membrane-Cloaked Nanoparticles For Drug Delivery

    Science.gov (United States)

    Carpenter, Cody Westcott

    Herein we describe the development of the Red Blood Cell coated nanoparticle, RBC-NP. Purified natural erythrocyte membrane is used to coat drug-loaded poly(lacticco-glycolic acid) (PLGA). Synthetic PLGA co-polymer is biocompatible and biodegradable and has already received US FDA approval for drug-delivery and diagnostics. This work looks specifically at the retention of immunosuppressive proteins on RBC-NPs, right-sidedness of natural RBC membranes interfacing with synthetic polymer nanoparticles, sustained and retarded drug release of RBC-NPs as well as further surface modification of RBC-NPs for increased targeting of model cancer cell lines.

  12. Increased phorbol 12,13-dibutyrate (PDBu) receptor function associated with sickle red cell membrane ghosts

    International Nuclear Information System (INIS)

    Ramachandran, M.; Nair, C.N.; Abraham, E.C.

    1987-01-01

    The biological receptor for tumor-promoting phorbol esters has been identified as the Ca 2+ /phospholipid dependent enzyme, protein kinase C. In the red cell, this enzyme is mainly cytosolic but becomes translocated to the membrane if the cellular Ca 2+ is allowed to rise. Since cellular Ca 2+ in sickle red cells is high, it was reasoned that this enzyme may become more membrane-bound. In fact, the authors noticed a four-fold increase in the binding of 3 H-PDBu by membrane ghosts isolated from sickle red cells compared to normal red cells (pmoles PDBu bound/mg protein; normal = 0.3 vs sickle cell = 1.4). Attempts to assay the enzyme directly as phospholipid-activated 32 P incorporation into the acid-precipitable membrane proteins also indicated a two-fold increase in the radiolabelling of sickle cell membrane ghosts. Autophosphorylation of membrane proteins and analysis of the phosphorylation profile by SDS-PAGE and autoradiography revealed phosphorylation predominantly of bands 3, 4.1 and 4.9 which are known protein kinase C substrates for the red cell enzyme. The increased membrane-associated protein kinase C in sickle red cells may have a bearing on the altered membrane properties reported in this condition

  13. Synthesis and structural characterization of a mimetic membrane-anchored prion protein

    OpenAIRE

    Hicks, M R; Gill, A C; Bath, I K; Rullay, A K; Sylvester, I D; Crout, D H; Pinheiro, T J T

    2006-01-01

    During pathogenesis of transmissible spongiform encephalopathies (TSEs) an abnormal form (PrPSc) of the host encoded prion protein (PrPC) accumulates in insoluble fibrils and plaques. The two forms of PrP appear to have identical covalent structures, but differ in secondary and tertiary structure. Both PrPC and PrPSc have glycosylphospatidylinositol (GPI) anchors through which the protein is tethered to cell membranes. Membrane attachment has been suggested to play a role in the conversion of...

  14. Present and future of membrane protein structure determination by electron crystallography.

    Science.gov (United States)

    Ubarretxena-Belandia, Iban; Stokes, David L

    2010-01-01

    Membrane proteins are critical to cell physiology, playing roles in signaling, trafficking, transport, adhesion, and recognition. Despite their relative abundance in the proteome and their prevalence as targets of therapeutic drugs, structural information about membrane proteins is in short supply. This chapter describes the use of electron crystallography as a tool for determining membrane protein structures. Electron crystallography offers distinct advantages relative to the alternatives of X-ray crystallography and NMR spectroscopy. Namely, membrane proteins are placed in their native membranous environment, which is likely to favor a native conformation and allow changes in conformation in response to physiological ligands. Nevertheless, there are significant logistical challenges in finding appropriate conditions for inducing membrane proteins to form two-dimensional arrays within the membrane and in using electron cryo-microscopy to collect the data required for structure determination. A number of developments are described for high-throughput screening of crystallization trials and for automated imaging of crystals with the electron microscope. These tools are critical for exploring the necessary range of factors governing the crystallization process. There have also been recent software developments to facilitate the process of structure determination. However, further innovations in the algorithms used for processing images and electron diffraction are necessary to improve throughput and to make electron crystallography truly viable as a method for determining atomic structures of membrane proteins. Copyright © 2010 Elsevier Inc. All rights reserved.

  15. Binding and Fusion of Extracellular Vesicles to the Plasma Membrane of Their Cell Targets.

    Science.gov (United States)

    Prada, Ilaria; Meldolesi, Jacopo

    2016-08-09

    Exosomes and ectosomes, extracellular vesicles of two types generated by all cells at multivesicular bodies and the plasma membrane, respectively, play critical roles in physiology and pathology. A key mechanism of their function, analogous for both types of vesicles, is the fusion of their membrane to the plasma membrane of specific target cells, followed by discharge to the cytoplasm of their luminal cargo containing proteins, RNAs, and DNA. Here we summarize the present knowledge about the interactions, binding and fusions of vesicles with the cell plasma membrane. The sequence initiates with dynamic interactions, during which vesicles roll over the plasma membrane, followed by the binding of specific membrane proteins to their cell receptors. Membrane binding is then converted rapidly into fusion by mechanisms analogous to those of retroviruses. Specifically, proteins of the extracellular vesicle membranes are structurally rearranged, and their hydrophobic sequences insert into the target cell plasma membrane which undergoes lipid reorganization, protein restructuring and membrane dimpling. Single fusions are not the only process of vesicle/cell interactions. Upon intracellular reassembly of their luminal cargoes, vesicles can be regenerated, released and fused horizontally to other target cells. Fusions of extracellular vesicles are relevant also for specific therapy processes, now intensely investigated.

  16. Nanodisc-based Co-immunoprecipitation for Mass Spectrometric Identification of Membrane-interacting Proteins

    DEFF Research Database (Denmark)

    Borch-Jensen, Jonas; Roepstorff, Peter; Møller-Jensen, Jakob

    2011-01-01

    Proteomic identification of protein interactions with membrane associated molecules in their native membrane environment pose a challenge because of technical problems of membrane handling. We investigate the possibility of employing membrane nanodiscs for harboring the membrane associated molecu...

  17. Folding Membrane Proteins by Deep Transfer Learning

    KAUST Repository

    Wang, Sheng

    2017-08-29

    Computational elucidation of membrane protein (MP) structures is challenging partially due to lack of sufficient solved structures for homology modeling. Here, we describe a high-throughput deep transfer learning method that first predicts MP contacts by learning from non-MPs and then predicts 3D structure models using the predicted contacts as distance restraints. Tested on 510 non-redundant MPs, our method has contact prediction accuracy at least 0.18 better than existing methods, predicts correct folds for 218 MPs, and generates 3D models with root-mean-square deviation (RMSD) less than 4 and 5 Å for 57 and 108 MPs, respectively. A rigorous blind test in the continuous automated model evaluation project shows that our method predicted high-resolution 3D models for two recent test MPs of 210 residues with RMSD ∼2 Å. We estimated that our method could predict correct folds for 1,345–1,871 reviewed human multi-pass MPs including a few hundred new folds, which shall facilitate the discovery of drugs targeting at MPs.

  18. Cooperative tumour cell membrane targeted phototherapy

    Science.gov (United States)

    Kim, Heegon; Lee, Junsung; Oh, Chanhee; Park, Ji-Ho

    2017-06-01

    The targeted delivery of therapeutics using antibodies or nanomaterials has improved the precision and safety of cancer therapy. However, the paucity and heterogeneity of identified molecular targets within tumours have resulted in poor and uneven distribution of targeted agents, thus compromising treatment outcomes. Here, we construct a cooperative targeting system in which synthetic and biological nanocomponents participate together in the tumour cell membrane-selective localization of synthetic receptor-lipid conjugates (SR-lipids) to amplify the subsequent targeting of therapeutics. The SR-lipids are first delivered selectively to tumour cell membranes in the perivascular region using fusogenic liposomes. By hitchhiking with extracellular vesicles secreted by the cells, the SR-lipids are transferred to neighbouring cells and further spread throughout the tumour tissues where the molecular targets are limited. We show that this tumour cell membrane-targeted delivery of SR-lipids leads to uniform distribution and enhanced phototherapeutic efficacy of the targeted photosensitizer.

  19. ARAMEMNON, a novel database for Arabidopsis integral membrane proteins

    DEFF Research Database (Denmark)

    Schwacke, Rainer; Schneider, Anja; van der Graaff, Eric

    2003-01-01

    A specialized database (DB) for Arabidopsis membrane proteins, ARAMEMNON, was designed that facilitates the interpretation of gene and protein sequence data by integrating features that are presently only available from individual sources. Using several publicly available prediction programs......, putative integral membrane proteins were identified among the approximately 25,500 proteins in the Arabidopsis genome DBs. By averaging the predictions from seven programs, approximately 6,500 proteins were classified as transmembrane (TM) candidate proteins. Some 1,800 of these contain at least four TM...

  20. Membrane dynamics

    DEFF Research Database (Denmark)

    Bendix, Pól Martin

    2015-01-01

    Current topics include membrane-protein interactions with regard to membrane deformation or curvature sensing by BAR domains. Also, we study the dynamics of membrane tubes of both cells and simple model membrane tubes. Finally, we study membrane phase behavior which has important implications...

  1. Polyclonal Antibody Production for Membrane Proteins via Genetic Immunization.

    Science.gov (United States)

    Hansen, Debra T; Robida, Mark D; Craciunescu, Felicia M; Loskutov, Andrey V; Dörner, Katerina; Rodenberry, John-Charles; Wang, Xiao; Olson, Tien L; Patel, Hetal; Fromme, Petra; Sykes, Kathryn F

    2016-02-24

    Antibodies are essential for structural determinations and functional studies of membrane proteins, but antibody generation is limited by the availability of properly-folded and purified antigen. We describe the first application of genetic immunization to a structurally diverse set of membrane proteins to show that immunization of mice with DNA alone produced antibodies against 71% (n = 17) of the bacterial and viral targets. Antibody production correlated with prior reports of target immunogenicity in host organisms, underscoring the efficiency of this DNA-gold micronanoplex approach. To generate each antigen for antibody characterization, we also developed a simple in vitro membrane protein expression and capture method. Antibody specificity was demonstrated upon identifying, for the first time, membrane-directed heterologous expression of the native sequences of the FopA and FTT1525 virulence determinants from the select agent Francisella tularensis SCHU S4. These approaches will accelerate future structural and functional investigations of therapeutically-relevant membrane proteins.

  2. Rasagiline prevents apoptosis induced by PK11195, a ligand of the outer membrane translocator protein (18 kDa), in SH-SY5Y cells through suppression of cytochrome c release from mitochondria.

    Science.gov (United States)

    Naoi, Makoto; Maruyama, Wakako; Yi, Hong

    2013-11-01

    Rasagiline protects neuronal cells from cell death caused by various lines of insults. Its neuroprotective function is due to suppression of mitochondrial apoptosis signaling and induction of neuroprotective genes, including Bcl-2 and neurotrophic factors. Rasagiline inhibits the mitochondrial membrane permeabilization, an initial stage in apoptosis, but the mechanism has been elusive. In this paper, it was investigated how rasagiline regulates mitochondrial death cascade in apoptosis induced in SH-SY5Y cells by PK11195, a ligand of the outer membrane translocator protein of 18 kDa. Rasagiline prevented release of cytochrome c (Cyt-c), and the following caspase 3 activation, ATP depletion and apoptosis, but did not inhibit the mitochondrial membrane potential collapse, in contrast to Bcl-2 overexpression. Rasagiline stabilized the mitochondrial contact site and suppressed Cyt-c release into cytoplasm, which should be the critical point for the regulation of apoptosis. Monoamine oxidase was not associated with anti-apoptotic activity of rasagiline in PK11195-induced apoptosis.

  3. Lipidic cubic phase injector facilitates membrane protein serial femtosecond crystallography.

    Science.gov (United States)

    Weierstall, Uwe; James, Daniel; Wang, Chong; White, Thomas A; Wang, Dingjie; Liu, Wei; Spence, John C H; Bruce Doak, R; Nelson, Garrett; Fromme, Petra; Fromme, Raimund; Grotjohann, Ingo; Kupitz, Christopher; Zatsepin, Nadia A; Liu, Haiguang; Basu, Shibom; Wacker, Daniel; Han, Gye Won; Katritch, Vsevolod; Boutet, Sébastien; Messerschmidt, Marc; Williams, Garth J; Koglin, Jason E; Marvin Seibert, M; Klinker, Markus; Gati, Cornelius; Shoeman, Robert L; Barty, Anton; Chapman, Henry N; Kirian, Richard A; Beyerlein, Kenneth R; Stevens, Raymond C; Li, Dianfan; Shah, Syed T A; Howe, Nicole; Caffrey, Martin; Cherezov, Vadim

    2014-01-01

    Lipidic cubic phase (LCP) crystallization has proven successful for high-resolution structure determination of challenging membrane proteins. Here we present a technique for extruding gel-like LCP with embedded membrane protein microcrystals, providing a continuously renewed source of material for serial femtosecond crystallography. Data collected from sub-10-μm-sized crystals produced with less than 0.5 mg of purified protein yield structural insights regarding cyclopamine binding to the Smoothened receptor.

  4. Specific Adhesion of Lipid Membranes Can Simultaneously Produce Two Types of Lipid and Protein Heterogeneities

    Science.gov (United States)

    Shindell, Orrin; Micah, Natalie; Ritzer, Max; Gordon, Vernita

    2015-03-01

    Living cells adhere to one another and their environment. Adhesion is associated with re-organization of the lipid and protein components of the cell membrane. The resulting heterogeneities are functional structures involved in biological processes. We use artificial lipid membranes that contain a single type of binding protein. Before adhesion, the lipid, protein, and dye components in the membrane are well-mixed and constitute a single disordered-liquid phase (Ld) . After adhesion, two distinct types of heterogeneities coexist in the adhesion zone: a central domain of ordered lipid phase that excludes both binding proteins and membrane dye, and a peripheral domain of disordered lipid phase that is densely packed with adhesion proteins and enriched in membrane dye relative to the non-adhered portion of the vesicle. Thus, we show that adhesion that is mediated by only one type of protein can organize the lipid and protein components of the membranes into heterogeneities that resemble those found in biology, for example the immune synapse.

  5. Fuel cell subassemblies incorporating subgasketed thrifted membranes

    Science.gov (United States)

    Iverson, Eric J.; Pierpont, Daniel M.; Yandrasits, Michael A.; Hamrock, Steven J.; Obradovich, Stephan J.; Peterson, Donald G.

    2016-03-01

    A fuel cell roll good subassembly is described that includes a plurality of individual electrolyte membranes. One or more first subgaskets are attached to the individual electrolyte membranes. Each of the first subgaskets has at least one aperture and the first subgaskets are arranged so the center regions of the individual electrolyte membranes are exposed through the apertures of the first subgaskets. A second subgasket comprises a web having a plurality of apertures. The second subgasket web is attached to the one or more first subgaskets so the center regions of the individual electrolyte membranes are exposed through the apertures of the second subgasket web. The second subgasket web may have little or no adhesive on the subgasket surface facing the electrolyte membrane.

  6. Cell membrane softening in human breast and cervical cancer cells

    Science.gov (United States)

    Händel, Chris; Schmidt, B. U. Sebastian; Schiller, Jürgen; Dietrich, Undine; Möhn, Till; Kießling, Tobias R.; Pawlizak, Steve; Fritsch, Anatol W.; Horn, Lars-Christian; Briest, Susanne; Höckel, Michael; Zink, Mareike; Käs, Josef A.

    2015-08-01

    Biomechanical properties are key to many cellular functions such as cell division and cell motility and thus are crucial in the development and understanding of several diseases, for instance cancer. The mechanics of the cellular cytoskeleton have been extensively characterized in cells and artificial systems. The rigidity of the plasma membrane, with the exception of red blood cells, is unknown and membrane rigidity measurements only exist for vesicles composed of a few synthetic lipids. In this study, thermal fluctuations of giant plasma membrane vesicles (GPMVs) directly derived from the plasma membranes of primary breast and cervical cells, as well as breast cell lines, are analyzed. Cell blebs or GPMVs were studied via thermal membrane fluctuations and mass spectrometry. It will be shown that cancer cell membranes are significantly softer than their non-malignant counterparts. This can be attributed to a loss of fluid raft forming lipids in malignant cells. These results indicate that the reduction of membrane rigidity promotes aggressive blebbing motion in invasive cancer cells.

  7. 3D pressure field in lipid membranes and membrane-protein complexes

    DEFF Research Database (Denmark)

    Ollila, O H Samuli; Risselada, H Jelger; Louhivuori, Martti

    2009-01-01

    We calculate full 3D pressure fields for inhomogeneous nanoscale systems using molecular dynamics simulation data. The fields represent systems with increasing level of complexity, ranging from semivesicles and vesicles to membranes characterized by coexistence of two phases, including also...... a protein-membrane complex. We show that the 3D pressure field is distinctly different for curved and planar bilayers, the pressure field depends strongly on the phase of the membrane, and that an integral protein modulates the tension and elastic properties of the membrane....

  8. Annexin A2 Mediates the Localization of Measles Virus Matrix Protein at the Plasma Membrane.

    Science.gov (United States)

    Koga, Ritsuko; Kubota, Marie; Hashiguchi, Takao; Yanagi, Yusuke; Ohno, Shinji

    2018-02-28

    Annexins are a family of structurally related proteins that bind negatively charged membrane phospholipids in a Ca 2+ -dependent manner. Annexin A2 (AnxA2), a member of the family, has been implicated in a variety of cellular functions including the organization of membrane domains, vesicular trafficking and cell-cell adhesion. AnxA2 generally forms the heterotetrameric complex with a small Ca 2+ -binding protein S100A10. Measles virus (MV), a member of the family Paramyxoviridae , is an enveloped virus with a nonsegmented negative strand RNA genome. Knockdown of AnxA2 greatly reduced MV growth in cells, without affecting its entry and viral RNA production. In MV-infected, AnxA2-knockdown cells, the expression level of the matrix (M) protein, but not other viral proteins, was reduced compared with that in control cells, and the distribution of the M protein at the plasma membrane was decreased. The M protein lines the inner surface of the envelope and plays an important role in virus assembly by connecting the nucleocapsid to the envelope proteins. The M protein bound to AnxA2 independently of AnxA2's phosphorylation or its association with S100A10, and was co-localized with AnxA2 within cells. Truncation of the N-terminal 10 amino acid residues, but not the N-terminal 5 residues, compromised the ability of the M protein to interact with AnxA2 and localize at the plasma membrane. These results indicate that AnxA2 mediates the localization of the MV M protein at the plasma membrane by interacting with its N-terminal region (especially residues at positions 6-10), thereby aiding in MV assembly. IMPORTANCE Measles virus (MV) is an important human pathogen, still claiming ∼ 100,000 lives per year despite the presence of effective vaccines, and causes occasional outbreaks even in developed countries. Replication of viruses largely relies on the functions of host cells. Our study revealed that the reduction of the host protein annexin A2 compromises the replication of

  9. High-yield membrane protein expression from E. coli using an engineered outer membrane protein F fusion.

    Science.gov (United States)

    Su, Pin-Chuan; Si, William; Baker, Deidre L; Berger, Bryan W

    2013-04-01

    Obtaining high yields of membrane proteins necessary to perform detailed structural study is difficult due to poor solubility and variability in yields from heterologous expression systems. To address this issue, an Escherichia coli-based membrane protein overexpression system utilizing an engineered bacterial outer membrane protein F (pOmpF) fusion has been developed. Full-length human receptor activity-modifying protein 1 (RAMP1) was expressed using pOmpF, solubilized in FC15 and purified to homogeneity. Using circular dichroism and fluorescence spectroscopy, purified full-length RAMP1 is composed of approximately 90% α-helix, and retains its solubility and structure in FC15 over a wide range of temperatures (20-60°C). Thus, our approach provides a useful, complementary approach to achieve high-yield, full-length membrane protein overexpression for biophysical studies. Copyright © 2013 The Protein Society.

  10. MEMBRANE-FUSION OF SEMLIKI FOREST VIRUS INVOLVES HOMOTRIMERS OF THE FUSION PROTEIN

    NARCIS (Netherlands)

    WAHLBERG, JM; WILSCHUT, J; GAROFF, H

    1992-01-01

    Infection of cells with enveloped viruses is accomplished through membrane fusion. The binding and fusion Processes are mediated by the spike proteins in the envelope of the virus particle and usually involve a series of conformational changes in these proteins. We have studied the low-pH-mediated

  11. Structural Aspects of Bacterial Outer Membrane Protein Assembly.

    Science.gov (United States)

    Calmettes, Charles; Judd, Andrew; Moraes, Trevor F

    2015-01-01

    The outer membrane of Gram-negative bacteria is predominantly populated by β-Barrel proteins and lipid anchored proteins that serve a variety of biological functions. The proper folding and assembly of these proteins is essential for bacterial viability and often plays a critical role in virulence and pathogenesis. The β-barrel assembly machinery (Bam) complex is responsible for the proper assembly of β-barrels into the outer membrane of Gram-negative bacteria, whereas the localization of lipoproteins (Lol) system is required for proper targeting of lipoproteins to the outer membrane.

  12. Tuning membrane protein mobility by confinement into nanodomains

    Science.gov (United States)

    Karner, Andreas; Nimmervoll, Benedikt; Plochberger, Birgit; Klotzsch, Enrico; Horner, Andreas; Knyazev, Denis G.; Kuttner, Roland; Winkler, Klemens; Winter, Lukas; Siligan, Christine; Ollinger, Nicole; Pohl, Peter; Preiner, Johannes

    2017-03-01

    High-speed atomic force microscopy (HS-AFM) can be used to visualize function-related conformational changes of single soluble proteins. Similar studies of single membrane proteins are, however, hampered by a lack of suitable flat, non-interacting membrane supports and by high protein mobility. Here we show that streptavidin crystals grown on mica-supported lipid bilayers can be used as porous supports for membranes containing biotinylated lipids. Using SecYEG (protein translocation channel) and GlpF (aquaglyceroporin), we demonstrate that the platform can be used to tune the lateral mobility of transmembrane proteins to any value within the dynamic range accessible to HS-AFM imaging through glutaraldehyde-cross-linking of the streptavidin. This allows HS-AFM to study the conformation or docking of spatially confined proteins, which we illustrate by imaging GlpF at sub-molecular resolution and by observing the motor protein SecA binding to SecYEG.

  13. Integral membrane protein structure determination using pseudocontact shifts

    Energy Technology Data Exchange (ETDEWEB)

    Crick, Duncan J.; Wang, Jue X. [University of Cambridge, Department of Biochemistry (United Kingdom); Graham, Bim; Swarbrick, James D. [Monash University, Monash Institute of Pharmaceutical Sciences (Australia); Mott, Helen R.; Nietlispach, Daniel, E-mail: dn206@cam.ac.uk [University of Cambridge, Department of Biochemistry (United Kingdom)

    2015-04-15

    Obtaining enough experimental restraints can be a limiting factor in the NMR structure determination of larger proteins. This is particularly the case for large assemblies such as membrane proteins that have been solubilized in a membrane-mimicking environment. Whilst in such cases extensive deuteration strategies are regularly utilised with the aim to improve the spectral quality, these schemes often limit the number of NOEs obtainable, making complementary strategies highly beneficial for successful structure elucidation. Recently, lanthanide-induced pseudocontact shifts (PCSs) have been established as a structural tool for globular proteins. Here, we demonstrate that a PCS-based approach can be successfully applied for the structure determination of integral membrane proteins. Using the 7TM α-helical microbial receptor pSRII, we show that PCS-derived restraints from lanthanide binding tags attached to four different positions of the protein facilitate the backbone structure determination when combined with a limited set of NOEs. In contrast, the same set of NOEs fails to determine the correct 3D fold. The latter situation is frequently encountered in polytopical α-helical membrane proteins and a PCS approach is thus suitable even for this particularly challenging class of membrane proteins. The ease of measuring PCSs makes this an attractive route for structure determination of large membrane proteins in general.

  14. A split ubiquitin system to reveal topology and released peptides of membrane proteins.

    Science.gov (United States)

    Li, Qiu-Ping; Wang, Shuai; Gou, Jin-Ying

    2017-09-02

    Membrane proteins define biological functions of membranes in cells. Extracellular peptides of transmembrane proteins receive signals from pathogens or environments, and are the major targets of drug developments. Despite of their essential roles, membrane proteins remain elusive in topological studies due to technique difficulties in their expressions and purifications. First, the target gene is cloned into a destination vector to fuse with C terminal ubiquitin at the N or C terminus. Then, Cub vector with target gene and Nub WT or Nub G vectors are transformed into AP4 or AP5 yeast cells, respectively. After mating, the diploid cells are dipped onto selection medium to check the growth. Topology of the target protein is determined according to Table 1. We present a split ubiquitin topology (SUT) analysis system to study the topology and truncation peptide of membrane proteins in a simple yeast experiment. In the SUT system, transcription activator (TA) fused with a nucleo-cytoplasmic protein shows strong auto-activation with both positive and negative control vectors. TA fused with the cytoplasmic end of membrane proteins activates reporter genes only with positive control vector with a wild type N terminal ubiquitin (Nub WT ). However, TA fused with the extracellular termini of membrane proteins can't activate reporter genes even with Nub WT . Interestingly,TA fused with the released peptide of a membrane protein shows autoactivation in the SUT system. The SUT system is a simple and fast experimental procedure complementary to computational predictions and large scale proteomic techniques. The preliminary data from SUT are valuable for pathogen recognitions and new drug developments.

  15. Detecting protein-protein interactions in living cells

    DEFF Research Database (Denmark)

    Gottschalk, Marie; Bach, Anders; Hansen, Jakob Lerche

    2009-01-01

    -terminal of the NMDA receptor and PDZ2 of PSD-95 were fused to green fluorescent protein (GFP) and Renilla luciferase (Rluc) and expressed in COS7 cells. A robust and specific BRET signal was obtained by expression of the appropriate partner proteins and subsequently, the assay was used to evaluate a Tat......The PDZ domain mediated interaction between the NMDA receptor and its intracellular scaffolding protein, PSD-95, is a potential target for treatment of ischemic brain diseases. We have recently developed a number of peptide analogues with improved affinity for the PDZ domains of PSD-95 compared...... to the endogenous C-terminal peptide of the NMDA receptor, as evaluated by a cell-free protein-protein interaction assay. However, it is important to address both membrane permeability and effect in living cells. Therefore a bioluminescence resonance energy transfer (BRET) assay was established, where the C...

  16. Factors Determining the Oxygen Permeability of Biological Membranes: Oxygen Transport Across Eye Lens Fiber-Cell Plasma Membranes.

    Science.gov (United States)

    Subczynski, Witold Karol; Widomska, Justyna; Mainali, Laxman

    2017-01-01

    Electron paramagnetic resonance (EPR) spin-label oximetry allows the oxygen permeability coefficient to be evaluated across homogeneous lipid bilayer membranes and, in some cases, across coexisting membrane domains without their physical separation. The most pronounced effect on oxygen permeability is observed for cholesterol, which additionally induces the formation of membrane domains. In intact biological membranes, integral proteins induce the formation of boundary and trapped lipid domains with a low oxygen permeability. The effective oxygen permeability coefficient across the intact biological membrane is affected not only by the oxygen permeability coefficients evaluated for each lipid domain but also by the surface area occupied by these domains in the membrane. All these factors observed in fiber cell plasma membranes of clear human eye lenses are reviewed here.

  17. How curved membranes recruit amphipathic helices and protein anchoring motifs.

    Science.gov (United States)

    Hatzakis, Nikos S; Bhatia, Vikram K; Larsen, Jannik; Madsen, Kenneth L; Bolinger, Pierre-Yves; Kunding, Andreas H; Castillo, John; Gether, Ulrik; Hedegård, Per; Stamou, Dimitrios

    2009-11-01

    Lipids and several specialized proteins are thought to be able to sense the curvature of membranes (MC). Here we used quantitative fluorescence microscopy to measure curvature-selective binding of amphipathic motifs on single liposomes 50-700 nm in diameter. Our results revealed that sensing is predominantly mediated by a higher density of binding sites on curved membranes instead of higher affinity. We proposed a model based on curvature-induced defects in lipid packing that related these findings to lipid sorting and accurately predicted the existence of a new ubiquitous class of curvature sensors: membrane-anchored proteins. The fact that unrelated structural motifs such as alpha-helices and alkyl chains sense MC led us to propose that MC sensing is a generic property of curved membranes rather than a property of the anchoring molecules. We therefore anticipate that MC will promote the redistribution of proteins that are anchored in membranes through other types of hydrophobic moieties.

  18. The exocyst at the interface between cytoskeleton and membranes in eukaryotic cells

    Directory of Open Access Journals (Sweden)

    Lukas eSynek

    2014-01-01

    Full Text Available Delivery and final fusion of the secretory vesicles with the relevant target membrane are hierarchically organized and reciprocally interconnected multi-step processes involving not only specific protein-protein interactions, but also specific protein-phospholipid interactions. The exocyst was discovered as a tethering complex mediating initial encounter of arriving exocytic vesicles with the plasma membrane. The exocyst complex is regulated by Rab and Rho small GTPases, resulting in docking of exocytic vesicles to the plasma membrane and finally their fusion mediated by specific SNARE complexes. In model Opisthokont cells, the exocyst was shown to directly interact with both microtubule and microfilament cytoskeleton and related motor proteins as well as with the plasma membrane via phosphatidylinositol 4,5-bisphosphate specific binding, which directly affects cortical cytoskeleton and plasma membrane dynamics. Here we summarize the current knowledge on exocyst-cytoskeleton-plasma membrane interactions in order to open a perspective for future research in this area in plant cells.

  19. Membrane progesterone receptor beta (mPRβ/Paqr8) promotes progesterone-dependent neurite outgrowth in PC12 neuronal cells via non-G protein-coupled receptor (GPCR) signaling.

    Science.gov (United States)

    Kasubuchi, Mayu; Watanabe, Keita; Hirano, Kanako; Inoue, Daisuke; Li, Xuan; Terasawa, Kazuya; Konishi, Morichika; Itoh, Nobuyuki; Kimura, Ikuo

    2017-07-12

    Recently, sex steroid membrane receptors garnered world-wide attention because they may be related to sex hormone-mediated unknown rapid non-genomic action that cannot be currently explained by their genomic action via nuclear receptors. Progesterone affects cell proliferation and survival via non-genomic effects. In this process, membrane progesterone receptors (mPRα, mPRβ, mPRγ, mPRδ, and mPRε) were identified as putative G protein-coupled receptors (GPCRs) for progesterone. However, the structure, intracellular signaling, and physiological functions of these progesterone receptors are still unclear. Here, we identify a molecular mechanism by which progesterone promotes neurite outgrowth through mPRβ (Paqr8) activation. Mouse mPRβ mRNA was specifically expressed in the central nervous system. It has an incomplete GPCR topology, presenting 6 transmembrane domains and did not exhibit typical GPCR signaling. Progesterone-dependent neurite outgrowth was exhibited by the promotion of ERK phosphorylation via mPRβ, but not via other progesterone receptors such as progesterone membrane receptor 1 (PGRMC-1) and nuclear progesterone receptor in nerve growth factor-induced neuronal PC12 cells. These findings provide new insights of regarding the non-genomic action of progesterone in the central nervous system.

  20. Protein-lipid interactions: from membrane domains to cellular networks

    National Research Council Canada - National Science Library

    Tamm, Lukas K

    2005-01-01

    ... membranes is the lipid bilayer. Embedded in the fluid lipid bilayer are proteins of various shapes and traits. This volume illuminates from physical, chemical and biological angles the numerous - mostly quite weak - interactions between lipids, proteins, and proteins and lipids that define the delicate, highly dynamic and yet so stable fabri...

  1. Dynamics of Membrane Proteins within Synthetic Polymer Membranes with Large Hydrophobic Mismatch.

    Science.gov (United States)

    Itel, Fabian; Najer, Adrian; Palivan, Cornelia G; Meier, Wolfgang

    2015-06-10

    The functioning of biological membrane proteins (MPs) within synthetic block copolymer membranes is an intriguing phenomenon that is believed to offer great potential for applications in life and medical sciences and engineering. The question why biological MPs are able to function in this completely artificial environment is still unresolved by any experimental data. Here, we have analyzed the lateral diffusion properties of different sized MPs within poly(dimethylsiloxane) (PDMS)-containing amphiphilic block copolymer membranes of membrane thicknesses between 9 and 13 nm, which results in a hydrophobic mismatch between the membrane thickness and the size of the proteins of 3.3-7.1 nm (3.5-5 times). We show that the high flexibility of PDMS, which provides membrane fluidities similar to phospholipid bilayers, is the key-factor for MP incorporation.

  2. Lactococcus lactis as host for overproduction of functional membrane proteins

    NARCIS (Netherlands)

    Kunji, ERS; Slotboom, DJ; Poolman, B

    2003-01-01

    Lactococcus lactis has many properties that are ideal for enhanced expression of membrane proteins. The organism is easy and inexpensive to culture, has a single membrane and relatively mild proteolytic activity. Methods for genetic manipulation are fully established and a tightly controlled

  3. Efficient cellular solid-state NMR of membrane proteins by targeted protein labeling

    International Nuclear Information System (INIS)

    Baker, Lindsay A.; Daniëls, Mark; Cruijsen, Elwin A. W. van der; Folkers, Gert E.; Baldus, Marc

    2015-01-01

    Solid-state NMR spectroscopy (ssNMR) has made significant progress towards the study of membrane proteins in their native cellular membranes. However, reduced spectroscopic sensitivity and high background signal levels can complicate these experiments. Here, we describe a method for ssNMR to specifically label a single protein by repressing endogenous protein expression with rifampicin. Our results demonstrate that treatment of E. coli with rifampicin during induction of recombinant membrane protein expression reduces background signals for different expression levels and improves sensitivity in cellular membrane samples. Further, the method reduces the amount of time and resources needed to produce membrane protein samples, enabling new strategies for studying challenging membrane proteins by ssNMR

  4. Chemical synthesis and biophysical applications of membrane proteins.

    Science.gov (United States)

    Zuo, Chao; Tang, Shan; Zheng, Ji-Shen

    2015-07-01

    Chemical synthesis or semi-synthesis of membrane proteins can provide unique molecular tools, such as site-specific isotope labeling or post-translationally modified membrane proteins to gain insight into their biophysical and functional characteristics. However, during preparation, purification, and ligation of transmembrane peptides, tremendous challenges are encountered owing to their hydrophobic nature. This review focuses on the recent advances in chemical synthesis strategies of membrane proteins. These strategies help to solubilize the hydrophobic transmembrane peptide sequences under standard purification and chemical ligation conditions to improve their handling properties. Biophysical and functional studies of synthetic membrane proteins are reviewed as well. Copyright © 2014 European Peptide Society and John Wiley & Sons, Ltd.

  5. Defining thermostability of membrane proteins by western blotting.

    Science.gov (United States)

    Ashok, Y; Nanekar, R; Jaakola, V-P

    2015-12-01

    Membrane proteins are relatively challenging targets for structural and other biophysical studies. Insufficient expression in various expression systems, inherent flexibility, and instability in the detergents that are required for membrane extraction are the main reasons for this limited success. Therefore, identification of suitable conditions and membrane protein variants that can help stabilize functional protein for extended periods of time is critical for structural studies. Here, we describe a western blot-based assay that simplifies identification of thermostabilizing conditions for membrane proteins. We show successful testing of a variety of parameters such as additive lipids, ligands and detergents. © The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

  6. Imaging and quantification of trans-membrane protein diffusion in living bacteria.

    NARCIS (Netherlands)

    Oswald, F.; Bank, E.; Bollen, Y.J.M.; Peterman, E.J.G.

    2014-01-01

    The cytoplasmic membrane forms the barrier between any cell's interior and the outside world. It contains many proteins that enable essential processes such as the transmission of signals, the uptake of nutrients, and cell division. In the case of prokaryotes, which do not contain intracellular

  7. Alternate Fuel Cell Membranes for Energy Independence

    Energy Technology Data Exchange (ETDEWEB)

    Storey, Robson, F.; Mauritz, Kenneth, A.; Patton, Derek, L.; Savin, Daniel, A.

    2012-12-18

    The overall objective of this project was the development and evaluation of novel hydrocarbon fuel cell (FC) membranes that possess high temperature performance and long term chemical/mechanical durability in proton exchange membrane (PEM) fuel cells (FC). The major research theme was synthesis of aromatic hydrocarbon polymers of the poly(arylene ether sulfone) (PAES) type containing sulfonic acid groups tethered to the backbone via perfluorinated alkylene linkages and in some cases also directly attached to the phenylene groups along the backbone. Other research themes were the use of nitrogen-based heterocyclics instead of acid groups for proton conduction, which provides high temperature, low relative humidity membranes with high mechanical/thermal/chemical stability and pendant moieties that exhibit high proton conductivities in the absence of water, and synthesis of block copolymers consisting of a proton conducting block coupled to poly(perfluorinated propylene oxide) (PFPO) blocks. Accomplishments of the project were as follows: 1) establishment of a vertically integrated program of synthesis, characterization, and evaluation of FC membranes, 2) establishment of benchmark membrane performance data based on Nafion for comparison to experimental membrane performance, 3) development of a new perfluoroalkyl sulfonate monomer, N,N-diisopropylethylammonium 2,2-bis(p-hydroxyphenyl) pentafluoropropanesulfonate (HPPS), 4) synthesis of random and block copolymer membranes from HPPS, 5) synthesis of block copolymer membranes containing high-acid-concentration hydrophilic blocks consisting of HPPS and 3,3'-disulfonate-4,4'-dichlorodiphenylsulfone (sDCDPS), 6) development of synthetic routes to aromatic polymer backbones containing pendent 1H-1,2,3-triazole moieties, 7) development of coupling strategies to create phase-separated block copolymers between hydrophilic sulfonated prepolymers and commodity polymers such as PFPO, 8) establishment of basic

  8. Multidimensional profiling of cell surface proteins and nuclear markers

    Energy Technology Data Exchange (ETDEWEB)

    Han, Ju; Chang, Hang; Andarawewa, Kumari; Yaswen, Paul; Helen Barcellos-Hoff, Mary; Parvin, Bahram

    2009-01-30

    Cell membrane proteins play an important role in tissue architecture and cell-cell communication. We hypothesize that segmentation and multidimensional characterization of the distribution of cell membrane proteins, on a cell-by-cell basis, enable improved classification of treatment groups and identify important characteristics that can otherwise be hidden. We have developed a series of computational steps to (i) delineate cell membrane protein signals and associate them with a specific nucleus; (ii) compute a coupled representation of the multiplexed DNA content with membrane proteins; (iii) rank computed features associated with such a multidimensional representation; (iv) visualize selected features for comparative evaluation through heatmaps; and (v) discriminate between treatment groups in an optimal fashion. The novelty of our method is in the segmentation of the membrane signal and the multidimensional representation of phenotypic signature on a cell-by-cell basis. To test the utility of this method, the proposed computational steps were applied to images of cells that have been irradiated with different radiation qualities in the presence and absence of other small molecules. These samples are labeled for their DNA content and E-cadherin membrane proteins. We demonstrate that multidimensional representations of cell-by-cell phenotypes improve predictive and visualization capabilities among different treatment groups, and identify hidden variables.

  9. Transport proteins of the plant plasma membrane

    Science.gov (United States)

    Assmann, S. M.; Haubrick, L. L.; Evans, M. L. (Principal Investigator)

    1996-01-01

    Recently developed molecular and genetic approaches have enabled the identification and functional characterization of novel genes encoding ion channels, ion carriers, and water channels of the plant plasma membrane.

  10. The Tobacco mosaic virus Movement Protein Associates with but Does Not Integrate into Biological Membranes

    Science.gov (United States)

    Peiró, Ana; Martínez-Gil, Luis; Tamborero, Silvia; Pallás, Vicente

    2014-01-01

    ABSTRACT Plant positive-strand RNA viruses require association with plant cell endomembranes for viral translation and replication, as well as for intra- and intercellular movement of the viral progeny. The membrane association and RNA binding of the Tobacco mosaic virus (TMV) movement protein (MP) are vital for orchestrating the macromolecular network required for virus movement. A previously proposed topological model suggests that TMV MP is an integral membrane protein with two putative α-helical transmembrane (TM) segments. Here we tested this model using an experimental system that measured the efficiency with which natural polypeptide segments were inserted into the ER membrane under conditions approximating the in vivo situation, as well as in planta. Our results demonstrated that the two hydrophobic regions (HRs) of TMV MP do not span biological membranes. We further found that mutations to alter the hydrophobicity of the first HR modified membrane association and precluded virus movement. We propose a topological model in which the TMV MP HRs intimately associate with the cellular membranes, allowing maximum exposure of the hydrophilic domains of the MP to the cytoplasmic cellular components. IMPORTANCE To facilitate plant viral infection and spread, viruses encode one or more movement proteins (MPs) that interact with ER membranes. The present work investigated the membrane association of the 30K MP of Tobacco mosaic virus (TMV), and the results challenge the previous topological model, which predicted that the TMV MP behaves as an integral membrane protein. The current data provide greatly needed clarification of the topological model and provide substantial evidence that TMV MP is membrane associated only at the cytoplasmic face of the membrane and that neither of its domains is integrated into the membrane or translocated into the lumen. Understanding the topology of MPs in the ER is vital for understanding the role of the ER in plant virus transport

  11. Cell-geometry-dependent changes in plasma membrane order direct stem cell signalling and fate.

    Science.gov (United States)

    von Erlach, Thomas C; Bertazzo, Sergio; Wozniak, Michele A; Horejs, Christine-Maria; Maynard, Stephanie A; Attwood, Simon; Robinson, Benjamin K; Autefage, Hélène; Kallepitis, Charalambos; Del Río Hernández, Armando; Chen, Christopher S; Goldoni, Silvia; Stevens, Molly M

    2018-03-01

    Cell size and shape affect cellular processes such as cell survival, growth and differentiation 1-4 , thus establishing cell geometry as a fundamental regulator of cell physiology. The contributions of the cytoskeleton, specifically actomyosin tension, to these effects have been described, but the exact biophysical mechanisms that translate changes in cell geometry to changes in cell behaviour remain mostly unresolved. Using a variety of innovative materials techniques, we demonstrate that the nanostructure and lipid assembly within the cell plasma membrane are regulated by cell geometry in a ligand-independent manner. These biophysical changes trigger signalling events involving the serine/threonine kinase Akt/protein kinase B (PKB) that direct cell-geometry-dependent mesenchymal stem cell differentiation. Our study defines a central regulatory role by plasma membrane ordered lipid raft microdomains in modulating stem cell differentiation with potential translational applications.

  12. Structuring detergents for extracting and stabilizing functional membrane proteins.

    Directory of Open Access Journals (Sweden)

    Rima Matar-Merheb

    Full Text Available BACKGROUND: Membrane proteins are privileged pharmaceutical targets for which the development of structure-based drug design is challenging. One underlying reason is the fact that detergents do not stabilize membrane domains as efficiently as natural lipids in membranes, often leading to a partial to complete loss of activity/stability during protein extraction and purification and preventing crystallization in an active conformation. METHODOLOGY/PRINCIPAL FINDINGS: Anionic calix[4]arene based detergents (C4Cn, n=1-12 were designed to structure the membrane domains through hydrophobic interactions and a network of salt bridges with the basic residues found at the cytosol-membrane interface of membrane proteins. These compounds behave as surfactants, forming micelles of 5-24 nm, with the critical micellar concentration (CMC being as expected sensitive to pH ranging from 0.05 to 1.5 mM. Both by 1H NMR titration and Surface Tension titration experiments, the interaction of these molecules with the basic amino acids was confirmed. They extract membrane proteins from different origins behaving as mild detergents, leading to partial extraction in some cases. They also retain protein functionality, as shown for BmrA (Bacillus multidrug resistance ATP protein, a membrane multidrug-transporting ATPase, which is particularly sensitive to detergent extraction. These new detergents allow BmrA to bind daunorubicin with a Kd of 12 µM, a value similar to that observed after purification using dodecyl maltoside (DDM. They preserve the ATPase activity of BmrA (which resets the protein to its initial state after drug efflux much more efficiently than SDS (sodium dodecyl sulphate, FC12 (Foscholine 12 or DDM. They also maintain in a functional state the C4Cn-extracted protein upon detergent exchange with FC12. Finally, they promote 3D-crystallization of the membrane protein. CONCLUSION/SIGNIFICANCE: These compounds seem promising to extract in a functional state

  13. The membrane stress response buffers lethal effects of lipid disequilibrium by reprogramming the protein homeostasis network.

    Science.gov (United States)

    Thibault, Guillaume; Shui, Guanghou; Kim, Woong; McAlister, Graeme C; Ismail, Nurzian; Gygi, Steven P; Wenk, Markus R; Ng, Davis T W

    2012-10-12

    Lipid composition can differ widely among organelles and even between leaflets of a membrane. Lipid homeostasis is critical because disequilibrium can have disease outcomes. Despite their importance, mechanisms maintaining lipid homeostasis remain poorly understood. Here, we establish a model system to study the global effects of lipid imbalance. Quantitative lipid profiling was integral to monitor changes to lipid composition and for system validation. Applying global transcriptional and proteomic analyses, a dramatically altered biochemical landscape was revealed from adaptive cells. The resulting composite regulation we term the "membrane stress response" (MSR) confers compensation, not through restoration of lipid composition, but by remodeling the protein homeostasis network. To validate its physiological significance, we analyzed the unfolded protein response (UPR), one facet of the MSR and a key regulator of protein homeostasis. We demonstrate that the UPR maintains protein biogenesis, quality control, and membrane integrity-functions otherwise lethally compromised in lipid dysregulated cells. Copyright © 2012 Elsevier Inc. All rights reserved.

  14. Remodelling of primary human CD4+ T cell plasma membrane order by n-3 PUFA.

    Science.gov (United States)

    Fan, Yang-Yi; Fuentes, Natividad R; Hou, Tim Y; Barhoumi, Rola; Li, Xian C; Deutz, Nicolaas E P; Engelen, Marielle P K J; McMurray, David N; Chapkin, Robert S

    2018-01-01

    Cell membrane fatty acids influence fundamental properties of the plasma membrane, including membrane fluidity, protein functionality, and lipid raft signalling. Evidence suggests that dietary n-3 PUFA may target the plasma membrane of immune cells by altering plasma membrane lipid dynamics, thereby regulating the attenuation of immune cell activation and suppression of inflammation. As lipid-based immunotherapy might be a promising new clinical strategy for the treatment of inflammatory disorders, we conducted in vitro and in vivo experiments to examine the effects of n-3 PUFA on CD4+ T cell membrane order, mitochondrial bioenergetics and lymphoproliferation. n-3 PUFA were incorporated into human primary CD4+ T cells phospholipids in vitro in a dose-dependent manner, resulting in a reduction in whole cell membrane order, oxidative phosphorylation and proliferation. At higher doses, n-3 PUFA induced unique phase separation in T cell-derived giant plasma membrane vesicles. Similarly, in a short-term human pilot study, supplementation of fish oil (4 g n-3 PUFA/d) for 6 weeks in healthy subjects significantly elevated EPA (20 : 5n-3) levels in CD4+ T cell membrane phospholipids, and reduced membrane lipid order. These results demonstrate that the dynamic reshaping of human CD4+ T cell plasma membrane organisation by n-3 PUFA may modulate down-stream clonal expansion.

  15. Noncontact microsurgery of cell membranes using femtosecond laser pulses for optoinjection of specified substances into cells

    Science.gov (United States)

    Il'ina, I. V.; Ovchinnikov, A. V.; Chefonov, O. V.; Sitnikov, D. S.; Agranat, Mikhail B.; Mikaelyan, A. S.

    2013-04-01

    IR femtosecond laser pulses were used for microsurgery of a cell membrane aimed at local and short-duration change in its permeability and injection of specified extracellular substances into the cells. The possibility of noncontact laser delivery of the propidium iodide fluorescent dye and the pEGFP plasmid, encoding the green fluorescent protein, into the cells with preservation of the cell viability was demonstrated.

  16. Isolation of Protein Storage Vacuoles and Their Membranes.

    Science.gov (United States)

    Shimada, Tomoo; Hara-Nishimura, Ikuko

    2017-01-01

    Protein-storage vacuoles (PSVs) are specialized vacuoles that sequester large amounts of storage proteins. During seed development, PSVs are formed de novo and/or from preexisting lytic vacuoles. Seed PSVs can be subdivided into four distinct compartments: membrane, globoid, matrix, and crystalloid. In this chapter, we introduce easy methods for isolation of PSVs and their membranes from pumpkin seeds. These methods facilitate the identification and characterization of PSV components.

  17. Development of immobilized membrane-based affinity columns for use in the online characterization of membrane bound proteins and for targeted affinity isolations

    International Nuclear Information System (INIS)

    Moaddel, Ruin; Wainer, Irving W.

    2006-01-01

    Membranes obtained from cell lines that express or do not express a target membrane bound protein have been immobilized on a silica-based liquid chromatographic support or on the surface of an activated glass capillary. The resulting chromatographic columns have been placed in liquid chromatographic systems and used to characterize the target proteins and to identify small molecules that bind to the target. Membranes containing ligand gated ion channels, G-protein coupled receptors and drug transporters have been prepared and characterized. If a marker ligand has been identified for the target protein, frontal or zonal displacement chromatographic techniques can be used to determine binding affinities (K d values) and non-linear chromatography can be used to assess the association (k on ) and dissociation (k off ) rate constants and the thermodynamics of the binding process. Membrane-based affinity columns have been created using membranes from a cell line that does not express the target protein (control) and the same cell line that expresses the target protein (experimental) after genomic transfection. The resulting columns can be placed in a parallel chromatography system and the differential retention between the control and experimental columns can be used to identify small molecules and protein that bind to the target protein. These applications will be illustrated using columns created using cellular membranes containing nicotinic acetylcholine receptors and the drug transporter P-glycoprotein

  18. Development of immobilized membrane-based affinity columns for use in the online characterization of membrane bound proteins and for targeted affinity isolations

    Energy Technology Data Exchange (ETDEWEB)

    Moaddel, Ruin [Gerontology Research Center, National Institute on Aging, National Institutes of Health, 5600 Nathan Shock Drive, Baltimore, MD 21224-6825 (United States); Wainer, Irving W. [Gerontology Research Center, National Institute on Aging, National Institutes of Health, 5600 Nathan Shock Drive, Baltimore, MD 21224-6825 (United States)]. E-mail: Wainerir@grc.nia.nih.gov

    2006-03-30

    Membranes obtained from cell lines that express or do not express a target membrane bound protein have been immobilized on a silica-based liquid chromatographic support or on the surface of an activated glass capillary. The resulting chromatographic columns have been placed in liquid chromatographic systems and used to characterize the target proteins and to identify small molecules that bind to the target. Membranes containing ligand gated ion channels, G-protein coupled receptors and drug transporters have been prepared and characterized. If a marker ligand has been identified for the target protein, frontal or zonal displacement chromatographic techniques can be used to determine binding affinities (K {sub d} values) and non-linear chromatography can be used to assess the association (k {sub on}) and dissociation (k {sub off}) rate constants and the thermodynamics of the binding process. Membrane-based affinity columns have been created using membranes from a cell line that does not express the target protein (control) and the same cell line that expresses the target protein (experimental) after genomic transfection. The resulting columns can be placed in a parallel chromatography system and the differential retention between the control and experimental columns can be used to identify small molecules and protein that bind to the target protein. These applications will be illustrated using columns created using cellular membranes containing nicotinic acetylcholine receptors and the drug transporter P-glycoprotein.

  19. Determination of the topology of endoplasmic reticulum membrane proteins using redox-sensitive green-fluorescence protein fusions.

    Science.gov (United States)

    Tsachaki, Maria; Birk, Julia; Egert, Aurélie; Odermatt, Alex

    2015-07-01

    Membrane proteins of the endoplasmic reticulum (ER) are involved in a wide array of essential cellular functions. Identification of the topology of membrane proteins can provide significant insight into their mechanisms of action and biological roles. This is particularly important for membrane enzymes, since their topology determines the subcellular site where a biochemical reaction takes place and the dependence on luminal or cytosolic co-factor pools and substrates. The methods currently available for the determination of topology of proteins are rather laborious and require post-lysis or post-fixation manipulation of cells. In this work, we have developed a simple method for defining intracellular localization and topology of ER membrane proteins in living cells, based on the fusion of the respective protein with redox-sensitive green-fluorescent protein (roGFP). We validated the method and demonstrated that roGFP fusion proteins constitute a reliable tool for the study of ER membrane protein topology, using as control microsomal 11β-hydroxysteroid dehydrogenase (11β-HSD) proteins whose topology has been resolved, and comparing with an independent approach. We then implemented this method to determine the membrane topology of six microsomal members of the 17β-hydroxysteroid dehydrogenase (17β-HSD) family. The results revealed a luminal orientation of the catalytic site for three enzymes, i.e. 17β-HSD6, 7 and 12. Knowledge of the intracellular location of the catalytic site of these enzymes will enable future studies on their biological functions and on the role of the luminal co-factor pool. Copyright © 2015 Elsevier B.V. All rights reserved.

  20. Assembly of β-barrel proteins in the mitochondrial outer membrane.

    Science.gov (United States)

    Höhr, Alexandra I C; Straub, Sebastian P; Warscheid, Bettina; Becker, Thomas; Wiedemann, Nils

    2015-01-01

    Mitochondria evolved through endosymbiosis of a Gram-negative progenitor with a host cell to generate eukaryotes. Therefore, the outer membrane of mitochondria and Gram-negative bacteria contain pore proteins with β-barrel topology. After synthesis in the cytosol, β-barrel precursor proteins are first transported into the mitochondrial intermembrane space. Folding and membrane integration of β-barrel proteins depend on the mitochondrial sorting and assembly machinery (SAM) located in the outer membrane, which is related to the β-barrel assembly machinery (BAM) in bacteria. The SAM complex recognizes β-barrel proteins by a β-signal in the C-terminal β-strand that is required to initiate β-barrel protein insertion into the outer membrane. In addition, the SAM complex is crucial to form membrane contacts with the inner mitochondrial membrane by interacting with the mitochondrial contact site and cristae organizing system (MICOS) and shares a subunit with the endoplasmic reticulum-mitochondria encounter structure (ERMES) that links the outer mitochondrial membrane to the endoplasmic reticulum (ER). Copyright © 2014 Elsevier B.V. All rights reserved.

  1. Detergent selection for enhanced extraction of membrane proteins.

    Science.gov (United States)

    Arachea, Buenafe T; Sun, Zhen; Potente, Nina; Malik, Radhika; Isailovic, Dragan; Viola, Ronald E

    2012-11-01

    Generating stable conditions for membrane proteins after extraction from their lipid bilayer environment is essential for subsequent characterization. Detergents are the most widely used means to obtain this stable environment; however, different types of membrane proteins have been found to require detergents with varying properties for optimal extraction efficiency and stability after extraction. The extraction profiles of several detergent types have been examined for membranes isolated from bacteria and yeast, and for a set of recombinant target proteins. The extraction efficiencies of these detergents increase at higher concentrations, and were shown to correlate with their respective CMC values. Two alkyl sugar detergents, octyl-β-d-glucoside (OG) and 5-cyclohexyl-1-pentyl-β-d-maltoside (Cymal-5), and a zwitterionic surfactant, N-decylphosphocholine (Fos-choline-10), were generally effective in the extraction of a broad range of membrane proteins. However, certain detergents were more effective than others in the extraction of specific classes of integral membrane proteins, offering guidelines for initial detergent selection. The differences in extraction efficiencies among this small set of detergents supports the value of detergent screening and optimization to increase the yields of targeted membrane proteins. Copyright © 2012 Elsevier Inc. All rights reserved.

  2. Optimal separation of jojoba protein using membrane processes

    Energy Technology Data Exchange (ETDEWEB)

    Nabetani, Hiroshi; Abbott, T.P.; Kleiman, R. [National Center for Agricultural Utilization Research, Peoria, IL (United States)

    1995-05-01

    The efficiency of a pilot-scale membrane system for purifying and concentrating jojoba protein was estimated. In this system, a jojoba extract was first clarified with a microfiltration membrane. The clarified extract was diafiltrated and the protein was purified with an ultrafiltration membrane. Then the protein solution was concentrated with the ultrafiltration membrane. Permeate flux during microfiltration was essentially independent of solids concentration in the feed, in contrast with the permeate flux during ultrafiltration which was a function of protein concentration. Based on these results, a mathematical model which describes the batchwise concentration process with ultrafiltration membranes was developed. Using this model, the combination of batchwise concentration with diafiltration was optimized, and an industrial-scale process was designed. The effect of ethylenediaminetetraacetic acid (EDTA) on the performance of the membrane system was also investigated. The addition of EDTA increased the concentration of protein in the extract and improved the recovery of protein in the final products. The quality of the final product (color and solubility) was also improved. However, EDTA decreased permeate flux during ultrafiltration.

  3. Critical determinants of the interactions of capsule-expressing Neisseria meningitidis with host cells: the role of receptor density in increased cellular targeting via the outer membrane Opa proteins.

    Science.gov (United States)

    Bradley, Christopher J; Griffiths, Natalie J; Rowe, Helen A; Heyderman, Robert S; Virji, Mumtaz

    2005-10-01

    Neisseria meningitidis capsule is an important virulence determinant required for survival in the blood but is reportedly involved in inhibiting cellular interactions mediated by meningococcal outer membrane adhesins. However, evidence from our previous studies suggested that target receptor density on host cells may determine whether or not capsulate bacteria can adhere via outer membrane proteins such as Opa. To confirm this and evaluate the impact of capsulation on bacterial interactions, we used Opa(+) and Opa(-) derivatives of capsulate and acapsulate meningococcal isolates and transfected cell lines expressing CEACAM1, a receptor targeted by Opa proteins. To assess the extent and rate of cell association, subpopulations of stably transfected Chinese hamster ovary cells with different receptor levels were derived. A quantitative correlation of CEACAM1 levels and Opa-dependent binding of both capsulate and acapsulate bacteria was demonstrated, which was accelerated at high receptor densities. However, it appears that invasion by Opa(+) capsulate bacteria only occurs when a threshold level of CEACAM density has been reached. Target cells expressing high levels of CEACAM1 (MFI c. 400) bound threefold more, but internalized 20-fold more Opa(+) capsulate bacteria than those with intermediate expression (MFI c. 100). No overall selection of acapsulate phenotype was observed in the internalized population. These observations confirm that capsule may not be an adequate barrier for cellular interactions and demonstrate the role of a host factor that may determine capsulate bacterial invasion potential. Upregulation of CEACAMs, which can occur in response to inflammatory cytokines, could lead to translocation of a small number of fully capsulate bacteria across mucosal epithelium into the bloodstream sufficient to cause a rapid onset of disseminated disease. Thus the data also suggest a novel rationale for the epidemiological observations that individuals with prior

  4. Membrane remodeling by amyloidogenic and non-amyloidogenic proteins studied by EPR

    Science.gov (United States)

    Varkey, Jobin; Langen, Ralf

    2017-07-01

    The advancement in site-directed spin labeling of proteins has enabled EPR studies to expand into newer research areas within the umbrella of protein-membrane interactions. Recently, membrane remodeling by amyloidogenic and non-amyloidogenic proteins has gained a substantial interest in relation to driving and controlling vital cellular processes such as endocytosis, exocytosis, shaping of organelles like endoplasmic reticulum, Golgi and mitochondria, intracellular vesicular trafficking, formation of filopedia and multivesicular bodies, mitochondrial fusion and fission, and synaptic vesicle fusion and recycling in neurotransmission. Misregulation in any of these processes due to an aberrant protein (mutation or misfolding) or alteration of lipid metabolism can be detrimental to the cell and cause disease. Dissection of the structural basis of membrane remodeling by proteins is thus quite necessary for an understanding of the underlying mechanisms, but it remains a formidable task due to the difficulties of various common biophysical tools in monitoring the dynamic process of membrane binding and bending by proteins. This is largely since membranes generally complicate protein structure analysis and this problem is amplified for structural analysis in the presence of different types of membrane curvatures. Recent EPR studies on membrane remodeling by proteins show that a significant structural information can be generated to delineate the role of different protein modules, domains and individual amino acids in the generation of membrane curvature. These studies also show how EPR can complement the data obtained by high resolution techniques such as X-ray and NMR. This perspective covers the application of EPR in recent studies for understanding membrane remodeling by amyloidogenic and non-amyloidogenic proteins that is useful for researchers interested in using or complimenting EPR to gain better understanding of membrane remodeling. We also discuss how a single

  5. The Xylella fastidiosa PD1063 protein is secreted in association with outer membrane vesicles.

    Science.gov (United States)

    Pierce, Brittany K; Voegel, Tanja; Kirkpatrick, Bruce C

    2014-01-01

    Xylella fastidiosa is a gram-negative, xylem-limited plant pathogenic bacterium that causes disease in a variety of economically important agricultural crops including Pierce's disease of grapevines. Xylella fastidiosa biofilms formed in the xylem vessels of plants play a key role in early colonization and pathogenicity by providing a protected niche and enhanced cell survival. Here we investigate the role of Xylella fastidiosa PD1063, the predicted ortholog of Xanthomonas oryzae pv. oryzae PXO_03968, which encodes an outer membrane protein. To assess the function of the Xylella fastidiosa ortholog, we created Xylella fastidiosa mutants deleted for PD1063 and then assessed biofilm formation, cell-cell aggregation and cell growth in vitro. We also assessed disease severity and pathogen titers in grapevines mechanically inoculated with the Xylella fastidiosa PD1063 mutant. We found a significant decrease in cell-cell aggregation among PD1063 mutants but no differences in cell growth, biofilm formation, disease severity or titers in planta. Based on the demonstration that Xanthomonas oryzae pv. oryzae PXO_03968 encodes an outer membrane protein, secreted in association with outer membrane vesicles, we predicted that PD1063 would also be secreted in a similar manner. Using anti-PD1063 antibodies, we found PD1063 in the supernatant and secreted in association with outer membrane vesicles. PD1063 purified from the supernatant, outer membrane fractions and outer membrane vesicles was 19.2 kD, corresponding to the predicted size of the processed protein. Our findings suggest Xylella fastidiosa PD1063 is not essential for development of Pierce's disease in Vitis vinifera grapevines although further research is required to determine the function of the PD1063 outer membrane protein in Xylella fastidiosa.

  6. A Plasmodium falciparum copper-binding membrane protein with copper transport motifs

    Directory of Open Access Journals (Sweden)

    Choveaux David L

    2012-11-01

    Full Text Available Abstract Background Copper is an essential catalytic co-factor for metabolically important cellular enzymes, such as cytochrome-c oxidase. Eukaryotic cells acquire copper through a copper transport protein and distribute intracellular copper using molecular chaperones. The copper chelator, neocuproine, inhibits Plasmodium falciparum ring-to-trophozoite transition in vitro, indicating a copper requirement for malaria parasite development. How the malaria parasite acquires or secretes copper still remains to be fully elucidated. Methods PlasmoDB was searched for sequences corresponding to candidate P. falciparum copper-requiring proteins. The amino terminal domain of a putative P. falciparum copper transport protein was cloned and expressed as a maltose binding fusion protein. The copper binding ability of this protein was examined. Copper transport protein-specific anti-peptide antibodies were generated in chickens and used to establish native protein localization in P. falciparum parasites by immunofluorescence microscopy. Results Six P. falciparum copper-requiring protein orthologs and a candidate P. falciparum copper transport protein (PF14_0369, containing characteristic copper transport protein features, were identified in PlasmoDB. The recombinant amino terminal domain of the transport protein bound reduced copper in vitro and within Escherichia coli cells during recombinant expression. Immunolocalization studies tracked the copper binding protein translocating from the erythrocyte plasma membrane in early ring stage to a parasite membrane as the parasites developed to schizonts. The protein appears to be a PEXEL-negative membrane protein. Conclusion Plasmodium falciparum parasites express a native protein with copper transporter characteristics that binds copper in vitro. Localization of the protein to the erythrocyte and parasite plasma membranes could provide a mechanism for the delivery of novel anti-malarial compounds.

  7. Integral membrane proteins in proteomics. How to break open the black box?

    Science.gov (United States)

    Vit, O; Petrak, J

    2017-02-05

    Integral membrane proteins (IMPs) are coded by 20-30% of human genes and execute important functions - transmembrane transport, signal transduction, cell-cell communication, cell adhesion to the extracellular matrix, and many other processes. Due to their hydrophobicity, low expression and lack of trypsin cleavage sites in their transmembrane segments, IMPs have been generally under-represented in routine proteomic analyses. However, the field of membrane proteomics has changed markedly in the past decade, namely due to the introduction of filter assisted sample preparation (FASP), the establishment of cell surface capture (CSC) protocols, and the development of methods that enable analysis of the hydrophobic transmembrane segments. This review will summarize the recent developments in the field and outline the most successful strategies for the analysis of integral membrane proteins. Integral membrane proteins (IMPs) are attractive therapeutic targets mostly due to their many important functions. However, our knowledge of the membrane proteome is severely limited to effectively exploit their potential. This is mostly due to the lack of appropriate techniques or methods compatible with the typical features of IMPs, namely hydrophobicity, low expression and lack of trypsin cleavage sites. This review summarizes the most recent development in membrane proteomics and outlines the most successful strategies for their large-scale analysis. Copyright © 2016 Elsevier B.V. All rights reserved.

  8. Membrane lipidome of an epithelial cell line

    DEFF Research Database (Denmark)

    Sampaio, Julio L; Gerl, Mathias J; Klose, Christian

    2011-01-01

    Tissue differentiation is an important process that involves major cellular membrane remodeling. We used Madin-Darby canine kidney cells as a model for epithelium formation and investigated the remodeling of the total cell membrane lipidome during the transition from a nonpolarized morphology...... to an epithelial morphology and vice versa. To achieve this, we developed a shotgun-based lipidomics workflow that enabled the absolute quantification of mammalian membrane lipidomes with minimal sample processing from low sample amounts. Epithelial morphogenesis was accompanied by a major shift from sphingomyelin...... to glycosphingolipid, together with an increase in plasmalogen, phosphatidylethanolamine, and cholesterol content, whereas the opposite changes took place during an epithelial-to-mesenchymal transition. Moreover, during polarization, the sphingolipids became longer, more saturated, and more hydroxylated as required...

  9. Proteomic analysis of isolated membrane fractions from superinvasive cancer cells

    OpenAIRE

    Dowling, Paul; Meleady, Paula; Dowd, Andrew; Henry, Michael; Glynn, Sharon; Clynes, Martin

    2007-01-01

    The superinvasive phenotype exhibited by paclitaxel-selected variants of an in vitro invasive clonal population of the human cancer cell line, MDA-MB-435S were examined using DIGE (Fluorescence 2-D Difference Gel Electrophoresis) and mass spectrometry. Isolation of membrane proteins from the MDA-MB-435S-F/Taxol-10p4p and parental populations was performed by temperature-dependent phase partitioning using the detergent Triton X-114. Subsequent DIGE-generated data analysed using Decyde...

  10. Membrane Protein Properties Revealed through Data-Rich Electrostatics Calculations.

    Science.gov (United States)

    Marcoline, Frank V; Bethel, Neville; Guerriero, Christopher J; Brodsky, Jeffrey L; Grabe, Michael

    2015-08-04

    The electrostatic properties of membrane proteins often reveal many of their key biophysical characteristics, such as ion channel selectivity and the stability of charged membrane-spanning segments. The Poisson-Boltzmann (PB) equation is the gold standard for calculating protein electrostatics, and the software APBSmem enables the solution of the PB equation in the presence of a membrane. Here, we describe significant advances to APBSmem, including full automation of system setup, per-residue energy decomposition, incorporation of PDB2PQR, calculation of membrane-induced pKa shifts, calculation of non-polar energies, and command-line scripting for large-scale calculations. We highlight these new features with calculations carried out on a number of membrane proteins, including the recently solved structure of the ion channel TRPV1 and a large survey of 1,614 membrane proteins of known structure. This survey provides a comprehensive list of residues with large electrostatic penalties for being embedded in the membrane, potentially revealing interesting functional information. Copyright © 2015 Elsevier Ltd. All rights reserved.

  11. RPE cell surface proteins in normal and dystrophic rats

    International Nuclear Information System (INIS)

    Clark, V.M.; Hall, M.O.

    1986-01-01

    Membrane-bound proteins in plasma membrane enriched fractions from cultured rat RPE were analyzed by two-dimensional gel electrophoresis. Membrane proteins were characterized on three increasingly specific levels. Total protein was visualized by silver staining. A maximum of 102 separate proteins were counted in silver-stained gels. Glycoproteins were labeled with 3H-glucosamine or 3H-fucose and detected by autoradiography. Thirty-eight fucose-labeled and 61-71 glucosamine-labeled proteins were identified. All of the fucose-labeled proteins were labeled with glucosamine-derived radioactivity. Proteins exposed at the cell surface were labeled by lactoperoxidase-catalyzed radioiodination prior to preparation of membranes for two-dimensional analysis. Forty separate 125I-labeled surface proteins were resolved by two-dimensional electrophoresis/autoradiography. Comparison with the glycoprotein map showed that a number of these surface labeled proteins were glycoproteins. Two-dimensional maps of total protein, fucose-labeled, and glucosamine-labeled glycoproteins, and 125I-labeled surface proteins of membranes from dystrophic (RCS rdy-p+) and normal (Long Evans or RCS rdy+p+) RPE were compared. No differences in the total protein or surface-labeled proteins were observed. However, the results suggest that a 183K glycoprotein is more heavily glycosylated with glucosamine and fucose in normal RPE membranes as compared to membranes from dystrophic RPE

  12. Structural Insights into the Yersinia pestis Outer Membrane Protein Ail in Lipid Bilayers.

    Science.gov (United States)

    Dutta, Samit Kumar; Yao, Yong; Marassi, Francesca M

    2017-08-17

    Yersinia pestis the causative agent of plague, is highly pathogenic and poses very high risk to public health. The outer membrane protein Ail (Adhesion invasion locus) is one of the most highly expressed proteins on the cell surface of Y. pestis, and a major target for the development of medical countermeasures. Ail is essential for microbial virulence and is critical for promoting the survival of Y. pestis in serum. Structures of Ail have been determined by X-ray diffraction and solution NMR spectroscopy, but the protein's activity is influenced by the detergents in these samples, underscoring the importance of the surrounding environment for structure-activity studies. Here we describe the backbone structure of Ail, determined in lipid bilayer nanodiscs, using solution NMR spectroscopy. We also present solid-state NMR data obtained for Ail in membranes containing lipopolysaccharide (LPS), a major component of the bacterial outer membranes. The protein in lipid bilayers, adopts the same eight-stranded β-barrel fold observed in the crystalline and micellar states. The membrane composition, however, appears to have a marked effect on protein dynamics, with LPS enhancing conformational order and slowing down the 15 N transverse relaxation rate. The results provide information about the way in which an outer membrane protein inserts and functions in the bacterial membrane.

  13. Membrane vesiculation induced by proteins of the dengue virus envelope studied by molecular dynamics simulations

    Science.gov (United States)

    de Oliveira dos Santos Soares, Ricardo; Oliveira Bortot, Leandro; van der Spoel, David; Caliri, Antonio

    2017-12-01

    Biological membranes are continuously remodeled in the cell by specific membrane-shaping machineries to form, for example, tubes and vesicles. We examine fundamental mechanisms involved in the vesiculation processes induced by a cluster of envelope (E) and membrane (M) proteins of the dengue virus (DENV) using molecular dynamics simulations and a coarse-grained model. We show that an arrangement of three E-M heterotetramers (EM3) works as a bending unit and an ordered cluster of five such units generates a closed vesicle, reminiscent of the virus budding process. In silico mutagenesis of two charged residues of the anchor helices of the envelope proteins of DENV shows that Arg-471 and Arg-60 are fundamental to produce bending stress on the membrane. The fine-tuning between the size of the EM3 unit and its specific bending action suggests this protein unit is an important factor in determining the viral particle size.

  14. Garbage on, garbage off: new insights into plasma membrane protein quality control.

    Science.gov (United States)

    MacGurn, Jason A

    2014-08-01

    Maintenance of cellular protein quality - by restoring misfolded proteins to their native state and by targeting terminally misfolded or damaged proteins for degradation - is a critical function of all cells. To ensure protein quality, cells have evolved various organelle-specific quality control mechanisms responsible for recognizing and responding to misfolded proteins at different subcellular locations of the cell. Recently, several publications have begun to elucidate mechanisms of quality control that operate at the plasma membrane (PM), recognizing misfolded PM proteins and targeting their endocytic trafficking and lysosomal degradation. Here, I discuss these recent developments in our understanding of PM quality control mechanisms and how they relate to global protein quality control strategies in the cell. Copyright © 2014 Elsevier Ltd. All rights reserved.

  15. Lithium. Effects on excitable cell membranes

    NARCIS (Netherlands)

    Ploeger, Egbert Johan

    1974-01-01

    LITHIUM: Effects on excitable cell membranes. Lithium salts have been used in the treatment of manic-depressive psychosis for many years but their mechanism of action is not well understood. Many workers assume that the action of lithium on catecholamine metabolism and/or on electrolyte distribution

  16. Alignment of helical membrane protein sequences using AlignMe.

    Directory of Open Access Journals (Sweden)

    Marcus Stamm

    Full Text Available Few sequence alignment methods have been designed specifically for integral membrane proteins, even though these important proteins have distinct evolutionary and structural properties that might affect their alignments. Existing approaches typically consider membrane-related information either by using membrane-specific substitution matrices or by assigning distinct penalties for gap creation in transmembrane and non-transmembrane regions. Here, we ask whether favoring matching of predicted transmembrane segments within a standard dynamic programming algorithm can improve the accuracy of pairwise membrane protein sequence alignments. We tested various strategies using a specifically designed program called AlignMe. An updated set of homologous membrane protein structures, called HOMEP2, was used as a reference for optimizing the gap penalties. The best of the membrane-protein optimized approaches were then tested on an independent reference set of membrane protein sequence alignments from the BAliBASE collection. When secondary structure (S matching was combined with evolutionary information (using a position-specific substitution matrix (P, in an approach we called AlignMePS, the resultant pairwise alignments were typically among the most accurate over a broad range of sequence similarities when compared to available methods. Matching transmembrane predictions (T, in addition to evolutionary information, and secondary-structure predictions, in an approach called AlignMePST, generally reduces the accuracy of the alignments of closely-related proteins in the BAliBASE set relative to AlignMePS, but may be useful in cases of extremely distantly related proteins for which sequence information is less informative. The open source AlignMe code is available at https://sourceforge.net/projects/alignme/, and at http://www.forrestlab.org, along with an online server and the HOMEP2 data set.

  17. Induction of the lac carrier and an associated membrane protein in Escherichia coli

    International Nuclear Information System (INIS)

    Lagarias, D.M.

    1985-01-01

    Induction of the lac operon in wild type Escherichia coli strains results in synthesis of a 16 kilodalton inner membrane protein in addition to the known products of the lacZ, lacY and lacA genes. Cells carrying the lacY gene on a plasmid over produce this 16 kilodalton polypeptide as well as the Lac carrier, the membrane protein product of the lacY gene. However, [ 35 S]methionine labeling of minicells carrying the lacY plasmid shows that the 16 kDa protein is not synthesized from the plasmid DNA. The 16 kDa protein was purified and partially characterized. It is an acidic membrane protein of apparent molecular weight 15,800 whose amino terminal sequence (NH 2 -Met-Arg-Asn-Phe-Asp-Leu-) does not correspond to any nucleotide sequence known in lac operon DNA. Using antibody prepared to the purified 16 kDa protein, a quantitative analysis of conditions under which this protein is made was accomplished, and reveals that the amount of 16 kDa protein which appears in the membrane is proportional to lac operon expression. Hybridization of a synthetic oligonucleotide probe complementary to the 5' end of 16 kDa protein mRNA shows that its synthesis is regulated at the level of transcription. A description of attempts to clone this gene is given. Possible functional roles for the 16 kDa protein are discussed

  18. High-level production of membrane proteins in E. coli BL21(DE3) by omitting the inducer IPTG.

    Science.gov (United States)

    Zhang, Zhe; Kuipers, Grietje; Niemiec, Łukasz; Baumgarten, Thomas; Slotboom, Dirk Jan; de Gier, Jan-Willem; Hjelm, Anna

    2015-09-16

    For membrane protein production, the Escherichia coli T7 RNA polymerase (T7 RNAP)-based protein production strain BL21(DE3) in combination with T7-promoter based expression vectors is widely used. Cells are routinely cultured in Lysogeny broth (LB medium) and expression of the chromosomally localized t7rnap gene is governed by the isopropyl-β-D-1-thiogalactopyranoside (IPTG) inducible lacUV5 promoter. The T7 RNAP drives the expression of the plasmid borne gene encoding the recombinant membrane protein. Production of membrane proteins in the cytoplasmic membrane rather than in inclusion bodies in a misfolded state is usually preferred, but often hampered due to saturation of the capacity of the Sec-translocon, resulting in low yields. Contrary to expectation we observed that omission of IPTG from BL21(DE3) cells cultured in LB medium can lead to significantly higher membrane protein production yields than when IPTG is added. In the complete absence of IPTG cultures stably produce membrane proteins in the cytoplasmic membrane, whereas upon the addition of IPTG membrane proteins aggregate in the cytoplasm and non-producing clones are selected for. Furthermore, in the absence of IPTG, membrane proteins are produced at a lower rate than in the presence of IPTG. These observations indicate that in the absence of IPTG the Sec-translocon capacity is not/hardly saturated, leading to enhanced membrane protein production yields in the cytoplasmic membrane. Importantly, for more than half of the targets tested the yields obtained using un-induced BL21(DE3) cells were higher than the yields obtained in the widely used membrane protein production strains C41(DE3) and C43(DE3). Since most secretory proteins reach the periplasm via the Sec-translocon, we also monitored the production of three secretory recombinant proteins in the periplasm of BL21(DE3) cells in the presence and absence of IPTG. For all three targets tested omitting IPTG led to the highest production levels in the

  19. Electron paramagnetic resonance study of lipid and protein membrane components of erythrocytes oxidized with hydrogen peroxide

    Energy Technology Data Exchange (ETDEWEB)

    Mendanha, S.A.; Anjos, J.L.V.; Silva, A.H.M.; Alonso, A. [Instituto de Física, Universidade Federal de Goiás, Goiânia, GO (Brazil)

    2012-04-05

    Electron paramagnetic resonance (EPR) spectroscopy of spin labels was used to monitor membrane dynamic changes in erythrocytes subjected to oxidative stress with hydrogen peroxide (H{sub 2}O{sub 2}). The lipid spin label, 5-doxyl stearic acid, responded to dramatic reductions in membrane fluidity, which was correlated with increases in the protein content of the membrane. Membrane rigidity, associated with the binding of hemoglobin (Hb) to the erythrocyte membrane, was also indicated by a spin-labeled maleimide, 5-MSL, covalently bound to the sulfhydryl groups of membrane proteins. At 2% hematocrit, these alterations in membrane occurred at very low concentrations of H{sub 2}O{sub 2} (50 µM) after only 5 min of incubation at 37°C in azide phosphate buffer, pH 7.4. Lipid peroxidation, suggested by oxidative hemolysis and malondialdehyde formation, started at 300 µM H{sub 2}O{sub 2} (for incubation of 3 h), which is a concentration about six times higher than those detected with the probes. Ascorbic acid and α-tocopherol protected the membrane against lipoperoxidation, but did not prevent the binding of proteins to the erythrocyte membrane. Moreover, the antioxidant (+)-catechin, which also failed to prevent the cross-linking of cytoskeletal proteins with Hb, was very effective in protecting erythrocyte ghosts from lipid peroxidation induced by the Fenton reaction. This study also showed that EPR spectroscopy can be useful to assess the molecular dynamics of red blood cell membranes in both the lipid and protein domains and examine oxidation processes in a system that is so vulnerable to oxidation.

  20. Biomimetic interfaces based on S-layer proteins, lipid membranes and functional biomolecules

    Science.gov (United States)

    Schuster, Bernhard; Sleytr, Uwe B.

    2014-01-01

    Designing and utilization of biomimetic membrane systems generated by bottom-up processes is a rapidly growing scientific and engineering field. Elucidation of the supramolecular construction principle of archaeal cell envelopes composed of S-layer stabilized lipid membranes led to new strategies for generating highly stable functional lipid membranes at meso- and macroscopic scale. In this review, we provide a state-of-the-art survey of how S-layer proteins, lipids and polymers may be used as basic building blocks for the assembly of S-layer-supported lipid membranes. These biomimetic membrane systems are distinguished by a nanopatterned fluidity, enhanced stability and longevity and, thus, provide a dedicated reconstitution matrix for membrane-active peptides and transmembrane proteins. Exciting areas in the (lab-on-a-) biochip technology are combining composite S-layer membrane systems involving specific membrane functions with the silicon world. Thus, it might become possible to create artificial noses or tongues, where many receptor proteins have to be exposed and read out simultaneously. Moreover, S-layer-coated liposomes and emulsomes copying virus envelopes constitute promising nanoformulations for the production of novel targeting, delivery, encapsulation and imaging systems. PMID:24812051

  1. Multiscale molecular dynamics simulations of membrane remodeling by Bin/Amphiphysin/Rvs family proteins

    Science.gov (United States)

    Chun, Chan; Haohua, Wen; Lanyuan, Lu; Jun, Fan

    2016-01-01

    Membrane curvature is no longer thought of as a passive property of the membrane; rather, it is considered as an active, regulated state that serves various purposes in the cell such as between cells and organelle definition. While transport is usually mediated by tiny membrane bubbles known as vesicles or membrane tubules, such communication requires complex interplay between the lipid bilayers and cytosolic proteins such as members of the Bin/Amphiphysin/Rvs (BAR) superfamily of proteins. With rapid developments in novel experimental techniques, membrane remodeling has become a rapidly emerging new field in recent years. Molecular dynamics (MD) simulations are important tools for obtaining atomistic information regarding the structural and dynamic aspects of biological systems and for understanding the physics-related aspects. The availability of more sophisticated experimental data poses challenges to the theoretical community for developing novel theoretical and computational techniques that can be used to better interpret the experimental results to obtain further functional insights. In this review, we summarize the general mechanisms underlying membrane remodeling controlled or mediated by proteins. While studies combining experiments and molecular dynamics simulations recall existing mechanistic models, concurrently, they extend the role of different BAR domain proteins during membrane remodeling processes. We review these recent findings, focusing on how multiscale molecular dynamics simulations aid in understanding the physical basis of BAR domain proteins, as a representative of membrane-remodeling proteins. Project supported by the National Natural Science Foundation of China (Grant No. 21403182) and the Research Grants Council of Hong Kong, China (Grant No. CityU 21300014).

  2. Lipopolysaccharide Membranes and Membrane Proteins of Pseudomonas aeruginosa Studied by Computer Simulation

    Energy Technology Data Exchange (ETDEWEB)

    Straatsma, TP

    2006-12-01

    Pseudomonas aeruginosa is a ubiquitous environmental Gram-negative bacterium with high metabolic versatility and an exceptional ability to adapt to a wide range of ecological environments, including soil, marches, coastal habitats, plant and animal tissues. Gram-negative microbes are characterized by the asymmetric lipopolysaccharide outer membrane, the study of which is important for a number of applications. The adhesion to mineral surfaces plays a central role in characterizing their contribution to the fate of contaminants in complex environmental systems by effecting microbial transport through soils, respiration redox chemistry, and ion mobility. Another important application stems from the fact that it is also a major opportunistic human pathogen that can result in life-threatening infections in many immunocompromised patients, such as lung infections in children with cystic fibrosis, bacteraemia in burn victims, urinary-tract infections in catheterized patients, hospital-acquired pneumonia in patients on respirators, infections in cancer patients receiving chemotherapy, and keratitis and corneal ulcers in users of extended-wear soft contact lenses. The inherent resistance against antibiotics which has been linked with the specific interactions in the outer membrane of P. aeruginosa makes these infections difficult to treat. Developments in simulation methodologies as well as computer hardware have enabled the molecular simulation of biological systems of increasing size and with increasing accuracy, providing detail that is difficult or impossible to obtain experimentally. Computer simulation studies contribute to our understanding of the behavior of proteins, protein-protein and protein-DNA complexes. In recent years, a number of research groups have made significant progress in applying these methods to the study of biological membranes. However, these applications have been focused exclusively on lipid bilayer membranes and on membrane proteins in lipid

  3. The lipid organisation of the cell membrane

    Directory of Open Access Journals (Sweden)

    Ladha, S.

    2000-04-01

    Full Text Available Lipids and proteins in biological membranes are arranged in a mosaic of domains in the membrane. These domains represent small-scale heterogeneities in composition, shape and fluidity within the plane of the membrane, over the range of hundreds of nanometers to a few micrometers. They arise from the complex interactions of the heterogeneous mixtures of phospholipids, sterols, and proteins that make up all biological membranes.Los lípidos y las proteínas en las membranas biológicas están dispuestos en un mosaico de campos en la membrana. Estos campos representan heterogeneidades a pequeña escala en la composición, forma y fluidez dentro del plano de la membrana, en un rango que va de los cientos de nanómetros a los pocos micrómetros. Estos campos se originan de las complejas interacciones de las mezclas heterogéneas de fosfolípidos, esteroles y proteínas de las que están hechas todas y cada una de las membranas biológicas.

  4. Selectivity of Direct Methanol Fuel Cell Membranes.

    Science.gov (United States)

    Aricò, Antonino S; Sebastian, David; Schuster, Michael; Bauer, Bernd; D'Urso, Claudia; Lufrano, Francesco; Baglio, Vincenzo

    2015-11-24

    Sulfonic acid-functionalized polymer electrolyte membranes alternative to Nafion(®) were developed. These were hydrocarbon systems, such as blend sulfonated polyetheretherketone (s-PEEK), new generation perfluorosulfonic acid (PFSA) systems, and composite zirconium phosphate-PFSA polymers. The membranes varied in terms of composition, equivalent weight, thickness, and filler and were investigated with regard to their methanol permeation characteristics and proton conductivity for application in direct methanol fuel cells. The behavior of the membrane electrode assemblies (MEA) was investigated in fuel cell with the aim to individuate a correlation between membrane characteristics and their performance in a direct methanol fuel cell (DMFC). The power density of the DMFC at 60 °C increased according to a square root-like function of the membrane selectivity. This was defined as the reciprocal of the product between area specific resistance and crossover. The power density achieved at 60 °C for the most promising s-PEEK-based membrane-electrode assembly (MEA) was higher than the benchmark Nafion(®) 115-based MEA (77 mW·cm(-2) vs. 64 mW·cm(-2)). This result was due to a lower methanol crossover (47 mA·cm(-2) equivalent current density for s-PEEK vs. 120 mA·cm(-2) for Nafion(®) 115 at 60 °C as recorded at OCV with 2 M methanol) and a suitable area specific resistance (0.15 Ohm cm² for s-PEEK vs. 0.22 Ohm cm² for Nafion(®) 115).

  5. Selectivity of Direct Methanol Fuel Cell Membranes

    Science.gov (United States)

    Aricò, Antonino S.; Sebastian, David; Schuster, Michael; Bauer, Bernd; D’Urso, Claudia; Lufrano, Francesco; Baglio, Vincenzo

    2015-01-01

    Sulfonic acid-functionalized polymer electrolyte membranes alternative to Nafion® were developed. These were hydrocarbon systems, such as blend sulfonated polyetheretherketone (s-PEEK), new generation perfluorosulfonic acid (PFSA) systems, and composite zirconium phosphate–PFSA polymers. The membranes varied in terms of composition, equivalent weight, thickness, and filler and were investigated with regard to their methanol permeation characteristics and proton conductivity for application in direct methanol fuel cells. The behavior of the membrane electrode assemblies (MEA) was investigated in fuel cell with the aim to individuate a correlation between membrane characteristics and their performance in a direct methanol fuel cell (DMFC). The power density of the DMFC at 60 °C increased according to a square root-like function of the membrane selectivity. This was defined as the reciprocal of the product between area specific resistance and crossover. The power density achieved at 60 °C for the most promising s-PEEK-based membrane-electrode assembly (MEA) was higher than the benchmark Nafion® 115-based MEA (77 mW·cm−2 vs. 64 mW·cm−2). This result was due to a lower methanol crossover (47 mA·cm−2 equivalent current density for s-PEEK vs. 120 mA·cm−2 for Nafion® 115 at 60 °C as recorded at OCV with 2 M methanol) and a suitable area specific resistance (0.15 Ohm cm2 for s-PEEK vs. 0.22 Ohm cm2 for Nafion® 115). PMID:26610582

  6. Selectivity of Direct Methanol Fuel Cell Membranes

    Directory of Open Access Journals (Sweden)

    Antonino S. Aricò

    2015-11-01

    Full Text Available Sulfonic acid-functionalized polymer electrolyte membranes alternative to Nafion® were developed. These were hydrocarbon systems, such as blend sulfonated polyetheretherketone (s-PEEK, new generation perfluorosulfonic acid (PFSA systems, and composite zirconium phosphate–PFSA polymers. The membranes varied in terms of composition, equivalent weight, thickness, and filler and were investigated with regard to their methanol permeation characteristics and proton conductivity for application in direct methanol fuel cells. The behavior of the membrane electrode assemblies (MEA was investigated in fuel cell with the aim to individuate a correlation between membrane characteristics and their performance in a direct methanol fuel cell (DMFC. The power density of the DMFC at 60 °C increased according to a square root-like function of the membrane selectivity. This was defined as the reciprocal of the product between area specific resistance and crossover. The power density achieved at 60 °C for the most promising s-PEEK-based membrane-electrode assembly (MEA was higher than the benchmark Nafion® 115-based MEA (77 mW·cm−2 vs. 64 mW·cm−2. This result was due to a lower methanol crossover (47 mA·cm−2 equivalent current density for s-PEEK vs. 120 mA·cm−2 for Nafion® 115 at 60 °C as recorded at OCV with 2 M methanol and a suitable area specific resistance (0.15 Ohm cm2 for s-PEEK vs. 0.22 Ohm cm2 for Nafion® 115.

  7. Biophysical characterization of membrane protein-small molecule interactions

    NARCIS (Netherlands)

    Chen, Dan

    2015-01-01

    Membrane proteins are account for up to two thirds of known druggable targets. Traditionally, new drugs against this class of proteins have been discovered through HTS. However, not all GPCRs are amenable to traditional screening methods. Recently, fragment-based drug discovery (FBDD) has emerged as

  8. Identification of outer membrane proteins of Yersinia pestis through biotinylation

    NARCIS (Netherlands)

    Smither, S.J.; Hill, J.; Baar, B.L.M. van; Hulst, A.G.; Jong, A.L. de; Titball, R.W.

    2007-01-01

    The outer membrane of Gram-negative bacteria contains proteins that might be good targets for vaccines, antimicrobials or detection systems. The identification of surface located proteins using traditional methods is often difficult. Yersinia pestis, the causative agent of plague, was labelled with

  9. Ultrafast permeation of water through protein-based membranes.

    Science.gov (United States)

    Peng, Xinsheng; Jin, Jian; Nakamura, Yoshimichi; Ohno, Takahisa; Ichinose, Izumi

    2009-06-01

    Pressure-driven filtration by porous membranes is widely used in the production of drinking water from ground and surface water. Permeation theory predicts that filtration rate is proportional to the pressure difference across the filtration membrane and inversely proportional to the thickness of the membrane. However, these membranes need to be able to withstand high water fluxes and pressures, which means that the active separation layers in commercial filtration systems typically have a thickness of a few tens to several hundreds of nanometres. Filtration performance might be improved by the use of ultrathin porous silicon membranes or carbon nanotubes immobilized in silicon nitride or polymer films, but these structures are difficult to fabricate. Here, we report a new type of filtration membrane made of crosslinked proteins that are mechanically robust and contain channels with diameters of less than 2.2 nm. We find that a 60-nm-thick membrane can concentrate aqueous dyes from fluxes up to 9,000 l h(-1) m(-2) bar(-1), which is approximately 1,000 times higher than the fluxes that can be withstood by commercial filtration membranes with similar rejection properties. Based on these results and molecular dynamics simulations, we propose that protein-surrounded channels with effective lengths of less than 5.8 nm can separate dye molecules while allowing the ultrafast permeation of water at applied pressures of less than 1 bar.

  10. High-resolution Structures of Protein-Membrane Complexes by Neutron Reflection and MD Simulation: Membrane Association of the PTEN Tumor Suppressor

    Science.gov (United States)

    Lösche, Matthias

    2012-02-01

    The lipid matrix of biomembranes is an in-plane fluid, thermally and compositionally disordered leaflet of 5 nm thickness and notoriously difficult to characterize in structural terms. Yet, biomembranes are ubiquitous in the cell, and membrane-bound proteins are implicated in a variety of signaling pathways and intra-cellular transport. We developed methodology to study proteins associated with model membranes using neutron reflection measurements and showed recently that this approach can resolve the penetration depth and orientation of membrane proteins with ångstrom resolution if their crystal or NMR structure is known. Here we apply this technology to determine the membrane bindung and unravel functional details of the PTEN phosphatase, a key player in the PI3K apoptosis pathway. PTEN is an important regulatory protein and tumor suppressor that performs its phosphatase activity as an interfacial enzyme at the plasma membrane-cytoplasm boundary. Acting as an antagonist to phosphoinositide-3-kinase (PI3K) in cell signaling, it is deleted in many human cancers. Despite its importance in regulating the levels of the phosphoinositoltriphosphate PI(3,4,5)P3, there is little understanding of how PTEN binds to membranes, is activated and then acts as a phosphatase. We investigated the structure and function of PTEN by studying its membrane affinity and localization on in-plane fluid, thermally disordered synthetic membrane models. The membrane association of the protein depends strongly on membrane composition, where phosphatidylserine (PS) and phosphatidylinositol diphosphate (PI(4,5)P2) act synergetically in attracting the enzyme to the membrane surface. Membrane affinities depend strongly on membrane fluidity, which suggests multiple binding sites on the protein for PI(4,5)P2. Neutron reflection measurements show that the PTEN phosphatase ``scoots'' along the membrane surface (penetration PTEN's regulatory C-terminal tail is displaced from the membrane and

  11. Chemical Imaging of the Cell Membrane by NanoSIMS

    Energy Technology Data Exchange (ETDEWEB)

    Weber, P K; Kraft, M L; Frisz, J F; Carpenter, K J; Hutcheon, I D

    2010-02-23

    The existence of lipid microdomains and their role in cell membrane organization are currently topics of great interest and controversy. The cell membrane is composed of a lipid bilayer with embedded proteins that can flow along the two-dimensional surface defined by the membrane. Microdomains, known as lipid rafts, are believed to play a central role in organizing this fluid system, enabling the cell membrane to carry out essential cellular processes, including protein recruitment and signal transduction. Lipid rafts are also implicated in cell invasion by pathogens, as in the case of the HIV. Therefore, understanding the role of lipid rafts in cell membrane organization not only has broad scientific implications, but also has practical implications for medical therapies. One of the major limitations on lipid organization research has been the inability to directly analyze lipid composition without introducing artifacts and at the relevant length-scales of tens to hundreds of nanometers. Fluorescence microscopy is widely used due to its sensitivity and specificity to the labeled species, but only the labeled components can be observed, fluorophores can alter the behavior of the lipids they label, and the length scales relevant to imaging cell membrane domains are between that probed by fluorescence resonance energy transfer (FRET) imaging (<10 nm) and the diffraction limit of light. Topographical features can be imaged on this length scale by atomic force microscopy (AFM), but the chemical composition of the observed structures cannot be determined. Immuno-labeling can be used to study the distribution of membrane proteins at high resolution, but not lipid composition. We are using imaging mass spectrometry by secondary ion mass spectrometry (SIMS) in concert with other high resolution imaging methods to overcome these limitations. The experimental approach of this project is to combine molecule-specific stable isotope labeling with high-resolution SIMS using a

  12. Amyloid protein unfolding and insertion kinetics on neuronal membrane mimics

    Science.gov (United States)

    Qiu, Liming; Buie, Creighton; Vaughn, Mark; Cheng, Kwan

    2010-03-01

    Atomistic details of beta-amyloid (Aβ ) protein unfolding and lipid interaction kinetics mediated by the neuronal membrane surface are important for developing new therapeutic strategies to prevent and cure Alzheimer's disease. Using all-atom MD simulations, we explored the early unfolding and insertion kinetics of 40 and 42 residue long Aβ in binary lipid mixtures with and without cholesterol that mimic the cholesterol-depleted and cholesterol-enriched lipid nanodomains of neurons. The protein conformational transition kinetics was evaluated from the secondary structure profile versus simulation time plot. The extent of membrane disruption was examined by the calculated order parameters of lipid acyl chains and cholesterol fused rings as well as the density profiles of water and lipid headgroups at defined regions across the lipid bilayer from our simulations. Our results revealed that both the cholesterol content and the length of the protein affect the protein-insertion and membrane stability in our model lipid bilayer systems.

  13. The endoplasmic reticulum membrane J protein C18 executes a distinct role in promoting simian virus 40 membrane penetration.

    Science.gov (United States)

    Bagchi, Parikshit; Walczak, Christopher Paul; Tsai, Billy

    2015-04-01

    The nonenveloped simian virus 40 (SV40) hijacks the three endoplasmic reticulum (ER) membrane-bound J proteins B12, B14, and C18 to escape from the ER into the cytosol en route to successful infection. How C18 controls SV40 ER-to-cytosol membrane penetration is the least understood of these processes. We previously found that SV40 triggers B12 and B14 to reorganize into discrete puncta in the ER membrane called foci, structures postulated to represent the cytosol entry site (C. P. Walczak, M. S. Ravindran, T. Inoue, and B. Tsai, PLoS Pathog 10: e1004007, 2014). We now find that SV40 also recruits C18 to the virus-induced B12/B14 foci. Importantly, the C18 foci harbor membrane penetration-competent SV40, further implicating this structure as the membrane penetration site. Consistent with this, a mutant SV40 that cannot penetrate the ER membrane and promote infection fails to induce C18 foci. C18 also regulates the recruitment of B12/B14 into the foci. In contrast to B14, C18's cytosolic Hsc70-binding J domain, but not the lumenal domain, is essential for its targeting to the foci; this J domain likewise is necessary to support SV40 infection. Knockdown-rescue experiments reveal that C18 executes a role that is not redundant with those of B12/B14 during SV40 infection. Collectively, our data illuminate C18's contribution to SV40 ER membrane penetration, strengthening the idea that SV40-triggered foci are critical for cytosol entry. Polyomaviruses (PyVs) cause devastating human diseases, particularly in immunocompromised patients. As this virus family continues to be a significant human pathogen, clarifying the molecular basis of their cellular entry pathway remains a high priority. To infect cells, PyV traffics from the cell surface to the ER, where it penetrates the ER membrane to reach the cytosol. In the cytosol, the virus moves to the nucleus to cause infection. ER-to-cytosol membrane penetration is a critical yet mysterious infection step. In this study, we

  14. Membrane and Protein Interactions of the Pleckstrin Homology Domain Superfamily

    Directory of Open Access Journals (Sweden)

    Marc Lenoir

    2015-10-01

    Full Text Available The human genome encodes about 285 proteins that contain at least one annotated pleckstrin homology (PH domain. As the first phosphoinositide binding module domain to be discovered, the PH domain recruits diverse protein architectures to cellular membranes. PH domains constitute one of the largest protein superfamilies, and have diverged to regulate many different signaling proteins and modules such as Dbl homology (DH and Tec homology (TH domains. The ligands of approximately 70 PH domains have been validated by binding assays and complexed structures, allowing meaningful extrapolation across the entire superfamily. Here the Membrane Optimal Docking Area (MODA program is used at a genome-wide level to identify all membrane docking PH structures and map their lipid-binding determinants. In addition to the linear sequence motifs which are employed for phosphoinositide recognition, the three dimensional structural features that allow peripheral membrane domains to approach and insert into the bilayer are pinpointed and can be predicted ab initio. The analysis shows that conserved structural surfaces distinguish which PH domains associate with membrane from those that do not. Moreover, the results indicate that lipid-binding PH domains can be classified into different functional subgroups based on the type of membrane insertion elements they project towards the bilayer.

  15. Membrane and Protein Interactions of the Pleckstrin Homology Domain Superfamily.

    Science.gov (United States)

    Lenoir, Marc; Kufareva, Irina; Abagyan, Ruben; Overduin, Michael

    2015-10-23

    The human genome encodes about 285 proteins that contain at least one annotated pleckstrin homology (PH) domain. As the first phosphoinositide binding module domain to be discovered, the PH domain recruits diverse protein architectures to cellular membranes. PH domains constitute one of the largest protein superfamilies, and have diverged to regulate many different signaling proteins and modules such as Dbl homology (DH) and Tec homology (TH) domains. The ligands of approximately 70 PH domains have been validated by binding assays and complexed structures, allowing meaningful extrapolation across the entire superfamily. Here the Membrane Optimal Docking Area (MODA) program is used at a genome-wide level to identify all membrane docking PH structures and map their lipid-binding determinants. In addition to the linear sequence motifs which are employed for phosphoinositide recognition, the three dimensional structural features that allow peripheral membrane domains to approach and insert into the bilayer are pinpointed and can be predicted ab initio. The analysis shows that conserved structural surfaces distinguish which PH domains associate with membrane from those that do not. Moreover, the results indicate that lipid-binding PH domains can be classified into different functional subgroups based on the type of membrane insertion elements they project towards the bilayer.

  16. Protein expression profiling of nuclear membrane protein reveals potential biomarker of human hepatocellular carcinoma

    OpenAIRE

    Khan, Rizma; Zahid, Saadia; Wan, Yu-Jui; Forster, Jameson; Karim, A-Bashar; Nawabi, Atta M; Azhar, Abid; Rahman, M; Ahmed, Nikhat

    2013-01-01

    Abstract Background Complex molecular events lead to development and progression of liver cirrhosis to HCC. Differentially expressed nuclear membrane associated proteins are responsible for the functional and structural alteration during the progression from cirrhosis to carcinoma. Although alterations/ post translational modifications in protein expression have been extensively quantified, complementary analysis of nuclear membrane proteome changes h...

  17. Neuron Membrane Trafficking and Protein Kinases Involved in Autism and ADHD

    Directory of Open Access Journals (Sweden)

    Yasuko Kitagishi

    2015-01-01

    Full Text Available A brain-enriched multi-domain scaffolding protein, neurobeachin has been identified as a candidate gene for autism patients. Mutations in the synaptic adhesion protein cell adhesion molecule 1 (CADM1 are also associated with autism spectrum disorder, a neurodevelopmental disorder of uncertain molecular origin. Potential roles of neurobeachin and CADM1 have been suggested to a function of vesicle transport in endosomal trafficking. It seems that protein kinase B (AKT and cyclic adenosine monophosphate (cAMP-dependent protein kinase A (PKA have key roles in the neuron membrane trafficking involved in the pathogenesis of autism. Attention deficit hyperactivity disorder (ADHD is documented to dopaminergic insufficiencies, which is attributed to synaptic dysfunction of dopamine transporter (DAT. AKT is also essential for the DAT cell-surface redistribution. In the present paper, we summarize and discuss the importance of several protein kinases that regulate the membrane trafficking involved in autism and ADHD, suggesting new targets for therapeutic intervention.

  18. Molecular characterization of a cold-induced plasma membrane protein gene from wheat.

    Science.gov (United States)

    Koike, Michiya; Sutoh, Keita; Kawakami, Akira; Torada, Atsushi; Oono, Kiyoharu; Imai, Ryozo

    2005-12-01

    As a means to study the function of plasma membrane proteins during cold acclimation, we have isolated a cDNA clone for wpi6 which encodes a putative plasma membrane protein from cold-acclimated winter wheat. The wpi6 gene encodes a putative 5.9 kDa polypeptide with two predicted membrane-spanning domains, the sequence of which shows high sequence similarity with BLT101-family proteins from plants and yeast. Strong induction of wpi6 mRNA was observed during an early stage of cold acclimation in root and shoot tissues of both winter and spring wheat cultivars. In contrast to blt101 in barley, wpi6 mRNA was also induced by drought and salinity stresses, and exogenous application of ABA. Expression of wpi6 in a Deltapmp3 mutant of Saccharomyces cerevisiae, which is disturbed in plasma membrane potential due to the lack of a BLT101-family protein, partially complemented NaCl sensitivity of the mutant. Transient expression analysis of a WPI6::GFP fusion protein in onion epidermal cells revealed that WPI6 is localized in the plasma membrane. Taken together, these data suggested that WPI6 may have a protective role in maintaining plasma membrane function during cold acclimation in wheat.

  19. Herpes Simplex Virus Membrane Proteins gE/gI and US9 Act Cooperatively To Promote Transport of Capsids and Glycoproteins from Neuron Cell Bodies into Initial Axon Segments

    Science.gov (United States)

    Howard, Paul W.; Howard, Tiffani L.

    2013-01-01

    Herpes simplex virus (HSV) and other alphaherpesviruses must move from sites of latency in ganglia to peripheral epithelial cells. How HSV navigates in neuronal axons is not well understood. Two HSV membrane proteins, gE/gI and US9, are key to understanding the processes by which viral glycoproteins, unenveloped capsids, and enveloped virions are transported toward axon tips. Whether gE/gI and US9 function to promote the loading of viral proteins onto microtubule motors in neuron cell bodies or to tether viral proteins onto microtubule motors within axons is not clear. One impediment to understanding how HSV gE/gI and US9 function in axonal transport relates to observations that gE−, gI−, or US9− mutants are not absolutely blocked in axonal transport. Mutants are significantly reduced in numbers of capsids and glycoproteins in distal axons, but there are less extensive effects in proximal axons. We constructed HSV recombinants lacking both gE and US9 that transported no detectable capsids and glycoproteins to distal axons and failed to spread from axon tips to adjacent cells. Live-cell imaging of a gE−/US9− double mutant that expressed fluorescent capsids and gB demonstrated >90% diminished capsids and gB in medial axons and no evidence for decreased rates of transport, stalling, or increased retrograde transport. Instead, capsids, gB, and enveloped virions failed to enter proximal axons. We concluded that gE/gI and US9 function in neuron cell bodies, in a cooperative fashion, to promote the loading of HSV capsids and vesicles containing glycoproteins and enveloped virions onto microtubule motors or their transport into proximal axons. PMID:23077321

  20. Solid-state nuclear magnetic resonance (NMR) spectroscopy of human immunodeficiency virus gp41 protein that includes the fusion peptide: NMR detection of recombinant Fgp41 in inclusion bodies in whole bacterial cells and structural characterization of purified and membrane-associated Fgp41.

    Science.gov (United States)

    Vogel, Erica P; Curtis-Fisk, Jaime; Young, Kaitlin M; Weliky, David P

    2011-11-22

    Human immunodeficiency virus (HIV) infection of a host cell begins with fusion of the HIV and host cell membranes and is mediated by the gp41 protein, a single-pass integral membrane protein of HIV. The 175 N-terminal residues make up the ectodomain that lies outside the virus. This work describes the production and characterization of an ectodomain construct containing the 154 N-terminal gp41 residues, including the fusion peptide (FP) that binds to target cell membranes. The Fgp41 sequence was derived from one of the African clade A strains of HIV-1 that have been less studied than European/North American clade B strains. Fgp41 expression at a level of ~100 mg/L of culture was evidenced by an approach that included amino acid type (13)CO and (15)N labeling of recombinant protein and solid-state NMR (SSNMR) spectroscopy of lyophilized whole cells. The approach did not require any protein solubilization or purification and may be a general approach for detection of recombinant protein. The purified Fgp41 yield was ~5 mg/L of culture. SSNMR spectra of membrane-associated Fgp41 showed high helicity for the residues C-terminal of the FP. This was consistent with a "six-helix bundle" (SHB) structure that is the final gp41 state during membrane fusion. This observation and negligible Fgp41-induced vesicle fusion supported a function for SHB gp41 of membrane stabilization and fusion arrest. SSNMR spectra of residues in the membrane-associated FP provided evidence of a mixture of molecular populations with either helical or β-sheet FP conformation. These and earlier SSNMR data strongly support the existence of these populations in the SHB state of membrane-associated gp41. © 2011 American Chemical Society

  1. An Improved 2-Dimensional Gel Electrophoresis Method for Resolving Human Erythrocyte Membrane Proteins.

    Science.gov (United States)

    Kumar, Manoj; Singh, Rajendra; Meena, Anil; Patidar, Bhagwan S; Prasad, Rajendra; Chhabra, Sunil K; Bansal, Surendra K

    2017-01-01

    The 2-dimensional gel electrophoresis (2-DE) technique is widely used for the analysis of complex protein mixtures extracted from biological samples. It is one of the most commonly used analytical techniques in proteomics to study qualitative and quantitative protein changes between different states of a cell or an organism (eg, healthy and diseased), conditionally expressed proteins, posttranslational modifications, and so on. The 2-DE technique is used for its unparalleled ability to separate thousands of proteins simultaneously. The resolution of the proteins by 2-DE largely depends on the quality of sample prepared during protein extraction which increases results in terms of reproducibility and minimizes protein modifications that may result in artifactual spots on 2-DE gels. The buffer used for the extraction and solubilization of proteins influences the quality and reproducibility of the resolution of proteins on 2-DE gel. The purification by cleanup kit is another powerful process to prevent horizontal streaking which occurs during isoelectric focusing due to the presence of contaminants such as salts, lipids, nucleic acids, and detergents. Erythrocyte membrane proteins serve as prototypes for multifunctional proteins in various erythroid and nonerythroid cells. In this study, we therefore optimized the selected major conditions of 2-DE for resolving various proteins of human erythrocyte membrane. The modification included the optimization of conditions for sample preparation, cleanup of protein sample, isoelectric focusing, equilibration, and storage of immobilized pH gradient strips, which were further carefully examined to achieve optimum conditions for improving the quality of protein spots on 2-DE gels. The present improved 2-DE analysis method enabled better detection of protein spots with higher quality and reproducibility. Therefore, the conditions established in this study may be used for the 2-DE analysis of erythrocyte membrane proteins for

  2. Selecting Targets for Tumor Imaging: An Overview of Cancer-Associated Membrane Proteins

    Directory of Open Access Journals (Sweden)

    Martin C. Boonstra

    2016-01-01

    Full Text Available Tumor targeting is a booming business: The global therapeutic monoclonal antibody market accounted for more than $78 billion in 2012 and is expanding exponentially. Tumors can be targeted with an extensive arsenal of monoclonal antibodies, ligand proteins, peptides, RNAs, and small molecules. In addition to therapeutic targeting, some of these compounds can also be applied for tumor visualization before or during surgery, after conjugation with radionuclides and/or near-infrared fluorescent dyes. The majority of these tumor-targeting compounds are directed against cell membrane-bound proteins. Various categories of targetable membrane-bound proteins, such as anchoring proteins, receptors, enzymes, and transporter proteins, exist. The functions and biological characteristics of these proteins determine their location and distribution on the cell membrane, making them more, or less, accessible, and therefore, it is important to understand these features. In this review, we evaluate the characteristics of cancer-associated membrane proteins and discuss their overall usability for cancer targeting, especially focusing on imaging applications.

  3. Lipid nanotechnologies for structural studies of membrane-associated proteins.

    Science.gov (United States)

    Stoilova-McPhie, Svetla; Grushin, Kirill; Dalm, Daniela; Miller, Jaimy

    2014-11-01

    We present a methodology of lipid nanotubes (LNT) and nanodisks technologies optimized in our laboratory for structural studies of membrane-associated proteins at close to physiological conditions. The application of these lipid nanotechnologies for structure determination by cryo-electron microscopy (cryo-EM) is fundamental for understanding and modulating their function. The LNTs in our studies are single bilayer galactosylceramide based nanotubes of ∼20 nm inner diameter and a few microns in length, that self-assemble in aqueous solutions. The lipid nanodisks (NDs) are self-assembled discoid lipid bilayers of ∼10 nm diameter, which are stabilized in aqueous solutions by a belt of amphipathic helical scaffold proteins. By combining LNT and ND technologies, we can examine structurally how the membrane curvature and lipid composition modulates the function of the membrane-associated proteins. As proof of principle, we have engineered these lipid nanotechnologies to mimic the activated platelet's phosphtaidylserine rich membrane and have successfully assembled functional membrane-bound coagulation factor VIII in vitro for structure determination by cryo-EM. The macromolecular organization of the proteins bound to ND and LNT are further defined by fitting the known atomic structures within the calculated three-dimensional maps. The combination of LNT and ND technologies offers a means to control the design and assembly of a wide range of functional membrane-associated proteins and complexes for structural studies by cryo-EM. The presented results confirm the suitability of the developed methodology for studying the functional structure of membrane-associated proteins, such as the coagulation factors, at a close to physiological environment. © 2014 Wiley Periodicals, Inc.

  4. SV40 late protein VP4 forms toroidal pores to disrupt membranes for viral release.

    Science.gov (United States)

    Raghava, Smita; Giorda, Kristina M; Romano, Fabian B; Heuck, Alejandro P; Hebert, Daniel N

    2013-06-04

    Nonenveloped viruses are generally released from the cell by the timely lysis of host cell membranes. SV40 has been used as a model virus for the study of the lytic nonenveloped virus life cycle. The expression of SV40 VP4 at later times during infection is concomitant with cell lysis. To investigate the role of VP4 in viral release and its mechanism of action, VP4 was expressed and purified from bacteria as a fusion protein for use in membrane disruption assays. Purified VP4 perforated membranes as demonstrated by the release of fluorescent markers encapsulated within large unilamellar vesicles or liposomes. Dynamic light scattering results revealed that VP4 treatment did not cause membrane lysis or change the size of the liposomes. Liposomes encapsulated with 4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-3-indacene-labeled streptavidin were used to show that VP4 formed stable pores in membranes. These VP4 pores had an inner diameter of 1-5 nm. Asymmetrical liposomes containing pyrene-labeled lipids in the outer monolayer were employed to monitor transbilayer lipid diffusion. Consistent with VP4 forming toroidal pore structures in membranes, VP4 induced transbilayer lipid diffusion or lipid flip-flop. Altogether, these studies support a central role for VP4 acting as a viroporin in the disruption of cellular membranes to trigger SV40 viral release by forming toroidal pores that unite the outer and inner leaflets of membrane bilayers.

  5. LDRD final report on imaging self-organization of proteins in membranes by photocatalytic nano-tagging.

    Energy Technology Data Exchange (ETDEWEB)

    Zavadil, Kevin Robert; Shelnutt, John Allen; Sasaki, Darryl Yoshio; Song, Yujiang; Medforth, Craig J.

    2005-11-01

    We have developed a new nanotagging technology for detecting and imaging the self-organization of proteins and other components of membranes at nanometer resolution for the purpose of investigating cell signaling and other membrane-mediated biological processes. We used protein-, lipid-, or drug-bound porphyrin photocatalysts to grow in-situ nanometer-sized metal particles, which reveal the location of the porphyrin-labeled molecules by electron microscopy. We initially used photocatalytic nanotagging to image assembled multi-component proteins and to monitor the distribution of lipids and porphyrin labels in liposomes. For example, by exchanging the heme molecules in hemoproteins with a photocatalytic tin porphyrin, a nanoparticle was grown at each heme site of the protein. The result obtained from electron microscopy for a tagged multi-subunit protein such as hemoglobin is a symmetric constellation of a specific number of nanoparticle tags, four in the case of the hemoglobin tetramer. Methods for covalently linking photocatalytic porphyrin labels to lipids and proteins were also developed to detect and image the self-organization of lipids, protein-protein supercomplexes, and membrane-protein complexes. Procedures for making photocatalytic porphyrin-drug, porphyrin-lipid, and porphyrin-protein hybrids for non-porphyrin-binding proteins and membrane components were pursued and the first porphyrin-labeled lipids was investigated in liposomal membrane models. Our photocatalytic nanotagging technique may ultimately allow membrane self-organization and cell signaling processes to be imaged in living cells. Fluorescence and plasmonic spectra of the tagged proteins might also provide additional information about protein association and membrane organization. In addition, a porphyrin-aspirin or other NSAID hybrid may be used to grow metal nanotags for the pharmacologically important COX enzymes in membranes so that the distribution of the protein can be imaged at the

  6. B-cell responses to pregnancy-restricted and -unrestricted Plasmodium falciparum erythrocyte membrane protein 1 antigens in Ghanaian women naturally exposed to malaria parasites

    DEFF Research Database (Denmark)

    Ampomah, Paulina; Stevenson, Liz; Ofori, Michael F

    2014-01-01

    -linked immunosorbent assay (ELISA) and memory B-cell frequencies by enzyme-linked immunosorbent spot (ELISPOT) assay in a cohort of P. falciparum-exposed nonpregnant Ghanaian women. The antigens used were a VAR2CSA-type PfEMP1 (IT4VAR04) with expression restricted to parasites infecting the placenta, as well as two...... immunity probably reflect the clonal antigenic variation and allelic polymorphism of PfEMP1. However, it is likely that other immune-evasive mechanisms are also involved, such as interference with formation and maintenance of immunological memory. We measured PfEMP1-specific antibody levels by enzyme...... commonly recognized PfEMP1 proteins (HB3VAR06 and IT4VAR60) implicated in rosetting and not pregnancy restricted. This enabled, for the first time, a direct comparison in the same individuals of immune responses specific for a clinically important parasite antigen expressed only during well-defined periods...

  7. A peptide derived from the rotavirus outer capsid protein VP7 permeabilizes artificial membranes.

    Science.gov (United States)

    Elaid, Sarah; Libersou, Sonia; Ouldali, Malika; Morellet, Nelly; Desbat, Bernard; Alves, Isabel D; Lepault, Jean; Bouaziz, Serge

    2014-08-01

    Biological membranes represent a physical barrier that most viruses have to cross for replication. While enveloped viruses cross membranes through a well-characterized membrane fusion mechanism, non-enveloped viruses, such as rotaviruses, require the destabilization of the host cell membrane by processes that are still poorly understood. We have identified, in the C-terminal region of the rotavirus glycoprotein VP7, a peptide that was predicted to contain a membrane domain and to fold into an amphipathic α-helix. Its structure was confirmed by circular dichroism in media mimicking the hydrophobic environment of the membrane at both acidic and neutral pHs. The helical folding of the peptide was corroborated by ATR-FTIR spectroscopy, which suggested a transmembrane orientation of the peptide. The interaction of this peptide with artificial membranes and its affinity were assessed by plasmon waveguide resonance. We have found that the peptide was able to insert into membranes and permeabilize them while the native protein VP7 did not. Finally, NMR studies revealed that in a hydrophobic environment, this helix has amphipathic properties characteristic of membrane-perforating peptides. Surprisingly, its structure varies from that of its counterpart in the structure of the native protein VP7, as was determined by X-ray. All together, our results show that a peptide released from VP7 is capable of changing its conformation and destabilizing artificial membranes. Such peptides could play an important role by facilitating membrane crossing by non-enveloped viruses during cell infection. Copyright © 2014 Elsevier B.V. All rights reserved.

  8. Molecular sieving action of the cell membrane during gradual osmotic hemolysis

    Energy Technology Data Exchange (ETDEWEB)

    MacGregor, R.D. II

    1977-05-01

    Rat erythrocytes were hemolyzed by controlled gradual osmotic hemolysis to study cell morphology and hemoglobin loss from individual cells. Results suggest that each increase in the