Kinetics of antibody-induced modulation of respiratory syncytial virus antigens in a human epithelial cell line

PubMed Central

Sarmiento, Rosa E; Tirado, Rocio G; Valverde, Laura E; Gómez-Garcia, Beatriz

2007-01-01

Background The binding of viral-specific antibodies to cell-surface antigens usually results in down modulation of the antigen through redistribution of antigens into patches that subsequently may be internalized by endocytosis or may form caps that can be expelled to the extracellular space. Here, by use of confocal-laser-scanning microscopy we investigated the kinetics of the modulation of respiratory syncytial virus (RSV) antigen by RSV-specific IgG. RSV-infected human epithelial cells (HEp-2) were incubated with anti-RSV polyclonal IgG and, at various incubation times, the RSV-cell-surface-antigen-antibody complexes (RSV Ag-Abs) and intracellular viral proteins were detected by indirect immunoflourescence. Results Interaction of anti-RSV polyclonal IgG with RSV HEp-2 infected cells induced relocalization and aggregation of viral glycoproteins in the plasma membrane formed patches that subsequently produced caps or were internalized through clathrin-mediated endocytosis participation. Moreover, the concentration of cell surface RSV Ag-Abs and intracellular viral proteins showed a time dependent cyclic variation and that anti-RSV IgG protected HEp-2 cells from viral-induced death. Conclusion The results from this study indicate that interaction between RSV cell surface proteins and specific viral antibodies alter the expression of viral antigens expressed on the cells surface and intracellular viral proteins; furthermore, interfere with viral induced destruction of the cell. PMID:17608950

Fate of prions in soil: trapped conformation of full-length ovine prion protein induced by adsorption on clays.

PubMed

Revault, M; Quiquampoix, H; Baron, M H; Noinville, S

2005-08-05

Studying the mechanism of retention of ovine prion protein in soils will tackle the environmental aspect of potential dissemination of scrapie infectious agent. We consider the surface-induced conformational changes that the recombinant ovine prion protein (ovPrP) may undergo under different pH conditions when interacting with soil minerals of highly adsorptive capacities such as montmorillonite. The conformational states of the full-length ovine prion protein adsorbed on the electronegative clay surface are compared to its solvated state in deuterated buffer in the pD range 3.5-9, using FTIR spectroscopy. The in vitro pH-induced conversion of the alpha-helical monomer of ovPrP into oligomers of beta-like structure prone to self-aggregation does not occur when the protein is adsorbed on the clay surface. The conformation of the trapped ovPrP molecules on montmorillonite is pH-independent and looks like that of the ovPrP solvated state at pD higher than 7, suggesting the major role of Arg and Lys residues in the electrostatic origin of adsorption. The uneven distribution of positively and negatively charged residues of the ovPrP protein would promote a favored orientation of the protein towards the clay, so that not only the basic residues embedded in the N-terminal flexible part but also external basic residues in the globular part of the protein might participate to the attractive interaction. From these results, it appears unlikely that the interaction of normal prions (PrP(C)) with soil clay surfaces could induce a change of conformation leading to the pathogenic form of prions (PrP(Sc)).

Steric Shielding of Surface Epitopes and Impaired Immune Recognition Induced by the Ebola Virus Glycoprotein

PubMed Central

Francica, Joseph R.; Varela-Rohena, Angel; Medvec, Andrew; Plesa, Gabriela; Riley, James L.; Bates, Paul

2010-01-01

Many viruses alter expression of proteins on the surface of infected cells including molecules important for immune recognition, such as the major histocompatibility complex (MHC) class I and II molecules. Virus-induced downregulation of surface proteins has been observed to occur by a variety of mechanisms including impaired transcription, blocks to synthesis, and increased turnover. Viral infection or transient expression of the Ebola virus (EBOV) glycoprotein (GP) was previously shown to result in loss of staining of various host cell surface proteins including MHC1 and β1 integrin; however, the mechanism responsible for this effect has not been delineated. In the present study we demonstrate that EBOV GP does not decrease surface levels of β1 integrin or MHC1, but rather impedes recognition by steric occlusion of these proteins on the cell surface. Furthermore, steric occlusion also occurs for epitopes on the EBOV glycoprotein itself. The occluded epitopes in host proteins and EBOV GP can be revealed by removal of the surface subunit of GP or by removal of surface N- and O- linked glycans, resulting in increased surface staining by flow cytometry. Importantly, expression of EBOV GP impairs CD8 T-cell recognition of MHC1 on antigen presenting cells. Glycan-mediated steric shielding of host cell surface proteins by EBOV GP represents a novel mechanism for a virus to affect host cell function, thereby escaping immune detection. PMID:20844579

Transfer of Fas (CD95) protein from the cell surface to the surface of polystyrene beads coated with anti-Fas antibody clone CH-11

PubMed Central

Sawai, H.; Domae, N.

2010-01-01

Mouse monoclonal anti-Fas (CD95) antibody clone CH-11 has been widely used in research on apoptosis. CH-11 has the ability to bind to Fas protein on cell surface and induce apoptosis. Here, we used polystyrene beads coated with CH-11 to investigate the role of lipid rafts in Fas-mediated apoptosis in SKW6.4 cells. Unexpectedly, by treatment of the cells with CH-11-coated beads Fas protein was detached from cell surface and transferred to the surface of CH-11-coated beads. Western blot analysis showed that Fas protein containing both extracellular and intracellular domains was attached to the beads. Fas protein was not transferred from the cells to the surface of the beads coated with other anti-Fas antibodies or Fas ligand. Similar phenomenon was observed in Jurkat T cells. Furthermore, CH-11-induced apoptosis was suppressed by pretreatment with CH-11-coated beads in Jurkat cells. These results suggest that CH-11 might possess distinct properties on Fas protein compared with other anti-Fas antibodies or Fas ligand, and also suggest that caution should be needed to use polystyrene beads coated with antibodies such as CH-11. PMID:20353915

Candida albicans Shaving to Profile Human Serum Proteins on Hyphal Surface

PubMed Central

Marín, Elvira; Parra-Giraldo, Claudia M.; Hernández-Haro, Carolina; Hernáez, María L.; Nombela, César; Monteoliva, Lucía; Gil, Concha

2015-01-01

Candida albicans is a human opportunistic fungus and it is responsible for a wide variety of infections, either superficial or systemic. C. albicans is a polymorphic fungus and its ability to switch between yeast and hyphae is essential for its virulence. Once C. albicans obtains access to the human body, the host serum constitutes a complex environment of interaction with C. albicans cell surface in bloodstream. To draw a comprehensive picture of this relevant step in host-pathogen interaction during invasive candidiasis, we have optimized a gel-free shaving proteomic strategy to identify both, human serum proteins coating C. albicans cells and fungi surface proteins simultaneously. This approach was carried out with normal serum (NS) and heat inactivated serum (HIS). We identified 214 human and 372 C. albicans unique proteins. Proteins identified in C. albicans included 147 which were described as located at the cell surface and 52 that were described as immunogenic. Interestingly, among these C. albicans proteins, we identified 23 GPI-anchored proteins, Gpd2 and Pra1, which are involved in complement system evasion and 7 other proteins that are able to attach plasminogen to C. albicans surface (Adh1, Eno1, Fba1, Pgk1, Tdh3, Tef1, and Tsa1). Furthermore, 12 proteins identified at the C. albicans hyphae surface induced with 10% human serum were not detected in other hypha-induced conditions. The most abundant human proteins identified are involved in complement and coagulation pathways. Remarkably, with this strategy, all main proteins belonging to complement cascades were identified on the C. albicans surface. Moreover, we identified immunoglobulins, cytoskeletal proteins, metabolic proteins such as apolipoproteins and others. Additionally, we identified more inhibitors of complement and coagulation pathways, some of them serpin proteins (serine protease inhibitors), in HIS vs. NS. On the other hand, we detected a higher amount of C3 at the C. albicans surface in NS than in HIS, as validated by immunofluorescence. PMID:26696967

A multichannel nanosensor for instantaneous readout of cancer drug mechanisms

NASA Astrophysics Data System (ADS)

Rana, Subinoy; Le, Ngoc D. B.; Mout, Rubul; Saha, Krishnendu; Tonga, Gulen Yesilbag; Bain, Robert E. S.; Miranda, Oscar R.; Rotello, Caren M.; Rotello, Vincent M.

2015-01-01

Screening methods that use traditional genomic, transcriptional, proteomic and metabonomic signatures to characterize drug mechanisms are known. However, they are time consuming and require specialized equipment. Here, we present a high-throughput multichannel sensor platform that can profile the mechanisms of various chemotherapeutic drugs in minutes. The sensor consists of a gold nanoparticle complexed with three different fluorescent proteins that can sense drug-induced physicochemical changes on cell surfaces. In the presence of cells, fluorescent proteins are rapidly displaced from the gold nanoparticle surface and fluorescence is restored. Fluorescence ‘turn on’ of the fluorescent proteins depends on the drug-induced cell surface changes, generating patterns that identify specific mechanisms of cell death induced by drugs. The nanosensor is generalizable to different cell types and does not require processing steps before analysis, offering an effective way to expedite research in drug discovery, toxicology and cell-based sensing.

Mechanisms of protein stabilization and prevention of protein aggregation by glycerol.

PubMed

Vagenende, Vincent; Yap, Miranda G S; Trout, Bernhardt L

2009-11-24

The stability of proteins in aqueous solution is routinely enhanced by cosolvents such as glycerol. Glycerol is known to shift the native protein ensemble to more compact states. Glycerol also inhibits protein aggregation during the refolding of many proteins. However, mechanistic insight into protein stabilization and prevention of protein aggregation by glycerol is still lacking. In this study, we derive mechanisms of glycerol-induced protein stabilization by combining the thermodynamic framework of preferential interactions with molecular-level insight into solvent-protein interactions gained from molecular simulations. Contrary to the common conception that preferential hydration of proteins in polyol/water mixtures is determined by the molecular size of the polyol and the surface area of the protein, we present evidence that preferential hydration of proteins in glycerol/water mixtures mainly originates from electrostatic interactions that induce orientations of glycerol molecules at the protein surface such that glycerol is further excluded. These interactions shift the native protein toward more compact conformations. Moreover, glycerol preferentially interacts with large patches of contiguous hydrophobicity where glycerol acts as an amphiphilic interface between the hydrophobic surface and the polar solvent. Accordingly, we propose that glycerol prevents protein aggregation by inhibiting protein unfolding and by stabilizing aggregation-prone intermediates through preferential interactions with hydrophobic surface regions that favor amphiphilic interface orientations of glycerol. These mechanisms agree well with experimental data available in the literature, and we discuss the extent to which these mechanisms apply to other cosolvents, including polyols, arginine, and urea.

Photonic activation of disulfide bridges achieves oriented protein immobilization on biosensor surfaces.

PubMed

Neves-Petersen, Maria Teresa; Snabe, Torben; Klitgaard, Søren; Duroux, Meg; Petersen, Steffen B

2006-02-01

Photonic induced immobilization is a novel technology that results in spatially oriented and spatially localized covalent coupling of biomolecules onto thiol-reactive surfaces. Immobilization using this technology has been achieved for a wide selection of proteins, such as hydrolytic enzymes (lipases/esterases, lysozyme), proteases (human plasminogen), alkaline phosphatase, immunoglobulins' Fab fragment (e.g., antibody against PSA [prostate specific antigen]), Major Histocompability Complex class I protein, pepsin, and trypsin. The reaction mechanism behind the reported new technology involves "photonic activation of disulfide bridges," i.e., light-induced breakage of disulfide bridges in proteins upon UV illumination of nearby aromatic amino acids, resulting in the formation of free, reactive thiol groups that will form covalent bonds with thiol-reactive surfaces (see Fig. 1). Interestingly, the spatial proximity of aromatic residues and disulfide bridges in proteins has been preserved throughout molecular evolution. The new photonic-induced method for immobilization of proteins preserves the native structural and functional properties of the immobilized protein, avoiding the use of one or more chemical/thermal steps. This technology allows for the creation of spatially oriented as well as spatially defined multiprotein/DNA high-density sensor arrays with spot size of 1 microm or less, and has clear potential for biomedical, bioelectronic, nanotechnology, and therapeutic applications.

Localization and Ordering of Lipids Around Aquaporin-0: Protein and Lipid Mobility Effects.

PubMed

Briones, Rodolfo; Aponte-Santamaría, Camilo; de Groot, Bert L

2017-01-01

Hydrophobic matching, lipid sorting, and protein oligomerization are key principles by which lipids and proteins organize in biological membranes. The Aquaporin-0 channel (AQP0), solved by electron crystallography (EC) at cryogenic temperatures, is one of the few protein-lipid complexes of which the structure is available in atomic detail. EC and room-temperature molecular dynamics (MD) of dimyristoylglycerophosphocholine (DMPC) annular lipids around AQP0 show similarities, however, crystal-packing and temperature might affect the protein surface or the lipids distribution. To understand the role of temperature, lipid phase, and protein mobility in the localization and ordering of AQP0-lipids, we used MD simulations of an AQP0-DMPC bilayer system. Simulations were performed at physiological and at DMPC gel-phase temperatures. To decouple the protein and lipid mobility effects, we induced gel-phase in the lipids or restrained the protein. We monitored the lipid ordering effects around the protein. Reducing the system temperature or inducing lipid gel-phase had a marginal effect on the annular lipid localization. However, restraining the protein mobility increased the annular lipid localization around the whole AQP0 surface, resembling EC. The distribution of the inter-phosphate and hydrophobic thicknesses showed that stretching of the DMPC annular layer around AQP0 surface is the mechanism that compensates the hydrophobic mismatch in this system. The distribution of the local area-per-lipid and the acyl-chain order parameters showed particular fluid- and gel-like areas that involved several lipid layers. These areas were in contact with the surfaces of higher and lower protein mobility, respectively. We conclude that the AQP0 surfaces induce specific fluid- and gel-phase prone areas. The presence of these areas might guide the AQP0 lipid sorting interactions with other membrane components, and is compatible with the squared array oligomerization of AQP0 tetramers separated by a layer of annular lipids.

Modulation of protein stability and aggregation properties by surface charge engineering.

PubMed

Raghunathan, Govindan; Sokalingam, Sriram; Soundrarajan, Nagasundarapandian; Madan, Bharat; Munussami, Ganapathiraman; Lee, Sun-Gu

2013-09-01

An attempt to alter protein surface charges through traditional protein engineering approaches often affects the native protein structure significantly and induces misfolding. This limitation is a major hindrance in modulating protein properties through surface charge variations. In this study, as a strategy to overcome such a limitation, we attempted to co-introduce stabilizing mutations that can neutralize the destabilizing effect of protein surface charge variation. Two sets of rational mutations were designed; one to increase the number of surface charged amino acids and the other to decrease the number of surface charged amino acids by mutating surface polar uncharged amino acids and charged amino acids, respectively. These two sets of mutations were introduced into Green Fluorescent Protein (GFP) together with or without stabilizing mutations. The co-introduction of stabilizing mutations along with mutations for surface charge modification allowed us to obtain functionally active protein variants (s-GFP(+15-17) and s-GFP(+5-6)). When the protein properties such as fluorescent activity, folding rate and kinetic stability were assessed, we found the possibility that the protein stability can be modulated independently of activity and folding by engineering protein surface charges. The aggregation properties of GFP could also be altered through the surface charge engineering.

Effect of hypoxia on lung gene expression and proteomic profile: insights into the pulmonary surfactant response

PubMed Central

Olmeda, Bárbara; Umstead, Todd M.; Silveyra, Patricia; Pascual, Alberto; López-Barneo, José; Phelps, David S.; Floros, Joanna; Pérez-Gil, Jesús

2014-01-01

Exposure of lung to hypoxia has been previously reported to be associated with significant alterations in the protein content of bronchoalveolar lavage (BAL) and lung tissue. In the present work we have used a proteomic approach to describe the changes in protein complement induced by moderate long-term hypoxia (rats exposed to 10% O2 for 72 hours) in BAL and lung tissue, with a special focus on the proteins associated with pulmonary surfactant, which could indicate adaptation of this system to limited oxygen availability. The analysis of the general proteomic profile indicates a hypoxia-induced increase in proteins associated with inflammation both in lavage and lung tissue. Analysis at mRNA and protein levels revealed no significant changes induced by hypoxia on the content in surfactant proteins or their apparent oligomeric state. In contrast, we detected a hypoxia-induced significant increase in the expression and accumulation of hemoglobin in lung tissue, at both mRNA and protein levels, as well as an accumulation of hemoglobin both in BAL and associated with surface-active membranes of the pulmonary surfactant complex. Evaluation of pulmonary surfactant surface activity from hypoxic rats showed no alterations in its spreading ability, ruling out inhibition by increased levels of serum or inflammatory proteins. PMID:24576641

Effect of homogenisation in formation of thermally induced aggregates in a non- and low- fat milk model system with microparticulated whey proteins.

PubMed

Torres, Isabel Celigueta; Nieto, Gema; Nylander, Tommy; Simonsen, Adam Cohen; Tolkach, Alexander; Ipsen, Richard

2017-05-01

The objective of the research presented in this paper was to investigate how different characteristics of whey protein microparticles (MWP) added to milk as fat replacers influence intermolecular interactions occurring with other milk proteins during homogenisation and heating. These interactions are responsible for the formation of heat-induced aggregates that influence the texture and sensory characteristics of the final product. The formation of heat-induced complexes was studied in non- and low-fat milk model systems, where microparticulated whey protein (MWP) was used as fat replacer. Five MWP types with different particle characteristics were utilised and three heat treatments used: 85 °C for 15 min, 90 °C for 5 min and 95 °C for 2 min. Surface characteristics of the protein aggregates were expressed as the number of available thiol groups and the surface net charge. Intermolecular interactions involved in the formation of protein aggregates were studied by polyacrylamide gel electrophoresis and the final complexes visualised by darkfield microscopy. Homogenisation of non-fat milk systems led to partial adsorption of caseins onto microparticles, independently of the type of microparticle. On the contrary, homogenisation of low-fat milk resulted in preferential adsorption of caseins onto fat globules, rather than onto microparticles. Further heating of the milk, led to the formation of heat induced complexes with different sizes and characteristics depending on the type of MWP and the presence or not of fat. The results highlight the importance of controlling homogenisation and heat processing in yoghurt manufacture in order to induce desired changes in the surface reactivity of the microparticles and thereby promote effective protein interactions.

Three steps to gold: mechanism of protein adsorption revealed by Brownian and molecular dynamics simulations.

PubMed

Ozboyaci, M; Kokh, D B; Wade, R C

2016-04-21

The addition of three N-terminal histidines to β-lactamase inhibitor protein was shown experimentally to increase its binding potency to an Au(111) surface substantially but the binding mechanism was not resolved. Here, we propose a complete adsorption mechanism for this fusion protein by means of a multi-scale simulation approach and free energy calculations. We find that adsorption is a three-step process: (i) recognition of the surface predominantly by the histidine fusion peptide and formation of an encounter complex facilitated by a reduced dielectric screening of water in the interfacial region, (ii) adsorption of the protein on the surface and adoption of a specific binding orientation, and (iii) adaptation of the protein structure on the metal surface accompanied by induced fit. We anticipate that the mechanistic features of protein adsorption to an Au(111) surface revealed here can be extended to other inorganic surfaces and proteins and will therefore aid the design of specific protein-surface interactions.

Nanoparticle-induced oxidation of corona proteins initiates an oxidative stress response in cells†

PubMed Central

Jayaram, Dhanya T.; Runa, Sabiha; Kemp, Melissa L.

2017-01-01

Titanium dioxide nanoparticles (TiO2 NPs), used as pigments and photocatalysts, are ubiquitous in our daily lives. Previous work has observed cellular oxidative stress in response to the UV-excitation of photocatalytic TiO2 NPs. In comparison, most human exposure to TiO2 NPs takes place in the dark, in the lung following inhalation or in the gut following consumption of TiO2 NP food pigment. Our spectroscopic characterization shows that both photocatalytic and food grade TiO2 NPs, in the dark, generate low levels of reactive oxygen species (ROS), specifically hydroxyl radicals and superoxides. These ROS oxidize serum proteins that form a corona of proteins on the NP surface. This protein layer is the interface between the NP and the cell. An oxidized protein corona triggers an oxidative stress response, detected with PCR and western blotting. Surface modification of TiO2 NPs to increase or decrease surface defects correlates with ROS generation and oxidative stress, suggesting that NP surface defects, likely oxygen vacancies, are the underlying cause of TiO2 NP-induced oxidative stress. PMID:28537609

Plasmonic Thermal Decomposition/Digestion of Proteins: A Rapid On-Surface Protein Digestion Technique for Mass Spectrometry Imaging.

PubMed

Zhou, Rong; Basile, Franco

2017-09-05

A method based on plasmon surface resonance absorption and heating was developed to perform a rapid on-surface protein thermal decomposition and digestion suitable for imaging mass spectrometry (MS) and/or profiling. This photothermal process or plasmonic thermal decomposition/digestion (plasmonic-TDD) method incorporates a continuous wave (CW) laser excitation and gold nanoparticles (Au-NPs) to induce known thermal decomposition reactions that cleave peptides and proteins specifically at the C-terminus of aspartic acid and at the N-terminus of cysteine. These thermal decomposition reactions are induced by heating a solid protein sample to temperatures between 200 and 270 °C for a short period of time (10-50 s per 200 μm segment) and are reagentless and solventless, and thus are devoid of sample product delocalization. In the plasmonic-TDD setup the sample is coated with Au-NPs and irradiated with 532 nm laser radiation to induce thermoplasmonic heating and bring about site-specific thermal decomposition on solid peptide/protein samples. In this manner the Au-NPs act as nanoheaters that result in a highly localized thermal decomposition and digestion of the protein sample that is independent of the absorption properties of the protein, making the method universally applicable to all types of proteinaceous samples (e.g., tissues or protein arrays). Several experimental variables were optimized to maximize product yield, and they include heating time, laser intensity, size of Au-NPs, and surface coverage of Au-NPs. Using optimized parameters, proof-of-principle experiments confirmed the ability of the plasmonic-TDD method to induce both C-cleavage and D-cleavage on several peptide standards and the protein lysozyme by detecting their thermal decomposition products with matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). The high spatial specificity of the plasmonic-TDD method was demonstrated by using a mask to digest designated sections of the sample surface with the heating laser and MALDI-MS imaging to map the resulting products. The solventless nature of the plasmonic-TDD method enabled the nonenzymatic on-surface digestion of proteins to proceed with undetectable delocalization of the resulting products from their precursor protein location. The advantages of this novel plasmonic-TDD method include short reaction times (<30 s/200 μm), compatibility with MALDI, universal sample compatibility, high spatial specificity, and localization of the digestion products. These advantages point to potential applications of this method for on-tissue protein digestion and MS-imaging/profiling for the identification of proteins, high-fidelity MS imaging of high molecular weight (>30 kDa) proteins, and the rapid analysis of formalin-fixed paraffin-embedded (FFPE) tissue samples.

Preparation of protein- and cell-resistant surfaces by hyperthermal hydrogen induced cross-linking of poly(ethylene oxide).

PubMed

Bonduelle, Colin V; Lau, Woon M; Gillies, Elizabeth R

2011-05-01

The functionalization of surfaces with poly(ethylene oxide) (PEO) is an effective means of imparting resistance to the adsorption of proteins and the attachment and growth of cells, properties that are critical for many biomedical applications. In this work, a new hyperthermal hydrogen induced cross-linking (HHIC) method was explored as a simple one-step approach for attaching PEO to surfaces through the selective cleavage of C-H bonds and subsequent cross-linking of the resulting carbon radicals. In order to study the effects of the process on the polymer, PEO-coated silicon wafers were prepared and the effects of different treatment times were investigated. Subsequently, using an optimized treatment time and a modified butyl polymer with increased affinity for PEO, the technique was applied to butyl rubber surfaces. All of the treated surfaces exhibited significantly reduced protein adsorption and cell growth relative to control surfaces and compared favorably with surfaces that were functionalized with PEO using conventional chemical methods. Thus HHIC is a simple and effective means of attaching PEO to non-functional polymer surfaces.

Membrane translocation of t-SNARE protein syntaxin-4 abrogates ground-state pluripotency in mouse embryonic stem cells

PubMed Central

Hagiwara-Chatani, Natsumi; Shirai, Kota; Kido, Takumi; Horigome, Tomoatsu; Yasue, Akihiro; Adachi, Naoki; Hirai, Yohei

2017-01-01

Embryonic stem (ES) and induced pluripotent stem (iPS) cells are attractive tools for regenerative medicine therapies. However, aberrant cell populations that display flattened morphology and lose ground-state pluripotency often appear spontaneously, unless glycogen synthase kinase 3β (GSK3β) and mitogen-activated protein kinase kinase (MEK1/2) are inactivated. Here, we show that membrane translocation of the t-SNARE protein syntaxin-4 possibly is involved in this phenomenon. We found that mouse ES cells cultured without GSK3β/MEK1/2 inhibitors (2i) spontaneously extrude syntaxin-4 at the cell surface and that artificial expression of cell surface syntaxin-4 induces appreciable morphological changes and mesodermal differentiation through dephosphorylation of Akt. Transcriptome analyses revealed several candidate elements responsible for this, specifically, an E-to P-cadherin switch and a marked downregulation of Zscan4 proteins, which are DNA-binding proteins essential for ES cell pluripotency. Embryonic carcinoma cell lines F9 and P19CL6, which maintain undifferentiated states independently of Zscan4 proteins, exhibited similar cellular behaviors upon stimulation with cell surface syntaxin-4. The functional ablation of E-cadherin and overexpression of P-cadherin reproduced syntaxin-4-induced cell morphology, demonstrating that the E- to P-cadherin switch executes morphological signals from cell surface syntaxin-4. Thus, spontaneous membrane translocation of syntaxin-4 emerged as a critical element for maintenance of the stem-cell niche. PMID:28057922

Insulin adsorption on crystalline SiO2: Comparison between polar and nonpolar surfaces using accelerated molecular-dynamics simulations

NASA Astrophysics Data System (ADS)

Nejad, Marjan A.; Mücksch, Christian; Urbassek, Herbert M.

2017-02-01

Adsorption of insulin on polar and nonpolar surfaces of crystalline SiO2 (cristobalite and α -quartz) is studied using molecular dynamics simulation. Acceleration techniques are used in order to sample adsorption phase space efficiently and to identify realistic adsorption conformations. We find major differences between the polar and nonpolar surfaces. Electrostatic interactions govern the adsorption on polar surfaces and can be described by the alignment of the protein dipole with the surface dipole; hence spreading of the protein on the surface is irrelevant. On nonpolar surfaces, on the other hand, van-der-Waals interaction dominates, inducing surface spreading of the protein.

Understanding the interfacial behavior of lysozyme on Au (111) surfaces with multiscale simulations

NASA Astrophysics Data System (ADS)

Samieegohar, Mohammadreza; Ma, Heng; Sha, Feng; Jahan Sajib, Md Symon; Guerrero-García, G. Iván; Wei, Tao

2017-02-01

The understanding of the adsorption and interfacial behavior of proteins is crucial to the development of novel biosensors and biomaterials. By using bottom-up atomistic multiscale simulations, we study here the adsorption of lysozyme on Au(111) surfaces in an aqueous environment. Atomistic simulations are used to calculate the inhomogeneous polarization of the gold surface, which is induced by the protein adsorption, and by the presence of an interfacial layer of water molecules and monovalent salts. The corresponding potential of mean force between the protein and the gold surface including polarization effects is used in Langevin Dynamics simulations to study the time dependent behavior of proteins at finite concentration. These simulations display a rapid adsorption and formation of a first-layer of proteins at the interface. Proteins are initially adsorbed directly on the gold surface due to the strong protein-surface attractive interaction. A subsequent interfacial weak aggregation of proteins leading to multilayer build-up is also observed at long times.

Quantum mechanical/molecular mechanical/continuum style solvation model: second order Møller-Plesset perturbation theory.

PubMed

Thellamurege, Nandun M; Si, Dejun; Cui, Fengchao; Li, Hui

2014-05-07

A combined quantum mechanical/molecular mechanical/continuum (QM/MM/C) style second order Møller-Plesset perturbation theory (MP2) method that incorporates induced dipole polarizable force field and induced surface charge continuum solvation model is established. The Z-vector method is modified to include induced dipoles and induced surface charges to determine the MP2 response density matrix, which can be used to evaluate MP2 properties. In particular, analytic nuclear gradient is derived and implemented for this method. Using the Assisted Model Building with Energy Refinement induced dipole polarizable protein force field, the QM/MM/C style MP2 method is used to study the hydrogen bonding distances and strengths of the photoactive yellow protein chromopore in the wild type and the Glu46Gln mutant.

PERP, a host tetraspanning membrane protein, is required for S almonella‐induced inflammation

PubMed Central

Hallstrom, Kelly N.; Srikanth, C. V.; Agbor, Terence A.; Dumont, Christopher M.; Peters, Kristen N.; Paraoan, Luminita; Casanova, James E.; Boll, Erik J.

2015-01-01

Summary S almonella enterica  Typhimurium induces intestinal inflammation through the activity of type III secreted effector (T3SE) proteins. Our prior results indicate that the secretion of the T3SE SipA and the ability of SipA to induce epithelial cell responses that lead to induction of polymorphonuclear transepithelial migration are not coupled to its direct delivery into epithelial cells from S almonella. We therefore tested the hypothesis that SipA interacts with a membrane protein located at the apical surface of intestinal epithelial cells. Employing a split ubiquitin yeast‐two‐hybrid screen, we identified the tetraspanning membrane protein, p53 effector related to PMP‐22 (PERP), as a SipA binding partner. SipA and PERP appear to have intersecting activities as we found PERP to be involved in proinflammatory pathways shown to be regulated by SipA. In sum, our studies reveal a critical role for PERP in the pathogenesis of S. Typhimurium, and for the first time demonstrate that SipA, a T3SE protein, can engage a host protein at the epithelial surface. PMID:25486861

Activation of cell-surface proteases promotes necroptosis, inflammation and cell migration.

PubMed

Cai, Zhenyu; Zhang, Anling; Choksi, Swati; Li, Weihua; Li, Tao; Zhang, Xue-Min; Liu, Zheng-Gang

2016-08-01

Necroptosis is a programmed, caspase-independent cell death that is morphologically similar to necrosis. TNF-induced necroptosis is mediated by receptor-interacting protein kinases, RIP1 and RIP3, and the mixed lineage kinase domain-like (MLKL). After being phosphorylated by RIP3, MLKL is translocated to the plasma membrane and mediates necroptosis. However, the execution of necroptosis and its role in inflammation and other cellular responses remain largely elusive. In this study, we report that MLKL-mediated activation of cell-surface proteases of the a disintegrin and metalloprotease (ADAM) family promotes necroptosis, inflammation and cell migration. ADAMs are specifically activated at the early stage of necroptosis when MLKL is phosphorylated and translocated to the cell plasma membrane. Activation of ADAMs induces ectodomain shedding of diverse cell-surface proteins including adhesion molecules, receptors, growth factors and cytokines. Importantly, the shedding of cell-surface proteins disrupts cell adhesion and accelerates necroptosis, while the soluble fragments of the cleaved proteins trigger the inflammatory responses. We also demonstrate that the shedding of E-cadherin ectodomain from necroptotic cells promotes cell migration. Thus, our study provides a novel mechanism of necroptosis-induced inflammation and new insights into the physiological and pathological functions of this unique form of cell death.

Activation of cell-surface proteases promotes necroptosis, inflammation and cell migration

PubMed Central

Cai, Zhenyu; Zhang, Anling; Choksi, Swati; Li, Weihua; Li, Tao; Zhang, Xue-Min; Liu, Zheng-Gang

2016-01-01

Necroptosis is a programmed, caspase-independent cell death that is morphologically similar to necrosis. TNF-induced necroptosis is mediated by receptor-interacting protein kinases, RIP1 and RIP3, and the mixed lineage kinase domain-like (MLKL). After being phosphorylated by RIP3, MLKL is translocated to the plasma membrane and mediates necroptosis. However, the execution of necroptosis and its role in inflammation and other cellular responses remain largely elusive. In this study, we report that MLKL-mediated activation of cell-surface proteases of the a disintegrin and metalloprotease (ADAM) family promotes necroptosis, inflammation and cell migration. ADAMs are specifically activated at the early stage of necroptosis when MLKL is phosphorylated and translocated to the cell plasma membrane. Activation of ADAMs induces ectodomain shedding of diverse cell-surface proteins including adhesion molecules, receptors, growth factors and cytokines. Importantly, the shedding of cell-surface proteins disrupts cell adhesion and accelerates necroptosis, while the soluble fragments of the cleaved proteins trigger the inflammatory responses. We also demonstrate that the shedding of E-cadherin ectodomain from necroptotic cells promotes cell migration. Thus, our study provides a novel mechanism of necroptosis-induced inflammation and new insights into the physiological and pathological functions of this unique form of cell death. PMID:27444869

Upregulation of BMSCs Osteogenesis by Positively-Charged Tertiary Amines on Polymeric Implants via Charge/iNOS Signaling Pathway

PubMed Central

Zhang, Wei; Liu, Na; Shi, Haigang; Liu, Jun; Shi, Lianxin; Zhang, Bo; Wang, Huaiyu; Ji, Junhui; Chu, Paul K.

2015-01-01

Positively-charged surfaces on implants have a similar potential to upregulate osteogenesis of bone marrow-derived mesenchymal stem cells (BMSCs) as electromagnetic therapy approved for bone regeneration. Generally, their osteogenesis functions are generally considered to stem from the charge-induced adhesion of extracellular matrix (ECM) proteins without exploring the underlying surface charge/cell signaling molecule pathways. Herein, a positively-charged surface with controllable tertiary amines is produced on a polymer implant by plasma surface modification. In addition to inhibiting the TNF-α expression, the positively-charged surface with tertiary amines exhibits excellent cytocompatibility as well as remarkably upregulated osteogenesis-related gene/protein expressions and calcification of the contacted BMSCs. Stimulated by the charged surface, these BMSCs display high iNOS expressions among the three NOS isoforms. Meanwhile, downregulation of the iNOS by L-Can or siRNA inhibit osteogenic differentiation in the BMSCs. These findings suggest that a positively-charged surface with tertiary amines induces osteogenesis of BMSCs via the surface charge/iNOS signaling pathway in addition to elevated ECM protein adhesion. Therefore, creating a positively-charged surface with tertiary amines is a promising approach to promote osseointegration with bone tissues. PMID:25791957

Quantitative proteomic view on secreted, cell surface-associated, and cytoplasmic proteins of the methicillin-resistant human pathogen Staphylococcus aureus under iron-limited conditions.

PubMed

Hempel, Kristina; Herbst, Florian-Alexander; Moche, Martin; Hecker, Michael; Becher, Dörte

2011-04-01

Staphylococcus aureus is capable of colonizing and infecting humans by its arsenal of surface-exposed and secreted proteins. Iron-limited conditions in mammalian body fluids serve as a major environmental signal to bacteria to express virulence determinants. Here we present a comprehensive, gel-free, and GeLC-MS/MS-based quantitative proteome profiling of S. aureus under this infection-relevant situation. (14)N(15)N metabolic labeling and three complementing approaches were combined for relative quantitative analyses of surface-associated proteins. The surface-exposed and secreted proteome profiling approaches comprise trypsin shaving, biotinylation, and precipitation of the supernatant. By analysis of the outer subproteomic and cytoplasmic protein fraction, 1210 proteins could be identified including 221 surface-associated proteins. Thus, access was enabled to 70% of the predicted cell wall-associated proteins, 80% of the predicted sortase substrates, two/thirds of lipoproteins and more than 50% of secreted and cytoplasmic proteins. For iron-deficiency, 158 surface-associated proteins were quantified. Twenty-nine proteins were found in altered amounts showing particularly surface-exposed proteins strongly induced, such as the iron-regulated surface determinant proteins IsdA, IsdB, IsdC and IsdD as well as lipid-anchored iron compound-binding proteins. The work presents a crucial subject for understanding S. aureus pathophysiology by the use of methods that allow quantitative surface proteome profiling.

What induces pocket openings on protein surface patches involved in protein-protein interactions?

PubMed

Eyrisch, Susanne; Helms, Volkhard

2009-02-01

We previously showed for the proteins BCL-X(L), IL-2, and MDM2 that transient pockets at their protein-protein binding interfaces can be identified by applying the PASS algorithm to molecular dynamics (MD) snapshots. We now investigated which aspects of the natural conformational dynamics of proteins induce the formation of such pockets. The pocket detection protocol was applied to three different conformational ensembles for the same proteins that were extracted from MD simulations of the inhibitor bound crystal conformation in water and the free crystal/NMR structure in water and in methanol. Additional MD simulations studied the impact of backbone mobility. The more efficient CONCOORD or normal mode analysis (NMA) techniques gave significantly smaller pockets than MD simulations, whereas tCONCOORD generated pockets comparable to those observed in MD simulations for two of the three systems. Our findings emphasize the influence of solvent polarity and backbone rearrangements on the formation of pockets on protein surfaces and should be helpful in future generation of transient pockets as putative ligand binding sites at protein-protein interfaces.

What induces pocket openings on protein surface patches involved in protein-protein interactions?

NASA Astrophysics Data System (ADS)

Eyrisch, Susanne; Helms, Volkhard

2009-02-01

We previously showed for the proteins BCL-XL, IL-2, and MDM2 that transient pockets at their protein-protein binding interfaces can be identified by applying the PASS algorithm to molecular dynamics (MD) snapshots. We now investigated which aspects of the natural conformational dynamics of proteins induce the formation of such pockets. The pocket detection protocol was applied to three different conformational ensembles for the same proteins that were extracted from MD simulations of the inhibitor bound crystal conformation in water and the free crystal/NMR structure in water and in methanol. Additional MD simulations studied the impact of backbone mobility. The more efficient CONCOORD or normal mode analysis (NMA) techniques gave significantly smaller pockets than MD simulations, whereas tCONCOORD generated pockets comparable to those observed in MD simulations for two of the three systems. Our findings emphasize the influence of solvent polarity and backbone rearrangements on the formation of pockets on protein surfaces and should be helpful in future generation of transient pockets as putative ligand binding sites at protein-protein interfaces.

Differential protein expression in tears of patients with primary open angle and pseudoexfoliative glaucoma.

PubMed

Pieragostino, Damiana; Bucci, Sonia; Agnifili, Luca; Fasanella, Vincenzo; D'Aguanno, Simona; Mastropasqua, Alessandra; Ciancaglini, Marco; Mastropasqua, Leonardo; Di Ilio, Carmine; Sacchetta, Paolo; Urbani, Andrea; Del Boccio, Piero

2012-04-01

Primary open angle (POAG) and pseudoexfoliative glaucoma (PXG) are the most common primary and secondary forms of glaucoma, respectively. Even though the patho-physiology, aqueous humor composition, risk factors, clinical features, therapy and drug induced ocular surface changes in POAG and PXG have been widely studied, to date information concerning tear protein characterization is lacking. Tears are a source of nourishment for ocular surface tissues and a vehicle to remove local waste products, metabolized drugs and inflammatory mediators produced in several ophthalmic diseases. In glaucoma, the proteomic definition of tears may provide insights concerning patho-physiology of the disease and ocular surface modifications induced by topical therapy. Our study aimed at characterizing protein patterns in tears of patients with medically controlled POAG and PXG. A comparative tears proteomic analysis by label-free LC-MS(E) highlighted differences in the expression of several proteins in the two glaucoma sub-types and control subjects, highlighting inflammation pathways expressed in both diseases. Results were independently reconfirmed by SDS-PAGE and linear MALDI-TOF MS, validating altered levels of Lysozyme C, Lipocalin-1, Protein S100, Immunoglobulins and Prolactin Inducible Protein. Moreover, we found a differential pattern of phosphorylated Cystatin-S that distinguishes the two pathologies. The most relevant results suggest that in both pathologies there may be active inflammation pathways related to the disease and/or induced by therapy. We show, for the first time, tear protein patterns expressed under controlled intraocular pressure conditions in POAG and PXG subjects. These findings could help in the understanding of molecular machinery underlying these ophthalmologic diseases, resulting in early diagnosis and more specific therapy.

Quantum mechanical/molecular mechanical/continuum style solvation model: Second order Møller-Plesset perturbation theory

DOE Office of Scientific and Technical Information (OSTI.GOV)

Thellamurege, Nandun M.; Si, Dejun; Cui, Fengchao

A combined quantum mechanical/molecular mechanical/continuum (QM/MM/C) style second order Møller-Plesset perturbation theory (MP2) method that incorporates induced dipole polarizable force field and induced surface charge continuum solvation model is established. The Z-vector method is modified to include induced dipoles and induced surface charges to determine the MP2 response density matrix, which can be used to evaluate MP2 properties. In particular, analytic nuclear gradient is derived and implemented for this method. Using the Assisted Model Building with Energy Refinement induced dipole polarizable protein force field, the QM/MM/C style MP2 method is used to study the hydrogen bonding distances and strengths ofmore » the photoactive yellow protein chromopore in the wild type and the Glu46Gln mutant.« less

Domain Formation Induced by the Adsorption of Charged Proteins on Mixed Lipid Membranes

PubMed Central

Mbamala, Emmanuel C.; Ben-Shaul, Avinoam; May, Sylvio

2005-01-01

Peripheral proteins can trigger the formation of domains in mixed fluid-like lipid membranes. We analyze the mechanism underlying this process for proteins that bind electrostatically onto a flat two-component membrane, composed of charged and neutral lipid species. Of particular interest are membranes in which the hydrocarbon lipid tails tend to segregate owing to nonideal chain mixing, but the (protein-free) lipid membrane is nevertheless stable due to the electrostatic repulsion between the charged lipid headgroups. The adsorption of charged, say basic, proteins onto a membrane containing anionic lipids induces local lipid demixing, whereby charged lipids migrate toward (or away from) the adsorption site, so as to minimize the electrostatic binding free energy. Apart from reducing lipid headgroup repulsion, this process creates a gradient in lipid composition around the adsorption zone, and hence a line energy whose magnitude depends on the protein's size and charge and the extent of lipid chain nonideality. Above a certain critical lipid nonideality, the line energy is large enough to induce domain formation, i.e., protein aggregation and, concomitantly, macroscopic lipid phase separation. We quantitatively analyze the thermodynamic stability of the dressed membrane based on nonlinear Poisson-Boltzmann theory, accounting for both the microscopic characteristics of the proteins and lipid composition modulations at and around the adsorption zone. Spinodal surfaces and critical points of the dressed membranes are calculated for several different model proteins of spherical and disk-like shapes. Among the models studied we find the most substantial protein-induced membrane destabilization for disk-like proteins whose charges are concentrated in the membrane-facing surface. If additional charges reside on the side faces of the proteins, direct protein-protein repulsion diminishes considerably the propensity for domain formation. Generally, a highly charged flat face of a macroion appears most efficient in inducing large compositional gradients, hence a large and unfavorable line energy and consequently lateral macroion aggregation and, concomitantly, macroscopic lipid phase separation. PMID:15626713

Ligand binding induces a sharp decrease in hydrophobicity of folate binding protein assessed by 1-anilinonaphthalene-8-sulphonate which suppresses self-association of the hydrophobic apo-protein.

PubMed

Holm, Jan; Lawaetz, Anders J; Hansen, Steen I

2012-08-17

High affinity folate binding protein (FBP) regulates as a soluble protein and as a cellular receptor intracellular trafficking of folic acid, a vitamin of great importance to cell growth and division. We addressed two issues of potential importance to the biological function of FBP, a possible decrease of the surface hydrophobicity associated with the ligand-induced conformation change of FBP, and protein-inter-protein interactions involved in self-association of hydrophobic apo-FBP. The extrinsic fluorescent apolar dye 1-anilinonaphthalene-8-sulphonate (ANS) exhibited enhanced fluorescence intensity and a blueshift of emission maximum from 510-520 nm to 460-470 nm upon addition of apo-FBP indicating binding to a strongly hydrophobic environment. Neither enhancement of fluorescence nor blueshift of ANS emission maximum occurred when folate-ligated holo-FBP replaced apo-FBP. The drastic decrease in surface hydrophobicity of holo-FBP could have bearings on the biological function of FBP since changes in surface hydrophobicity have critical effects on the biological function of receptors and transport proteins. ANS interacts with exposed hydrophobic surfaces on proteins and may thereby block and prevent aggregation of proteins (chaperone-like effect). Hence, hydrophobic interactions seemed to participate in the concentration-dependent self-association of apo-FBP which was suppressed by high ANS concentrations in light scatter measurements. Copyright © 2012 Elsevier Inc. All rights reserved.

Advanced Oxidation Protein Products-Modified Albumin Induces Differentiation of RAW264.7 Macrophages into Dendritic-Like Cells Which Is Modulated by Cell Surface Thiols.

PubMed

Garibaldi, Silvano; Barisione, Chiara; Marengo, Barbara; Ameri, Pietro; Brunelli, Claudio; Balbi, Manrico; Ghigliotti, Giorgio

2017-01-10

Local accumulation of Advanced Oxidation Protein Products (AOPP) induces pro-inflammatory and pro-fibrotic processes in kidneys and is an independent predictor of renal fibrosis and of rapid decline of eGFR in patients with chronic kidney disease (CKD). In addition to kidney damage, circulating AOPP may be regarded as mediators of systemic oxidative stress and, in this capacity, they might play a role in the progression of atherosclerotic damage of arterial walls. Atherosclerosis is a chronic inflammatory disease that involves activation of innate and adaptive immunity. Dendritic cells (DCs) are key cells in this process, due to their role in antigen presentation, inflammation resolution and T cell activation. AOPP consist in oxidative modifications of proteins (such as albumin and fibrinogen) that mainly occur through myeloperoxidase (MPO)-derived hypochlorite (HOCl). HOCl modified proteins have been found in atherosclerotic lesions. The oxidizing environment and the shifts in cellular redox equilibrium trigger inflammation, activate immune cells and induce immune responses. Thus, surface thiol groups contribute to the regulation of immune functions. The aims of this work are: (1) to evaluate whether AOPP-proteins induce activation and differentiation of mature macrophages into dendritic cells in vitro; and (2) to define the role of cell surface thiol groups and of free radicals in this process. AOPP-proteins were prepared by in vitro incubation of human serum albumin (HSA) with HOCl. Mouse macrophage-like RAW264.7 were treated with various concentrations of AOPP-HSA with or without the antioxidant N -acetyl cysteine (NAC). Following 48 h of HSA-AOPP treatment, RAW264.7 morphological changes were evaluated by microscopic observation, while markers of dendritic lineage and activation (CD40, CD86, and MHC class II) and allogeneic T cell proliferation were evaluated by flow cytometry. Cell surface thiols were measured by AlexaFluor-maleimide binding, and ROS production was assessed as DCF fluorescence by flow cytometry. HSA-AOPP induced the differentiation of RAW264.7 cells into a dendritic-like phenotype, as shown by morphological changes, by increased CD40, CD86 and MHC class II surface expression and by induction of T cell proliferation. The cell surface thiols dose dependently decreased following HSA-AOPP treatment, while ROS production increased. NAC pre-treatment enhanced the amount of cell surface thiols and prevented their reduction due to treatment with AOPP. Both ROS production and RAW264.7 differentiation into DC-like cells induced by HSA-AOPP were reduced by NAC. Our results highlight that oxidized plasma proteins modulate specific immune responses of macrophages through a process involving changes in the thiol redox equilibrium. We suggest that this mechanism may play a role in determining the rapid progression of the atherosclerotic process observed in CKD patients.

Improving the Immunogenicity of the Mycobacterium bovis BCG Vaccine by Non-Genetic Bacterial Surface Decoration Using the Avidin-Biotin System.

PubMed

Liao, Ting-Yu Angela; Lau, Alice; Joseph, Sunil; Hytönen, Vesa; Hmama, Zakaria

2015-01-01

Current strategies to improve the current BCG vaccine attempt to over-express genes encoding specific M. tuberculosis (Mtb) antigens and/or regulators of antigen presentation function, which indeed have the potential to reshape BCG in many ways. However, these approaches often face serious difficulties, in particular the efficiency and stability of gene expression via nucleic acid complementation and safety concerns associated with the introduction of exogenous DNA. As an alternative, we developed a novel non-genetic approach for rapid and efficient display of exogenous proteins on bacterial cell surface. The technology involves expression of proteins of interest in fusion with a mutant version of monomeric avidin that has the feature of reversible binding to biotin. Fusion proteins are then used to decorate the surface of biotinylated BCG. Surface coating of BCG with recombinant proteins was highly reproducible and stable. It also resisted to the freeze-drying shock routinely used in manufacturing conventional BCG. Modifications of BCG surface did not affect its growth in culture media neither its survival within the host cell. Macrophages phagocytized coated BCG bacteria, which efficiently delivered their surface cargo of avidin fusion proteins to MHC class I and class II antigen presentation compartments. Thereafter, chimeric proteins corresponding to a surrogate antigen derived from ovalbumin and the Mtb specific ESAT6 antigen were generated and tested for immunogenicity in vaccinated mice. We found that BCG displaying ovalbumin antigen induces an immune response with a magnitude similar to that induced by BCG genetically expressing the same surrogate antigen. We also found that BCG decorated with Mtb specific antigen ESAT6 successfully induces the expansion of specific T cell responses. This novel technology, therefore, represents a practical and effective alternative to DNA-based gene expression for upgrading the current BCG vaccine.

Influence of dynamic flow conditions on adsorbed plasma protein corona and surface-induced thrombus generation on antifouling brushes.

PubMed

Yu, Kai; Andruschak, Paula; Yeh, Han Hung; Grecov, Dana; Kizhakkedathu, Jayachandran N

2018-06-01

The information regarding the nature of protein corona (and its changes) and cell binding on biomaterial surface under dynamic conditions is critical to dissect the mechanism of surface-induced thrombosis. In this manuscript, we investigated the nature of protein corona and blood cell binding in heparinized recalcified human plasma, platelet rich plasma and whole blood on three highly hydrophilic antifouling polymer brushes, (poly(N, N-dimethylacrylamide) (PDMA), poly(2-methacryloyloxyethyl phosphorylcholine) (PMPC) and poly[N-(2-hydroxypropyl) methacrylamide] (PHPMA) using an in vitro blood loop model at comparable arterial and venous flow, and static conditions. A fluid dynamics model was used initially to better understand the resulting flow patterns in a vertical channel containing the substrates to arrive at the placement of the substrates within the blood loop. The protein binding on the brush modified substrates was determined using ellipsometry, fluorescence microscopy and the nature of the protein corona was investigated using mass spectrometry based proteomics. The flow elevated fouling on brush coated surface from blood. The extent of plasma protein adsorption and platelet adhesion onto PDMA brush was lower than other surfaces in both static and flow conditions. The profiles of adsorbed protein corona showed strong dependence on the test conditions (static vs. flow), and the chemistry of the polymer brushes. Specially, the PDMA brush under flow conditions was more enriched with coagulation proteins, complement proteins, vitronectin and fibronectin but was less enriched with serum albumin. Apolipoprotein B-100 and complement proteins were the most abundant proteins seen on PMPC and PHPMA surfaces under both flow and static conditions, respectively. Unlike PDMA brush, the flow conditions did not affect the composition of protein corona on PMPC and PHPMA brushes. The nature of the protein corona formed in flow conditions influenced the platelet and red blood cell binding. The dependence of shear stress on platelet adhesion from platelet rich plasma and whole blood highlights the contribution of red blood cells in enhancing platelet adhesion on the surface under high shear condition. Copyright © 2018 Elsevier Ltd. All rights reserved.

Effect of osmolytes on the thermal stability of proteins: replica exchange simulations of Trp-cage in urea and betaine solutions.

PubMed

Adamczak, Beata; Kogut, Mateusz; Czub, Jacek

2018-04-25

Although osmolytes are known to modulate the folding equilibrium, the molecular mechanism of their effect on thermal denaturation of proteins is still poorly understood. Here, we simulated the thermal denaturation of a small model protein (Trp-cage) in the presence of denaturing (urea) and stabilizing (betaine) osmolytes, using the all-atom replica exchange molecular dynamics simulations. We found that urea destabilizes Trp-cage by enthalpically-driven association with the protein, acting synergistically with temperature to induce unfolding. In contrast, betaine is sterically excluded from the protein surface thereby exerting entropic depletion forces that contribute to the stabilization of the native state. In fact, we find that while at low temperatures betaine slightly increases the folding free energy of Trp-cage by promoting another near-native conformation, it protects the protein against temperature-induced denaturation. This, in turn, can be attributed to enhanced exclusion of betaine at higher temperatures that arises from less attractive interactions with the protein surface.

Light-fuelled transport of large dendrimers and proteins.

PubMed

Koskela, Jenni E; Liljeström, Ville; Lim, Jongdoo; Simanek, Eric E; Ras, Robin H A; Priimagi, Arri; Kostiainen, Mauri A

2014-05-14

This work presents a facile water-based supramolecular approach for light-induced surface patterning. The method is based upon azobenzene-functionalized high-molecular weight triazine dendrimers up to generation 9, demonstrating that even very large globular supramolecular complexes can be made to move in response to light. We also demonstrate light-fuelled macroscopic movements in native biomolecules, showing that complexes of apoferritin protein and azobenzene can effectively form light-induced surface patterns. Fundamentally, the results establish that thin films comprising both flexible and rigid globular particles of large diameter can be moved with light, whereas the presented material concepts offer new possibilities for the yet marginally explored biological applications of azobenzene surface patterning.

Differential roles of auxin efflux carrier PIN proteins in hypocotyl phototropism of etiolated Arabidopsis seedlings depend on the direction of light stimulus.

PubMed

Haga, Ken; Sakai, Tatsuya

2013-01-01

In a recent study, we demonstrated that although the auxin efflux carrier PIN-FORMED (PIN) proteins, such as PIN3 and PIN7, are required for the pulse-induced first positive phototropism in etiolated Arabidopsis hypocotyls, they are not necessary for the continuous-light-induced second positive phototropism when the seedlings are grown on the surface of agar medium, which causes the hypocotyls to separate from the agar surface. Previous reports have shown that hypocotyl phototropism is slightly impaired in pin3 single mutants when they are grown along the surface of agar medium, where the hypocotyls always contact the agar, producing some friction. To clarify the possible involvement of PIN3 and PIN7 in continuous-light-induced phototropism, we investigated hypocotyl phototropism in the pin3 pin7 double mutant grown along the surface of agar medium. Intriguingly, the phototropic curvature was slightly impaired in the double mutant when the phototropic stimulus was presented on the adaxial side of the hook, but was not impaired when the phototropic stimulus was presented on the abaxial side of the hook. These results indicate that PIN proteins are required for continuous-light-induced second positive phototropism, depending on the direction of the light stimulus, when the seedlings are in contact with agar medium.

Differential roles of auxin efflux carrier PIN proteins in hypocotyl phototropism of etiolated Arabidopsis seedlings depend on the direction of light stimulus

PubMed Central

Haga, Ken; Sakai, Tatsuya

2013-01-01

In a recent study, we demonstrated that although the auxin efflux carrier PIN-FORMED (PIN) proteins, such as PIN3 and PIN7, are required for the pulse-induced first positive phototropism in etiolated Arabidopsis hypocotyls, they are not necessary for the continuous-light-induced second positive phototropism when the seedlings are grown on the surface of agar medium, which causes the hypocotyls to separate from the agar surface. Previous reports have shown that hypocotyl phototropism is slightly impaired in pin3 single mutants when they are grown along the surface of agar medium, where the hypocotyls always contact the agar, producing some friction. To clarify the possible involvement of PIN3 and PIN7 in continuous-light-induced phototropism, we investigated hypocotyl phototropism in the pin3 pin7 double mutant grown along the surface of agar medium. Intriguingly, the phototropic curvature was slightly impaired in the double mutant when the phototropic stimulus was presented on the adaxial side of the hook, but was not impaired when the phototropic stimulus was presented on the abaxial side of the hook. These results indicate that PIN proteins are required for continuous-light-induced second positive phototropism, depending on the direction of the light stimulus, when the seedlings are in contact with agar medium. PMID:23104115

Salt-induced enhancement of antifreeze protein activity: a salting-out effect.

PubMed

Kristiansen, Erlend; Pedersen, Sindre Andre; Zachariassen, Karl Erik

2008-10-01

Antifreeze proteins are a structurally diverse group of proteins characterized by their unique ability to cause a separation of the melting- and growth-temperatures of ice. These proteins have evolved independently in different kinds of cold-adapted ectothermic animals, including insects and fish, where they protect against lethal freezing of the body fluids. There is a great variability in the capacity of different kinds of antifreeze proteins to evoke the antifreeze effect, but the basis of these differences is not well understood. This study reports on salt-induced enhancement of the antifreeze activity of an antifreeze protein from the longhorn beetle Rhagium inquisitor (L.). The results imply that antifreeze activity is predetermined by a steady-state distribution of the antifreeze protein between the solution and the ice surface region. The observed salt-induced enhancement of the antifreeze activity compares qualitatively and quantitatively with salt-induced lowering of protein solubility. Thus, salts apparently enhance antifreeze activity by evoking a solubility-induced shift in the distribution pattern of the antifreeze proteins in favour of the ice. These results indicate that the solubility of antifreeze proteins in the solution surrounding the ice crystal is a fundamental physiochemical property in relation to their antifreeze potency.

Heterologous expression of surface-active proteins from barley and filamentous fungi in Pichia pastoris and characterization of their contribution to beer gushing.

PubMed

Lutterschmid, Georg; Muranyi, Monika; Stübner, Matthias; Vogel, Rudi F; Niessen, Ludwig

2011-05-14

The spontaneous over-foaming of beer upon opening, i.e. beer gushing, is an unwanted phenomenon for the brewing industry. Currently, surface-active proteins from filamentous fungi and non-specific lipid transfer proteins (nsLTP1) from barley are discussed as gushing inducers. In our study the class I hydrophobin FcHyd3p from Fusarium culmorum, the class II hydrophobin Hfb2 from Trichoderma reesei, the alkaline foam protein A (AfpA) from F. graminearum and nsLTP1 from Hordeum vulgare cv. Marnie (barley) were heterologously expressed in Pichia pastoris and used in gushing tests. The class I hydrophobin FcHyd3p was unable to induce gushing in beer. The class II hydrophobin Hfb2 was able to induce gushing in beer, but proved to be inhibited by heat treatment as well as by the presence of enriched hop compounds. Both resulted in a reduced gushing potential. AfpA and nsLTP1 exhibited no gushing-inducing potential at the amounts added to beer. Addition of these proteins to beer or carbonated water previously treated with class II hydrophobins revealed a gushing reducing character. Copyright © 2011 Elsevier B.V. All rights reserved.

Influence of the Surface Functional Group Density on the Carbon-Nanotube-Induced α-Chymotrypsin Structure and Activity Alterations.

PubMed

Zhao, Xingchen; Hao, Fang; Lu, Dawei; Liu, Wei; Zhou, Qunfang; Jiang, Guibin

2015-08-26

Because of the special properties of carbon nanotubes (CNTs), their applications have been introduced to many fields. The biosafety of these emerging materials is of high concern concomitantly. Because CNTs may initially bind with proteins in biofluids before they exert biological effects, it is of great importance to understand how the target proteins interact with these exogenous nanomaterials. Here we investigated the interaction between α-chymotrypsin (α-ChT) and carboxylized multiwalled CNTs in a simulated biophysical environment utilizing the techniques of fluorescence, UV-vis, circular dichroism spectroscopy, ζ potential, atomic force microscopy, and bicinchoninic acid analysis. It was demonstrated that CNTs interacted with α-ChT through electrostatic forces, causing a decrement in the α-helix and an increment in the β-sheet content of the protein. The protein fluorescence was quenched in a static mode. The increase in the surface modification density of CNTs enhanced the protein absorption and decreased the enzymatic activity correspondingly. α-ChT activity inhibition induced by CNTs with low surface modification density exhibited noncompetitive characteristics; however, a competitive feature was observed when CNTs with high surface modification density interacted with the protein. An increase of the ionic strength in the reaction buffer may help to reduce the interaction between CNTs and α-ChT because the high ionic strength may favor the release of the protein from binding on a CNT surface modified with functional groups. Accordingly, the functionalization density on the CNT surface plays an important role in the regulation of their biological effects and is worthy of concern when new modified CNTs are developed.

Calcium oxalate monohydrate aggregation induced by aggregation of desialylated Tamm-Horsfall protein

PubMed Central

Viswanathan, Pragasam; Rimer, Jeffrey D.; Kolbach, Ann M.; Kleinman, Jack G.

2011-01-01

Tamm-Horsfall protein (THP) is thought to protect against calcium oxalate monohydrate (COM) stone formation by inhibiting COM aggregation. Several studies reported that stone formers produce THP with reduced levels of glycosylation, particularly sialic acid levels, which leads to reduced negative charge. In this study, normal THP was treated with neuraminidase to remove sialic acid residues, confirmed by an isoelectric point shift to higher pH. COM aggregation assays revealed that desialylated THP (ds-THP) promoted COM aggregation, while normal THP inhibited aggregation. The appearance of protein aggregates in solutions at ds-THP concentrations ≥1 µg/mL in 150 mM NaCl correlated with COM aggregation promotion, implying that ds-THP aggregation induced COM aggregation. The aggregation-promoting effect of the ds-THP was independent of pH above its isoelectric point, but was substantially reduced at low ionic strength, where protein aggregation was much reduced. COM aggregation promotion was maximized at a ds-THP to COM mass ratio of ~0.025, which can be explained by a model wherein partial COM surface coverage by ds-THP aggregates promotes crystal aggregation by bridging opposing COM surfaces, whereas higher surface coverage leads to repulsion between adsorbed ds-THP aggregates. Thus, desialylation of THP apparently abrogates a normal defensive action of THP by inducing protein aggregation, and subsequently COM aggregation, a condition that favors kidney stone formation. PMID:21229239

Vanadium inhibits DNA-protein cross-links and ameliorates surface level changes of aberrant crypt foci during 1,2-dimethylhydrazine induced rat colon carcinogenesis.

PubMed

Kanna, P Suresh; Saralaya, M G; Samanta, K; Chatterjee, M

2005-01-01

The trace mineral vanadium inhibits cancer development in a variety of experimental animal models. The present study was to gain insight into a putative anticancer effect of vanadium in a rat model of colon carcinogenesis. The in vivo study was intended to clarify the effect of vanadium on DNA-protein cross-links (DPC), surface level changes of aberrant crypt foci (ACF) and biotransformation status during 1,2-dimethylhydrazine (1,2-DMH) induced preneoplastic rat colon carcinogenesis. The comet assay showed statistically higher mean base values of DNA-protein mass (p<0.01) and mean frequencies of tailed cells (p<0.001) in the carcinogen-induced group after treatment with proteinase K. Treatment with vanadium in the form of ammonium monovanadate supplemented ad libitum in drinking water for the entire experimental period caused a significant (p<0.02) reduction (40%) in DNA-protein cross-links in colon cells. Further, the biotransformation status of vanadium was ascertained measuring the drug metabolising enzymes, glutathione S-transferase (GST) and cytochrome P-450 (Cyt P-450). Significantly, there was an increase in glutathione S-transferase and cytochrome P-450 levels (p<0.01 and p<0.02, respectively) in rats supplemented with vanadium as compared to their carcinogen controls. As an endpoint marker, we also evaluated the effect of vanadium on surface level changes of aberrant crypt foci induced by 1,2-DMH by scanning electron microscopy. Animals induced with 1,2-DMH and supplemented with vanadium showed a marked improvement in colonic architecture with less number of aberrant crypt foci in contrast to the animals induced with 1,2-DMH alone, thereby exhibiting its anti-carcinogenicity by modulating the markers studied herein.

Bacterial Ice Crystal Controlling Proteins

PubMed Central

Lorv, Janet S. H.; Rose, David R.; Glick, Bernard R.

2014-01-01

Across the world, many ice active bacteria utilize ice crystal controlling proteins for aid in freezing tolerance at subzero temperatures. Ice crystal controlling proteins include both antifreeze and ice nucleation proteins. Antifreeze proteins minimize freezing damage by inhibiting growth of large ice crystals, while ice nucleation proteins induce formation of embryonic ice crystals. Although both protein classes have differing functions, these proteins use the same ice binding mechanisms. Rather than direct binding, it is probable that these protein classes create an ice surface prior to ice crystal surface adsorption. Function is differentiated by molecular size of the protein. This paper reviews the similar and different aspects of bacterial antifreeze and ice nucleation proteins, the role of these proteins in freezing tolerance, prevalence of these proteins in psychrophiles, and current mechanisms of protein-ice interactions. PMID:24579057

Bacterial ice crystal controlling proteins.

PubMed

Lorv, Janet S H; Rose, David R; Glick, Bernard R

2014-01-01

Across the world, many ice active bacteria utilize ice crystal controlling proteins for aid in freezing tolerance at subzero temperatures. Ice crystal controlling proteins include both antifreeze and ice nucleation proteins. Antifreeze proteins minimize freezing damage by inhibiting growth of large ice crystals, while ice nucleation proteins induce formation of embryonic ice crystals. Although both protein classes have differing functions, these proteins use the same ice binding mechanisms. Rather than direct binding, it is probable that these protein classes create an ice surface prior to ice crystal surface adsorption. Function is differentiated by molecular size of the protein. This paper reviews the similar and different aspects of bacterial antifreeze and ice nucleation proteins, the role of these proteins in freezing tolerance, prevalence of these proteins in psychrophiles, and current mechanisms of protein-ice interactions.

Surface Modification of Biomaterials: A Quest for Blood Compatibility

PubMed Central

de Mel, Achala; Cousins, Brian G.; Seifalian, Alexander M.

2012-01-01

Cardiovascular implants must resist thrombosis and intimal hyperplasia to maintain patency. These implants when in contact with blood face a challenge to oppose the natural coagulation process that becomes activated. Surface protein adsorption and their relevant 3D confirmation greatly determine the degree of blood compatibility. A great deal of research efforts are attributed towards realising such a surface, which comprise of a range of methods on surface modification. Surface modification methods can be broadly categorized as physicochemical modifications and biological modifications. These modifications aim to modulate platelet responses directly through modulation of thrombogenic proteins or by inducing antithrombogenic biomolecules that can be biofunctionalised onto surfaces or through inducing an active endothelium. Nanotechnology is recognising a great role in such surface modification of cardiovascular implants through biofunctionalisation of polymers and peptides in nanocomposites and through nanofabrication of polymers which will pave the way for finding a closer blood match through haemostasis when developing cardiovascular implants with a greater degree of patency. PMID:22693509

Exploitation of stable nanostructures based on the mouse polyomavirus for development of a recombinant vaccine against porcine circovirus 2

PubMed Central

Fraiberk, Martin; Hájková, Michaela; Krulová, Magdaléna; Kojzarová, Martina; Drda Morávková, Alena; Pšikal, Ivan

2017-01-01

The aim of this study was to develop a suitable vaccine antigen against porcine circovirus 2 (PCV2), the causative agent of post-weaning multi-systemic wasting syndrome, which causes significant economic losses in swine breeding. Chimeric antigens containing PCV2b Cap protein sequences based on the mouse polyomavirus (MPyV) nanostructures were developed. First, universal vectors for baculovirus-directed production of chimeric MPyV VLPs or pentamers of the major capsid protein, VP1, were designed for their exploitation as vaccines against other pathogens. Various strategies were employed based on: A) exposure of selected immunogenic epitopes on the surface of MPyV VLPs by insertion into a surface loop of the VP1 protein, B) insertion of foreign protein molecules inside the VLPs, or C) fusion of a foreign protein or its part with the C-terminus of VP1 protein, to form giant pentamers of a chimeric protein. We evaluated these strategies by developing a recombinant vaccine against porcine circovirus 2. All candidate vaccines induced the production of antibodies against the capsid protein of porcine circovirus after immunization of mice. The candidate vaccine, Var C, based on fusion of mouse polyomavirus and porcine circovirus capsid proteins, could induce the production of antibodies with the highest PCV2 neutralizing capacity. Its ability to induce the production of neutralization antibodies was verified after immunization of pigs. The advantage of this vaccine, apart from its efficient production in insect cells and easy purification, is that it represents a DIVA (differentiating infected from vaccinated animals) vaccine, which also induces an immune response against the mouse polyoma VP1 protein and is thus able to distinguish between vaccinated and naturally infected animals. PMID:28922413

Protein phosphatase 2A mediates resensitization of the neurokinin 1 receptor

PubMed Central

Murphy, Jane E.; Roosterman, Dirk; Cottrell, Graeme S.; Padilla, Benjamin E.; Feld, Micha; Brand, Eva; Cedron, Wendy J.; Steinhoff, Martin

2011-01-01

Activated G protein-coupled receptors (GPCRs) are phosphorylated and interact with β-arrestins, which mediate desensitization and endocytosis. Endothelin-converting enzyme-1 (ECE-1) degrades neuropeptides in endosomes and can promote recycling. Although endocytosis, dephosphorylation, and recycling are accepted mechanisms of receptor resensitization, a large proportion of desensitized receptors can remain at the cell surface. We investigated whether reactivation of noninternalized, desensitized (phosphorylated) receptors mediates resensitization of the substance P (SP) neurokinin 1 receptor (NK1R). Herein, we report a novel mechanism of resensitization by which protein phosphatase 2A (PP2A) is recruited to dephosphorylate noninternalized NK1R. A desensitizing concentration of SP reduced cell-surface SP binding sites by only 25%, and SP-induced Ca2+ signals were fully resensitized before cell-surface binding sites started to recover, suggesting resensitization of cell-surface-retained NK1R. SP induced association of β-arrestin1 and PP2A with noninternalized NK1R. β-Arrestin1 small interfering RNA knockdown prevented SP-induced association of cell-surface NK1R with PP2A, indicating that β-arrestin1 mediates this interaction. ECE-1 inhibition, by trapping β-arrestin1 in endosomes, also impeded SP-induced association of cell-surface NK1R with PP2A. Resensitization of NK1R signaling required both PP2A and ECE-1 activity. Thus, after stimulation with SP, PP2A interacts with noninternalized NK1R and mediates resensitization. PP2A interaction with NK1R requires β-arrestin1. ECE-1 promotes this process by releasing β-arrestin1 from NK1R in endosomes. These findings represent a novel mechanism of PP2A- and ECE-1-dependent resensitization of GPCRs. PMID:21795521

Studying protein structural changes based on surface plasmon resonance and surface-enhanced Raman scattering

NASA Astrophysics Data System (ADS)

Hu, Wen-Pin; Chen, Shean-Jen; Yih, Jenq-Nan; Lin, G.-Y.; Chang, Guan L.

2004-06-01

The ability to recognize the conformational changes and structural variations of a protein when immobilized in a solid surface is of great importance in a variety of applications. Surface plasmon resonance (SPR) sensing is an appropriate technique for investigating interfacial phenomena, and enables the conformational changes of proteins to be monitored through the variation in the SPR angle shift. Meanwhile, the surface-enhanced Raman scattering (SERS) system can also assist in clarifying the changes in protein structure. The present study utilizes a 1 mM CrO3 phosphate buffer solution (PBS) to induce conformational changes of human serum albumin (HSA). Monitoring the corresponding SPR angle shifts and the SPR reflectivity spectrum enables the relationships between the conformational changes of the surface-immobilized protein and the thickness and dielectric constants of the protein layer to be estimated. The experimental SPR results indicate that the Cr6+ ions cause significant conformational change of the protein. It is established that the ions are not merely absorbed into the protein as a result of electrostatic forces, but that complex protein refolding events also take place. Furthermore, the data acquired from the SERS system yield valuable information regarding the changes which take place in the protein structure.

Activation of m1 muscarinic acetylcholine receptor induces surface transport of KCNQ channels through a CRMP-2-mediated pathway.

PubMed

Jiang, Ling; Kosenko, Anastasia; Yu, Clinton; Huang, Lan; Li, Xuejun; Hoshi, Naoto

2015-11-15

Neuronal excitability is strictly regulated by various mechanisms, including modulation of ion channel activity and trafficking. Stimulation of m1 muscarinic acetylcholine receptor (also known as CHRM1) increases neuronal excitability by suppressing the M-current generated by the Kv7/KCNQ channel family. We found that m1 muscarinic acetylcholine receptor stimulation also triggers surface transport of KCNQ subunits. This receptor-induced surface transport was observed with KCNQ2 as well as KCNQ3 homomeric channels, but not with Kv3.1 channels. Deletion analyses identified that a conserved domain in a proximal region of the N-terminal tail of KCNQ protein is crucial for this surface transport--the translocation domain. Proteins that bind to this domain were identified as α- and β-tubulin and collapsin response mediator protein 2 (CRMP-2; also known as DPYSL2). An inhibitor of casein kinase 2 (CK2) reduced tubulin binding to the translocation domain, whereas an inhibitor of glycogen synthase kinase 3 (GSK3) facilitated CRMP-2 binding to the translocation domain. Consistently, treatment with the GSK3 inhibitor enhanced receptor-induced KCNQ2 surface transport. M-current recordings from neurons showed that treatment with a GSK3 inhibitor shortened the duration of muscarinic suppression and led to over-recovery of the M-current. These results suggest that m1 muscarinic acetylcholine receptor stimulates surface transport of KCNQ channels through a CRMP-2-mediated pathway. © 2015. Published by The Company of Biologists Ltd.

Effect of thermal stability on protein adsorption to silica using homologous aldo-keto reductases.

PubMed

Felsovalyi, Flora; Patel, Tushar; Mangiagalli, Paolo; Kumar, Sanat K; Banta, Scott

2012-08-01

Gaining more insight into the mechanisms governing the behavior of proteins at solid/liquid interfaces is particularly relevant in the interaction of high-value biologics with storage and delivery device surfaces, where adsorption-induced conformational changes may dramatically affect biocompatibility. The impact of structural stability on interfacial behavior has been previously investigated by engineering nonwild-type stability mutants. Potential shortcomings of such approaches include only modest changes in thermostability, and the introduction of changes in the topology of the proteins when disulfide bonds are incorporated. Here we employ two members of the aldo-keto reductase superfamily (alcohol dehydrogenase, AdhD and human aldose reductase, hAR) to gain a new perspective on the role of naturally occurring thermostability on adsorbed protein arrangement and its subsequent impact on desorption. Unexpectedly, we find that during initial adsorption events, both proteins have similar affinity to the substrate and undergo nearly identical levels of structural perturbation. Interesting differences between AdhD and hAR occur during desorption and both proteins exhibit some level of activity loss and irreversible conformational change upon desorption. Although such surface-induced denaturation is expected for the less stable hAR, it is remarkable that the extremely thermostable AdhD is similarly affected by adsorption-induced events. These results question the role of thermal stability as a predictor of protein adsorption/desorption behavior. Copyright © 2012 The Protein Society.

Electrostatic Interactions between OmpG Nanopore and Analyte Protein Surface Can Distinguish between Glycosylated Isoforms.

PubMed

Fahie, Monifa A; Chen, Min

2015-08-13

The flexible loops decorating the entrance of OmpG nanopore move dynamically during ionic current recording. The gating caused by these flexible loops changes when a target protein is bound. The gating is characterized by parameters including frequency, duration, and open-pore current, and these features combine to reveal the identity of a specific analyte protein. Here, we show that OmpG nanopore equipped with a biotin ligand can distinguish glycosylated and deglycosylated isoforms of avidin by their differences in surface charge. Our studies demonstrate that the direct interaction between the nanopore and analyte surface, induced by the electrostatic attraction between the two molecules, is essential for protein isoform detection. Our technique is remarkably sensitive to the analyte surface, which may provide a useful tool for glycoprotein profiling.

Adenovirus Particles that Display the Plasmodium falciparum Circumsporozoite Protein NANP Repeat Induce Sporozoite-Neutralizing Antibodies in Mice

PubMed Central

Palma, Christopher; Overstreet, Michael G.; Guedon, Jean-Marc; Hoiczyk, Egbert; Ward, Cameron; Karen, Kasey A.; Zavala, Fidel; Ketner, Gary

2011-01-01

Adenovirus particles can be engineered to display exogenous peptides on their surfaces by modification of viral capsid proteins, and particles that display pathogen-derived peptides can induce protective immunity. We constructed viable recombinant adenoviruses that display B-cell epitopes from the Plasmodium falciparum circumsporozoite protein (PfCSP) in the major adenovirus capsid protein, hexon. Recombinants induced high-titer antibodies against CSP when injected intraperitoneally into mice. Serum obtained from immunized mice recognized both recombinant PfCSP protein and P. falciparum sporozoites, and neutralized P. falciparum sporozoites in vitro. Replicating adenovirus vaccines have provided economical protection against adenovirus disease for over three decades. The recombinants described here may provide a path to an affordable malaria vaccine in the developing world. PMID:21199707

Effect of a pore-forming protein derived from Flammulina velutipes on the Caco-2 intestinal epithelial cell monolayer.

PubMed

Narai, Asako; Watanabe, Hirohito; Iwanaga, Toshihiko; Tomita, Toshio; Shimizu, Makoto

2004-11-01

We have previously found a transepithelial electrical resistance (TEER)-decreasing protein derived from Flammulina velutipes, which was revealed to be identical to flammutoxin (FTX) that is known as a hemolytic pore-forming protein. This protein induced a rapid decrease in TEER and parallel increase in paracellular permeability in the intestinal epithelial Caco-2 cell monolayer without any cytotoxicity. An immunoblotting analysis revealed that the FTX-induced decrease in TEER was accompanied by the formation of a high-molecular-weight complex on the surface of Caco-2 cells. Intracellular Ca(2+) imaging showed that exposure to FTX caused a rapid Ca(2+) influx. It was observed by electron microscopy that FTX induced swelling of microvilli and expansion of the cellular surface. Staining with fluorescent phalloidin showed a marked change to filamentous actin in the FTX-treated cells. These results suggest that TEER reduction could sensitively detect small membrane pore formation by FTX in the intestinal epithelium which causes a morphological alteration and disruption of the paracellular barrier function.

Subdominant outer membrane antigens in anaplasma marginale: conservation, antigenicity, and protective capacity using recombinant protein

USDA-ARS?s Scientific Manuscript database

Anaplasma marginale is a tick-borne rickettsial pathogen of cattle with a worldwide distribution. Currently a safe and efficacious vaccine is unavailable. Outer membrane protein (OMP) extracts or a well- defined surface protein complex reproducibly induce protective immunity. However, there are seve...

Counter effect of sucrose on ethanol-induced aggregation of protein.

PubMed

Yadav, Jay Kant; Chandani, N; Pande Prajakt, P R; Chauhan, Jyoti Bala

2010-12-01

The present paper is an attempt to study the mechanism of ethanol induced aggregation of chicken egg albumin and to stabilize the protein against ethanol induced aggregation. The protein aggregation was determined by monitoring the light scattering of protein aggregates spectrophotometrically. The protein undergoes certain structural changes in water-ethanol solution and the degree of aggregation was found to be linearly depending upon the concentration of alcohol used. The intrinsic fluorescence study showed a large blue shift in the λ(max) (16 nm) in the presence of 50% ethanol. The ANS fluorescence intensity was found to be gradually increasing with increasing concentration of ethanol. This indicates an increase in the hydrophobic cluster on the protein surface and as a result the hydrophobic interaction is the major driving force for the aggregate formation. Addition of sucrose significantly reduced the ethanol-induced protein aggregation. In presence of 50% sucrose the ethanol the aggregation was reduced to 5%. The study reveals that addition of sucrose brings out changes in the solvent distribution and prevents the structural changes in protein which lead the aggregation.

Functional dynamics of cell surface membrane proteins

NASA Astrophysics Data System (ADS)

Nishida, Noritaka; Osawa, Masanori; Takeuchi, Koh; Imai, Shunsuke; Stampoulis, Pavlos; Kofuku, Yutaka; Ueda, Takumi; Shimada, Ichio

2014-04-01

Cell surface receptors are integral membrane proteins that receive external stimuli, and transmit signals across plasma membranes. In the conventional view of receptor activation, ligand binding to the extracellular side of the receptor induces conformational changes, which convert the structure of the receptor into an active conformation. However, recent NMR studies of cell surface membrane proteins have revealed that their structures are more dynamic than previously envisioned, and they fluctuate between multiple conformations in an equilibrium on various timescales. In addition, NMR analyses, along with biochemical and cell biological experiments indicated that such dynamical properties are critical for the proper functions of the receptors. In this review, we will describe several NMR studies that revealed direct linkage between the structural dynamics and the functions of the cell surface membrane proteins, such as G-protein coupled receptors (GPCRs), ion channels, membrane transporters, and cell adhesion molecules.

Functional dynamics of cell surface membrane proteins.

PubMed

Nishida, Noritaka; Osawa, Masanori; Takeuchi, Koh; Imai, Shunsuke; Stampoulis, Pavlos; Kofuku, Yutaka; Ueda, Takumi; Shimada, Ichio

2014-04-01

Cell surface receptors are integral membrane proteins that receive external stimuli, and transmit signals across plasma membranes. In the conventional view of receptor activation, ligand binding to the extracellular side of the receptor induces conformational changes, which convert the structure of the receptor into an active conformation. However, recent NMR studies of cell surface membrane proteins have revealed that their structures are more dynamic than previously envisioned, and they fluctuate between multiple conformations in an equilibrium on various timescales. In addition, NMR analyses, along with biochemical and cell biological experiments indicated that such dynamical properties are critical for the proper functions of the receptors. In this review, we will describe several NMR studies that revealed direct linkage between the structural dynamics and the functions of the cell surface membrane proteins, such as G-protein coupled receptors (GPCRs), ion channels, membrane transporters, and cell adhesion molecules. Copyright © 2013 Elsevier Inc. All rights reserved.

Effect of mechanical denaturation on surface free energy of protein powders.

PubMed

Mohammad, Mohammad Amin; Grimsey, Ian M; Forbes, Robert T; Blagbrough, Ian S; Conway, Barbara R

2016-10-01

Globular proteins are important both as therapeutic agents and excipients. However, their fragile native conformations can be denatured during pharmaceutical processing, which leads to modification of the surface energy of their powders and hence their performance. Lyophilized powders of hen egg-white lysozyme and β-galactosidase from Aspergillus oryzae were used as models to study the effects of mechanical denaturation on the surface energies of basic and acidic protein powders, respectively. Their mechanical denaturation upon milling was confirmed by the absence of their thermal unfolding transition phases and by the changes in their secondary and tertiary structures. Inverse gas chromatography detected differences between both unprocessed protein powders and the changes induced by their mechanical denaturation. The surfaces of the acidic and basic protein powders were relatively basic, however the surface acidity of β-galactosidase was higher than that of lysozyme. Also, the surface of β-galactosidase powder had a higher dispersive energy compared to lysozyme. The mechanical denaturation decreased the dispersive energy and the basicity of the surfaces of both protein powders. The amino acid composition and molecular conformation of the proteins explained the surface energy data measured by inverse gas chromatography. The biological activity of mechanically denatured protein powders can either be reversible (lysozyme) or irreversible (β-galactosidase) upon hydration. Our surface data can be exploited to understand and predict the performance of protein powders within pharmaceutical dosage forms. Copyright © 2016 Elsevier B.V. All rights reserved.

Maillard Conjugation of Sodium Alginate to Whey Protein for Enhanced Resistance to Surfactant-Induced Competitive Displacement from Air-Water Interfaces.

PubMed

Cai, Bingqing; Saito, Anna; Ikeda, Shinya

2018-01-24

Whey protein adsorbed to an interface forms a viscoelastic interfacial film but is displaced competitively from the interface by a small-molecule surfactant added afterward. The present study evaluated the impact of the covalent conjugation of high- or low-molecular-weight sodium alginate (HA or LA) to whey protein isolate (WPI) via the Maillard reaction on the ability of whey protein to resist surfactant-induced competitive displacement from the air-water interface. Surfactant added after the pre-adsorption of conjugate to the interface increased surface pressure. At a given surface pressure, the WPI-LA conjugate showed a significantly higher interfacial area coverage and lower interfacial film thickness compared to those of the WPI-HA conjugate or unconjugated WPI. The addition of LA to the aqueous phase had little effect on the interfacial area and thickness of pre-adsorbed WPI. These results suggest the importance of the molecular weight of the polysaccharide moiety in determining interfacial properties of whey protein-alginate conjugates.

Protein adsorption on tailored substrates: long-range forces and conformational changes

NASA Astrophysics Data System (ADS)

Bellion, M.; Santen, L.; Mantz, H.; Hähl, H.; Quinn, A.; Nagel, A.; Gilow, C.; Weitenberg, C.; Schmitt, Y.; Jacobs, K.

2008-10-01

Adsorption of proteins onto solid surfaces is an everyday phenomenon that is not yet fully understood. To further the current understanding, we have performed in situ ellipsometry studies to reveal the adsorption kinetics of three different proteins, lysozyme, α-amylase and bovine serum albumin. As substrates we offer Si wafers with a controlled Si oxide layer thickness and a hydrophilic or hydrophobic surface functionalization, allowing the tailoring of the influence of short- and long-range interactions. Our studies show that not only the surface chemistry determines the properties of an adsorbed protein layer but also the van der Waals contributions of a composite substrate. We compare the experimental findings to results of a colloidal Monte Carlo approach that includes conformational changes of the adsorbed proteins induced by density fluctuations.

Down-regulation of lipid raft-associated onco-proteins via cholesterol-dependent lipid raft internalization in docosahexaenoic acid-induced apoptosis.

PubMed

Lee, Eun Jeong; Yun, Un-Jung; Koo, Kyung Hee; Sung, Jee Young; Shim, Jaegal; Ye, Sang-Kyu; Hong, Kyeong-Man; Kim, Yong-Nyun

2014-01-01

Lipid rafts, plasma membrane microdomains, are important for cell survival signaling and cholesterol is a critical lipid component for lipid raft integrity and function. DHA is known to have poor affinity for cholesterol and it influences lipid rafts. Here, we investigated a mechanism underlying the anti-cancer effects of DHA using a human breast cancer cell line, MDA-MB-231. We found that DHA decreased cell surface levels of lipid rafts via their internalization, which was partially reversed by cholesterol addition. With DHA treatment, caveolin-1, a marker for rafts, and EGFR were colocalized with LAMP-1, a lysosomal marker, in a cholesterol-dependent manner, indicating that DHA induces raft fusion with lysosomes. DHA not only displaced several raft-associated onco-proteins, including EGFR, Hsp90, Akt, and Src, from the rafts but also decreased total levels of those proteins via multiple pathways, including the proteasomal and lysosomal pathways, thereby decreasing their activities. Hsp90 overexpression maintained its client proteins, EGFR and Akt, and attenuated DHA-induced cell death. In addition, overexpression of Akt or constitutively active Akt attenuated DHA-induced apoptosis. All these data indicate that the anti-proliferative effect of DHA is mediated by targeting of lipid rafts via decreasing cell surface lipid rafts by their internalization, thereby decreasing raft-associated onco-proteins via proteasomal and lysosomal pathways and decreasing Hsp90 chaperone function. © 2013.

Effects of the conjugation of whey proteins with gellan polysaccharides on surfactant-induced competitive displacement from the air-water interface.

PubMed

Cai, B; Ikeda, S

2016-08-01

Whey proteins can be used to stabilize foams and emulsions against coalescence because of their ability to form viscoelastic films at the interface that resist film rupture on collision between colloidal particles. However, whey proteins are competitively displaced from the interface if small-molecule surfactants are added, leading to destabilization of the entire system. This is because surfactants are more effective in molecular packing at the interface, and they lower interfacial tension to a greater degree than whey proteins do, but their interfacial films are poor in viscoelasticity. We hypothesized that whey proteins would become more resistant to surfactant-induced competitive displacement if they were conjugated with network-forming polysaccharides. The protein moiety of the conjugate would be expected to enable its adsorption to the interface, and the polysaccharide moiety would be expected to form self-assembled networks, strengthening the interfacial film as a whole. In this study, whey proteins were conjugated with gellan polysaccharides using the Maillard reaction. Atomic force microscopy images of interfacial films formed by the whey protein-gellan conjugate at the air-water interface and transferred onto mica sheets using the Langmuir-Blodgett method revealed that gellan did form self-assembled networks at the interface and that interfacial films also contained a large number of unconjugated whey protein molecules. Following the addition of a small-molecule surfactant (Tween 20) to the sub-phase, surface pressure increased, indicating spontaneous adsorption of surfactants to the interface. Atomic force microscopy images showed decreases in interfacial area coverage by whey proteins as surface pressure increased. At a given surface pressure, the interfacial area coverage by whey protein-gellan conjugates was greater than coverage by unconjugated whey proteins, confirming that whey proteins became more resistant to surfactant-induced displacement after conjugation with gellan. Furthermore, gellan molecules added to the sub-phase after the formation of a monolayer of whey proteins at the air-water interface did not adsorb to the interfacial protein film. These results provide a molecular basis for designing interfacial structures to enhance the stability of colloidal systems. Copyright © 2016 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

Temperature-Triggered Protein Adsorption on Polymer-Coated Nanoparticles in Serum.

PubMed

Koshkina, Olga; Lang, Thomas; Thiermann, Raphael; Docter, Dominic; Stauber, Roland H; Secker, Christian; Schlaad, Helmut; Weidner, Steffen; Mohr, Benjamin; Maskos, Michael; Bertin, Annabelle

2015-08-18

The protein corona, which forms on the nanoparticle's surface in most biological media, determines the nanoparticle's physicochemical characteristics. The formation of the protein corona has a significant impact on the biodistribution and clearance of nanoparticles in vivo. Therefore, the ability to influence the formation of the protein corona is essential to most biomedical applications, including drug delivery and imaging. In this study, we investigate the protein adsorption on nanoparticles with a hydrodynamic radius of 30 nm and a coating of thermoresponsive poly(2-isopropyl-2-oxazoline) in serum. Using multiangle dynamic light scattering (DLS) we demonstrate that heating of the nanoparticles above their phase separation temperature induces the formation of agglomerates, with a hydrodynamic radius of 1 μm. In serum, noticeably stronger agglomeration occurs at lower temperatures compared to serum-free conditions. Cryogenic transmission electron microscopy (cryo-TEM) revealed a high packing density of agglomerates when serum was not present. In contrast, in the presence of serum, agglomerated nanoparticles were loosely packed, indicating that proteins are intercalated between them. Moreover, an increase in protein content is observed upon heating, confirming that protein adsorption is induced by the alteration of the surface during phase separation. After cooling and switching the surface back, most of the agglomerates were dissolved and the main fraction returned to the original size of approximately 30 nm as shown by asymmetrical flow-field flow fractionation (AF-FFF) and DLS. Furthermore, the amounts of adsorbed proteins are similar before and after heating the nanoparticles to above their phase-separation temperature. Overall, our results demonstrate that the thermoresponsivity of the polymer coating enables turning the corona formation on nanoparticles on and off in situ. As the local heating of body areas can be easily done in vivo, the thermoresponsive coating could potentially be used to induce the agglomeration of nanoparticles and proteins and the accumulation of nanoparticles in a targeted body region.

Effect of thermal stability on protein adsorption to silica using homologous aldo-keto reductases

PubMed Central

Felsovalyi, Flora; Patel, Tushar; Mangiagalli, Paolo; Kumar, Sanat K; Banta, Scott

2012-01-01

Gaining more insight into the mechanisms governing the behavior of proteins at solid/liquid interfaces is particularly relevant in the interaction of high-value biologics with storage and delivery device surfaces, where adsorption-induced conformational changes may dramatically affect biocompatibility. The impact of structural stability on interfacial behavior has been previously investigated by engineering nonwild-type stability mutants. Potential shortcomings of such approaches include only modest changes in thermostability, and the introduction of changes in the topology of the proteins when disulfide bonds are incorporated. Here we employ two members of the aldo-keto reductase superfamily (alcohol dehydrogenase, AdhD and human aldose reductase, hAR) to gain a new perspective on the role of naturally occurring thermostability on adsorbed protein arrangement and its subsequent impact on desorption. Unexpectedly, we find that during initial adsorption events, both proteins have similar affinity to the substrate and undergo nearly identical levels of structural perturbation. Interesting differences between AdhD and hAR occur during desorption and both proteins exhibit some level of activity loss and irreversible conformational change upon desorption. Although such surface-induced denaturation is expected for the less stable hAR, it is remarkable that the extremely thermostable AdhD is similarly affected by adsorption-induced events. These results question the role of thermal stability as a predictor of protein adsorption/desorption behavior. PMID:22619179

Interaction of nanoparticles with proteins: relation to bio-reactivity of the nanoparticle.

PubMed

Saptarshi, Shruti R; Duschl, Albert; Lopata, Andreas L

2013-07-19

Interaction of nanoparticles with proteins is the basis of nanoparticle bio-reactivity. This interaction gives rise to the formation of a dynamic nanoparticle-protein corona. The protein corona may influence cellular uptake, inflammation, accumulation, degradation and clearance of the nanoparticles. Furthermore, the nanoparticle surface can induce conformational changes in adsorbed protein molecules which may affect the overall bio-reactivity of the nanoparticle. In depth understanding of such interactions can be directed towards generating bio-compatible nanomaterials with controlled surface characteristics in a biological environment. The main aim of this review is to summarise current knowledge on factors that influence nanoparticle-protein interactions and their implications on cellular uptake.

Heterogeneous nucleation of hydroxyapatite on protein: structural effect of silk sericin

PubMed Central

Takeuchi, Akari; Ohtsuki, Chikara; Miyazaki, Toshiki; Kamitakahara, Masanobu; Ogata, Shin-ichi; Yamazaki, Masao; Furutani, Yoshiaki; Kinoshita, Hisao; Tanihara, Masao

2005-01-01

Acidic proteins play an important role during mineral formation in biological systems, but the mechanism of mineral formation is far from understood. In this paper, we report on the relationship between the structure of a protein and hydroxyapatite deposition under biomimetic conditions. Sericin, a type of silk protein, was adopted as a suitable protein for studying structural effect on hydroxyapatite deposition, since it forms a hydroxyapatite layer on its surface in a metastable calcium phosphate solution, and its structure has been reported. Sericin effectively induced hydroxyapatite nucleation when it has high molecular weight and a β sheet structure. This indicates that the specific structure of a protein can effectively induce heterogeneous nucleation of hydroxyapatite in a biomimetic solution, i.e. a metastable calcium phosphate solution. This finding is useful in understanding biomineralization, as well as for the design of organic polymers that can effectively induce hydroxyapatite nucleation. PMID:16849195

Targeting RAS-driven human cancer cells with antibodies to upregulated and essential cell-surface proteins.

PubMed

Martinko, Alexander J; Truillet, Charles; Julien, Olivier; Diaz, Juan E; Horlbeck, Max A; Whiteley, Gordon; Blonder, Josip; Weissman, Jonathan S; Bandyopadhyay, Sourav; Evans, Michael J; Wells, James A

2018-01-23

While there have been tremendous efforts to target oncogenic RAS signaling from inside the cell, little effort has focused on the cell-surface. Here, we used quantitative surface proteomics to reveal a signature of proteins that are upregulated on cells transformed with KRAS G12V , and driven by MAPK pathway signaling. We next generated a toolkit of recombinant antibodies to seven of these RAS-induced proteins. We found that five of these proteins are broadly distributed on cancer cell lines harboring RAS mutations. In parallel, a cell-surface CRISPRi screen identified integrin and Wnt signaling proteins as critical to RAS-transformed cells. We show that antibodies targeting CDCP1, a protein common to our proteomics and CRISPRi datasets, can be leveraged to deliver cytotoxic and immunotherapeutic payloads to RAS-transformed cancer cells and report for RAS signaling status in vivo. Taken together, this work presents a technological platform for attacking RAS from outside the cell. © 2018, Martinko et al.

Evodiamine Inhibits Angiotensin II-Induced Rat Cardiomyocyte Hypertrophy.

PubMed

He, Na; Gong, Qi-Hai; Zhang, Feng; Zhang, Jing-Yi; Lin, Shu-Xian; Hou, Hua-Hua; Wu, Qin; Sun, An-Sheng

2018-05-01

To investigate the effects of evodiamine (Evo), a component of Evodiaminedia rutaecarpa (Juss.) Benth, on cardiomyocyte hypertrophy induced by angiotensin II (Ang II) and further explore the potential mechanisms. Cardiomyocytes from neonatal Sprague Dawley rats were isolated and characterized, and then the cadiomyocyte cultures were randomly divided into control, model (Ang II 0.1 μmol/L), and Evo (0.03, 0.3, 3 μmol/L) groups. The cardiomyocyte surface area, protein level, intracellular free calcium ([Ca 2+ ] i ) concentration, activity of nitric oxide synthase (NOS) and content of nitric oxide (NO) were measured, respectively. The mRNA expressions of atrial natriuretic factor (ANF), calcineurin (CaN), extracellular signal-regulated kinase-2 (ERK-2), and endothelial nitric oxide synthase (eNOS) of cardiomyocytes were analyzed by real-time reverse transcriptionpolymerase chain reaction. The protein expressions of calcineurin catalytic subunit (CnA) and mitogen-activated protein kinase phosphatase-1 (MKP-1) were detected by Western blot analysis. Compared with the control group, Ang II induced cardiomyocytes hypertrophy, as evidenced by increased cardiomyocyte surface area, protein content, and ANF mRNA expression; increased intracellular free calcium ([Ca 2+ ] i ) concentration and expressions of CaN mRNA, CnA protein, and ERK-2 mRNA, but decreased MKP-1 protein expression (P<0.05 or P<0.01). Compared with Ang II, Evo (0.3, 3 μmol/L) significantly attenuated Ang II-induced cardiomyocyte hypertrophy, decreased the [Ca 2+ ] i concentration and expressions of CaN mRNA, CnA protein, and ERK-2 mRNA, but increased MKP-1 protein expression (P<0.05 or P<0.01). Most interestingly, Evo increased the NOS activity and NO production, and upregulated the eNOS mRNA expression (P<0.05). Evo signifificantly attenuated Ang II-induced cardiomyocyte hypertrophy, and this effect was partly due to promotion of NO production, reduction of [Ca 2+ ]i concentration, and inhibition of CaN and ERK-2 signal transduction pathways.

Flow cytometry: a promising technique for the study of silicone oil-induced particulate formation in protein formulations.

PubMed

Ludwig, D Brett; Trotter, Joseph T; Gabrielson, John P; Carpenter, John F; Randolph, Theodore W

2011-03-15

Subvisible particles in formulations intended for parenteral administration are of concern in the biopharmaceutical industry. However, monitoring and control of subvisible particulates can be complicated by formulation components, such as the silicone oil used for the lubrication of prefilled syringes, and it is difficult to differentiate microdroplets of silicone oil from particles formed by aggregated protein. In this study, we demonstrate the ability of flow cytometry to resolve mixtures comprising subvisible bovine serum albumin (BSA) aggregate particles and silicone oil emulsion droplets with adsorbed BSA. Flow cytometry was also used to investigate the effects of silicone oil emulsions on the stability of BSA, lysozyme, abatacept, and trastuzumab formulations containing surfactant, sodium chloride, or sucrose. To aid in particle characterization, the fluorescence detection capabilities of flow cytometry were exploited by staining silicone oil with BODIPY 493/503 and model proteins with Alexa Fluor 647. Flow cytometric analyses revealed that silicone oil emulsions induced the loss of soluble protein via protein adsorption onto the silicone oil droplet surface. The addition of surfactant prevented protein from adsorbing onto the surface of silicone oil droplets. There was minimal formation of homogeneous protein aggregates due to exposure to silicone oil droplets, although oil droplets with surface-adsorbed trastuzumab exhibited flocculation. The results of this study demonstrate the utility of flow cytometry as an analytical tool for monitoring the effects of subvisible silicone oil droplets on the stability of protein formulations. Copyright © 2010 Elsevier Inc. All rights reserved.

The Phospholipid:Diacylglycerol Acyltransferase Lro1 Is Responsible for Hepatitis C Virus Core-Induced Lipid Droplet Formation in a Yeast Model System

PubMed Central

Wang, Chao-Wen; Cheng, Yun-Hsin; Irokawa, Hayato; Hwang, Gi-Wook; Naganuma, Akira; Kuge, Shusuke

2016-01-01

Chronic infection with the hepatitis C virus frequently induces steatosis, which is a significant risk factor for liver pathogenesis. Steatosis is characterized by the accumulation of lipid droplets in hepatocytes. The structural protein core of the virus induces lipid droplet formation and localizes on the surface of the lipid droplets. However, the precise molecular mechanisms for the core-induced formation of lipid droplets remain elusive. Recently, we showed that the expression of the core protein in yeast as a model system could induce lipid droplet formation. In this study, we probed the cellular factors responsible for the formation of core-induced lipid-droplets in yeast cells. We demonstrated that one of the enzymes responsible for triglyceride synthesis, a phospholipid:diacylglycerol acyltransferase (Lro1), is required for the core-induced lipid droplet formation. While core proteins inhibit Lro1 degradation and alter Lro1 localization, the characteristic localization of Lro1 adjacent to the lipid droplets appeared to be responsible for the core-induced lipid droplet formation. RNA virus genomes have evolved using high mutation rates to maintain their ability to replicate. Our observations suggest a functional relationship between the core protein with hepatocytes and yeast cells. The possible interactions between core proteins and the endoplasmic reticulum membrane affect the mobilization of specific proteins. PMID:27459103

Partial molar volume of proteins studied by the three-dimensional reference interaction site model theory.

PubMed

Imai, Takashi; Kovalenko, Andriy; Hirata, Fumio

2005-04-14

The three-dimensional reference interaction site model (3D-RISM) theory is applied to the analysis of hydration effects on the partial molar volume of proteins. For the native structure of some proteins, the partial molar volume is decomposed into geometric and hydration contributions using the 3D-RISM theory combined with the geometric volume calculation. The hydration contributions are correlated with the surface properties of the protein. The thermal volume, which is the volume of voids around the protein induced by the thermal fluctuation of water molecules, is directly proportional to the accessible surface area of the protein. The interaction volume, which is the contribution of electrostatic interactions between the protein and water molecules, is apparently governed by the charged atomic groups on the protein surface. The polar atomic groups do not make any contribution to the interaction volume. The volume differences between low- and high-pressure structures of lysozyme are also analyzed by the present method.

Human Enterovirus 71 Protein Displayed on the Surface of Saccharomyces cerevisiae as an Oral Vaccine.

PubMed

Zhang, Congdang; Wang, Yi; Ma, Shuzhi; Li, Leike; Chen, Liyun; Yan, Huimin; Peng, Tao

2016-06-01

Human enterovirus 71 (EV-A71), a major agent of hand, foot, and mouth disease, has become an important public health issue in recent years. No effective antiviral or vaccines against EV-A71 infection are currently available. EV-A71 infection intrudes bodies through the gastric mucosal surface and it is necessary to enhance mucosal immune response to protect children from these pathogens. Recently, the majority of EV-A71 vaccine candidates have been developed for parenteral immunization. However, parenteral vaccine candidates often induce poor mucosal responses. On the other hand, oral vaccines could induce effective mucosal and systemic immunity, and could be easily and safely administered. Thus, proper oral vaccines have attached more interest compared with parenteral vaccine. In this study, the major immunogenic capsid protein of EV-A71 was displayed on the surface of Saccharomyces cerevisiae. Oral immunization of mice with surface-displayed VP1 S. cerevisiae induced systemic humoral and mucosal immune responses, including virus-neutralizing titers, VP1-specific antibody, and the induction of Th1 immune responses in the spleen. Furthermore, oral immunization of mother mice with surface-displayed VP1 S. cerevisiae conferred protection to neonatal mice against the lethal EV-A71 infection. Furthermore, we observed that multiple boost immunization as well as higher immunization dosage could induce higher EV-A71-specific immune response. Our results demonstrated that surface-displayed VP1 S. cerevisiae could be used as potential oral vaccine against EV-A71 infection.

HFE interacts with the BMP type I receptor ALK3 to regulate hepcidin expression

PubMed Central

Wu, Xing-gang; Wang, Yang; Wu, Qian; Cheng, Wai-Hang; Liu, Wenjing; Zhao, Yueshui; Mayeur, Claire; Schmidt, Paul J.; Yu, Paul B.; Wang, Fudi

2014-01-01

Mutations in HFE are the most common cause of hereditary hemochromatosis (HH). HFE mutations result in reduced expression of hepcidin, a hepatic hormone, which negatively regulates iron absorption from the duodenum and iron release from macrophages. However, the mechanism by which HFE regulates hepcidin expression in hepatocytes is not well understood. It is known that the bone morphogenetic protein (BMP) pathway plays a central role in controlling hepcidin expression in the liver. Here we show that HFE overexpression increased Smad1/5/8 phosphorylation and hepcidin expression, whereas inhibition of BMP signaling abolished HFE-induced hepcidin expression in Hep3B cells. HFE was found to associate with ALK3, inhibiting ALK3 ubiquitination and proteasomal degradation and increasing ALK3 protein expression and accumulation on the cell surface. The 2 HFE mutants associated with HH, HFE C282Y and HFE H63D, regulated ALK3 protein ubiquitination and trafficking differently, but both failed to increase ALK3 cell-surface expression. Deletion of Hfe in mice resulted in a decrease in hepatic ALK3 protein expression. Our results provide evidence that HFE induces hepcidin expression via the BMP pathway: HFE interacts with ALK3 to stabilize ALK3 protein and increase ALK3 expression at the cell surface. PMID:24904118

Vaccine approaches conferring cross-protection against influenza viruses

PubMed Central

Vemula, Sai V.; Sayedahmed, Ekramy E; Sambhara, Suryaprakash; Mittal, Suresh K.

2018-01-01

Introduction Annual vaccination is one of the most efficient and cost-effective strategies to prevent and control influenza epidemics. Most of currently available influenza vaccines are strong inducer of antibody responses against viral surface proteins, hemagglutinin (HA) and neuraminidase (NA), but are poor inducers of cell-mediated immune responses against conserved internal proteins. Moreover, due to the high variability of viral surface proteins because of antigenic drift or antigenic shift, many of the currently licensed vaccines confer little or no protection against drift or shift variants. Areas covered Next generation influenza vaccines that can induce humoral immune responses to receptor-binding epitopes as well as broadly neutralizing conserved epitopes, and cell-mediated immune responses against highly conserved internal proteins would be effective against variant viruses as well as a novel pandemic influenza until circulating strain-specific vaccines become available. Here we discuss vaccine approaches that have potential to provide broad spectrum protection against influenza viruses. Expert opinion Based on current progress in defining cross-protective influenza immunity, it seems that the development of a universal influenza vaccine is feasible. It would revolutionize the strategy for influenza pandemic preparedness, and significantly impact the shelf-life and protection efficacy of seasonal influenza vaccines. PMID:28925296

Upregulation of cell proliferation via Shc and ERK1/2 MAPK signaling in SaOS-2 osteoblasts grown on magnesium alloy surface coating with tricalcium phosphate.

PubMed

Jiang, Tianlong; Guo, Lei; Ni, Shenghui; Zhao, Yuyan

2015-04-01

Magnesium (Mg) alloys have been demonstrated to be viable orthopedic implants because of mechanical and biocompatible properties similar to natural bone. In order to improve its osteogenic properties, a porous β-tricalcium phosphate (β-TCP) was coated on the Mg-3AI-1Zn alloy by alkali-heat treatment technique. The human bone-derived cells (SaOS-2) were cultured on (β-TCP)-Mg-3AI-1Zn in vitro, and the osteoblast response, the morphology and the elements on this alloy surface were investigated. Also, the regulation of key intracellular signalling proteins was investigated in the SaOS-2 cells cultured on alloy surface. The results from scanning electron microscope and immunofluorescence staining demonstrated that (β-TCP)-Mg-3AI-1Zn induced significant osteogenesis. SaOS-2 cell proliferation was improved by β-TCP coating. Moreover, the (β-TCP)-Mg-3AI-1Zn surface induced activation of key intracellular signalling proteins in SaOS-2 cells. We observed an enhanced activation of Src homology and collagen (Shc), a common point of integration between bone morphogenetic protein 2, and the Ras/mitogen-activated protein kinase (MAPK) pathway. ERK1/2 MAP kinase activation was also upregulated, suggesting a role in mediating osteoblastic cell interactions with biomaterials. The signalling pathway involving c-fos (member of the activated protein-1) was also shown to be upregulated in osteoblasts cultured on the (β-TCP)-Mg-3AI-1Zn. These results suggest that β-TCP coating may contribute to successful osteoblast function on Mg alloy surface. (β-TCP)-Mg-3AI-1Zn may upregulate cell proliferation via Shc and ERK1/2 MAPK signaling in SaOS-2 osteoblasts grown on Mg alloy surface.

Surface-Induced Dissociation of Protein Complexes in a Hybrid Fourier Transform Ion Cyclotron Resonance Mass Spectrometer.

PubMed

Yan, Jing; Zhou, Mowei; Gilbert, Joshua D; Wolff, Jeremy J; Somogyi, Árpád; Pedder, Randall E; Quintyn, Royston S; Morrison, Lindsay J; Easterling, Michael L; Paša-Tolić, Ljiljana; Wysocki, Vicki H

2017-01-03

Mass spectrometry continues to develop as a valuable tool in the analysis of proteins and protein complexes. In protein complex mass spectrometry studies, surface-induced dissociation (SID) has been successfully applied in quadrupole time-of-flight (Q-TOF) instruments. SID provides structural information on noncovalent protein complexes that is complementary to other techniques. However, the mass resolution of Q-TOF instruments can limit the information that can be obtained for protein complexes by SID. Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS) provides ultrahigh resolution and ultrahigh mass accuracy measurements. In this study, an SID device was designed and successfully installed in a hybrid FT-ICR instrument in place of the standard gas collision cell. The SID-FT-ICR platform has been tested with several protein complex systems (homooligomers, a heterooligomer, and a protein-ligand complex, ranging from 53 to 85 kDa), and the results are consistent with data previously acquired on Q-TOF platforms, matching predictions from known protein interface information. SID fragments with the same m/z but different charge states are well-resolved based on distinct spacing between adjacent isotope peaks, and the addition of metal cations and ligands can also be isotopically resolved with the ultrahigh mass resolution available in FT-ICR.

Induction of Caveolae in the Apical Plasma Membrane of Madin-Darby Canine Kidney Cells

PubMed Central

Verkade, Paul; Harder, Thomas; Lafont, Frank; Simons, Kai

2000-01-01

In this paper, we have analyzed the behavior of antibody cross-linked raft-associated proteins on the surface of MDCK cells. We observed that cross-linking of membrane proteins gave different results depending on whether cross-linking occurred on the apical or basolateral plasma membrane. Whereas antibody cross-linking induced the formation of large clusters on the basolateral membrane, resembling those observed on the surface of fibroblasts (Harder, T., P. Scheiffele, P. Verkade, and K. Simons. 1998. J. Cell Biol. 929–942), only small (∼100 nm) clusters formed on the apical plasma membrane. Cross-linked apical raft proteins e.g., GPI-anchored placental alkaline phosphatase (PLAP), influenza hemagglutinin, and gp114 coclustered and were internalized slowly (∼10% after 60 min). Endocytosis occurred through surface invaginations that corresponded in size to caveolae and were labeled with caveolin-1 antibodies. Upon cholesterol depletion the internalization of PLAP was completely inhibited. In contrast, when a non-raft protein, the mutant LDL receptor LDLR-CT22, was cross-linked, it was excluded from the clusters of raft proteins and was rapidly internalized via clathrin-coated pits. Since caveolae are normally present on the basolateral membrane but lacking from the apical side, our data demonstrate that antibody cross-linking induced the formation of caveolae, which slowly internalized cross-linked clusters of raft-associated proteins. PMID:10684254

Imparting albumin-binding affinity to a human protein by mimicking the contact surface of a bacterial binding protein.

PubMed

Oshiro, Satoshi; Honda, Shinya

2014-04-18

Attachment of a bacterial albumin-binding protein module is an attractive strategy for extending the plasma residence time of protein therapeutics. However, a protein fused with such a bacterial module could induce unfavorable immune reactions. To address this, we designed an alternative binding protein by imparting albumin-binding affinity to a human protein using molecular surface grafting. The result was a series of human-derived 6 helix-bundle proteins, one of which specifically binds to human serum albumin (HSA) with adequate affinity (KD = 100 nM). The proteins were designed by transferring key binding residues of a bacterial albumin-binding module, Finegoldia magna protein G-related albumin-binding domain (GA) module, onto the human protein scaffold. Despite 13-15 mutations, the designed proteins maintain the original secondary structure by virtue of careful grafting based on structural informatics. Competitive binding assays and thermodynamic analyses of the best binders show that the binding mode resembles that of the GA module, suggesting that the contacting surface of the GA module is mimicked well on the designed protein. These results indicate that the designed protein may act as an alternative low-risk binding module to HSA. Furthermore, molecular surface grafting in combination with structural informatics is an effective approach for avoiding deleterious mutations on a target protein and for imparting the binding function of one protein onto another.

Isthmin is a novel vascular permeability inducer that functions through cell-surface GRP78-mediated Src activation.

PubMed

Venugopal, Shruthi; Chen, Mo; Liao, Wupeng; Er, Shi Yin; Wong, Wai-Shiu Fred; Ge, Ruowen

2015-07-01

Isthmin (ISM) is a recently identified 60 kDa secreted angiogenesis inhibitor. Two cell-surface receptors for ISM have been defined, the high-affinity glucose-regulated protein 78 kDa (GRP78) and the low-affinity αvβ5 integrin. As αvβ5 integrin plays an important role in pulmonary vascular permeability (VP) and ISM is highly expressed in mouse lung, we sought to clarify the role of ISM in VP. Recombinant ISM (rISM) dose-dependently enhances endothelial monolayer permeability in vitro and local dermal VP when administered intradermally in mice. Systemic rISM administration through intravenous injection leads to profound lung vascular hyperpermeability but not in other organs. Mechanistic investigations using molecular, biochemical approaches and specific chemical inhibitors revealed that ISM-GRP78 interaction triggers a direct interaction between GRP78 and Src, leading to Src activation and subsequent phosphorylation of adherens junction proteins and loss of junctional proteins from inter-endothelial junctions, resulting in enhanced VP. Dynamic studies of Src activation, VP and apoptosis revealed that ISM induces VP directly via Src activation while apoptosis contributes indirectly only after prolonged treatment. Furthermore, ISM is significantly up-regulated in lipopolysaccharide (LPS)-treated mouse lung. Blocking cell-surface GRP78 by systemic infusion of anti-GRP78 antibody significantly attenuates pulmonary vascular hyperpermeability in LPS-induced acute lung injury (ALI) in mice. ISM is a novel VP inducer that functions through cell-surface GRP78-mediated Src activation as well as induction of apoptosis. It induces a direct GRP78-Src interaction, leading to cytoplasmic Src activation. ISM contributes to pulmonary vascular hyperpermeability of LPS-induced ALI in mice. Published on behalf of the European Society of Cardiology. All rights reserved. © The Author 2015. For permissions please email: journals.permissions@oup.com.

Conformational transition free energy profiles of an adsorbed, lattice model protein by multicanonical Monte Carlo simulation

NASA Astrophysics Data System (ADS)

Castells, Victoria; Van Tassel, Paul R.

2005-02-01

Proteins often undergo changes in internal conformation upon interacting with a surface. We investigate the thermodynamics of surface induced conformational change in a lattice model protein using a multicanonical Monte Carlo method. The protein is a linear heteropolymer of 27 segments (of types A and B) confined to a cubic lattice. The segmental order and nearest neighbor contact energies are chosen to yield, in the absence of an adsorbing surface, a unique 3×3×3 folded structure. The surface is a plane of sites interacting either equally with A and B segments (equal affinity surface) or more strongly with the A segments (A affinity surface). We use a multicanonical Monte Carlo algorithm, with configuration bias and jump walking moves, featuring an iteratively updated sampling function that converges to the reciprocal of the density of states 1/Ω(E), E being the potential energy. We find inflection points in the configurational entropy, S(E)=klnΩ(E), for all but a strongly adsorbing equal affinity surface, indicating the presence of free energy barriers to transition. When protein-surface interactions are weak, the free energy profiles F(E)=E-TS(E) qualitatively resemble those of a protein in the absence of a surface: a free energy barrier separates a folded, lowest energy state from globular, higher energy states. The surface acts in this case to stabilize the globular states relative to the folded state. When the protein surface interactions are stronger, the situation differs markedly: the folded state no longer occurs at the lowest energy and free energy barriers may be absent altogether.

Nanoscale analysis of caspofungin-induced cell surface remodelling in Candida albicans

NASA Astrophysics Data System (ADS)

El-Kirat-Chatel, Sofiane; Beaussart, Audrey; Alsteens, David; Jackson, Desmond N.; Lipke, Peter N.; Dufrêne, Yves F.

2013-01-01

The advent of fungal pathogens that are resistant to the classic repertoire of antifungal drugs has increased the need for new therapeutic agents. A prominent example of such a novel compound is caspofungin, known to alter cell wall biogenesis by inhibiting β-1,3-d-glucan synthesis. Although much progress has been made in understanding the mechanism of action of caspofungin, little is known about its influence on the biophysical properties of the fungal cells. Here, we use atomic force microscopy (AFM) to demonstrate that caspofungin induces major remodelling of the cell surface properties of Candida albicans. Caspofungin causes major morphological and structural alterations of the cells, which correlate with a decrease of the cell wall mechanical strength. Moreover, we find that the drug induces the massive exposure of the cell adhesion protein Als1 on the cell surface and leads to increased cell surface hydrophobicity, two features that trigger cell aggregation. This behaviour is not observed in yeast species lacking Als1, demonstrating the key role that the protein plays in determining the aggregation phenotype of C. albicans. The results show that AFM opens up new avenues for understanding the molecular bases of microbe-drug interactions and for developing new therapeutic agents.The advent of fungal pathogens that are resistant to the classic repertoire of antifungal drugs has increased the need for new therapeutic agents. A prominent example of such a novel compound is caspofungin, known to alter cell wall biogenesis by inhibiting β-1,3-d-glucan synthesis. Although much progress has been made in understanding the mechanism of action of caspofungin, little is known about its influence on the biophysical properties of the fungal cells. Here, we use atomic force microscopy (AFM) to demonstrate that caspofungin induces major remodelling of the cell surface properties of Candida albicans. Caspofungin causes major morphological and structural alterations of the cells, which correlate with a decrease of the cell wall mechanical strength. Moreover, we find that the drug induces the massive exposure of the cell adhesion protein Als1 on the cell surface and leads to increased cell surface hydrophobicity, two features that trigger cell aggregation. This behaviour is not observed in yeast species lacking Als1, demonstrating the key role that the protein plays in determining the aggregation phenotype of C. albicans. The results show that AFM opens up new avenues for understanding the molecular bases of microbe-drug interactions and for developing new therapeutic agents. Electronic supplementary information (ESI) available. See DOI: 10.1039/c2nr33215a

Activation of p44/42 in Human Natural Killer Cells Decreases Cell-surface Protein Expression: Relationship to Tributyltin-induced alterations of protein expression

PubMed Central

Dudimah, Fred D.; Abraha, Abraham; Wang, Xiaofei; Whalen, Margaret M.

2010-01-01

Tributyltin (TBT) activates the mitogen activated protein kinase (MAPK), p44/42 in human natural killer (NK) cells. TBT also reduces NK cytotoxic function and decreases the expression of several NK-cell proteins. To understand the role that p44/42 activation plays in TBT-induced loss of NK cell function, we have investigated how selective activation of p44/42 by phorbol 12-myristate 13-acetate (PMA) affects NK cells. Previously we showed that PMA caused losses of lytic function similar to those seen with TBT exposures. Here we examined activation of p44/42 in the regulation of NK-cell protein expression and how this regulation may explain the protein expression changes seen with TBT exposures. NK cells exposed to PMA were examined for levels of cell-surface proteins, granzyme mRNA, and perforin mRNA expression. The expression of CD11a, CD16, CD18, and CD56 were reduced, perforin mRNA levels were unchanged and granzyme mRNA levels were increased. To verify that activation of p44/42 was responsible for the alterations seen in CD11a, CD16, CD18, and CD56 with PMA, NK cells were treated with the p44/42 pathway inhibitor (PD98059) prior to PMA exposures. In the presence of PD98059, PMA caused no decreases in the expression of the cell-surface proteins. Results of these studies indicate that the activation of p44/42 may lead to the loss of NK cell cytotoxic function by decreasing the expression of CD11a, CD16, CD18, and CD56. Further, activation of p44/42 appears to be at least in part responsible for the TBT-induced decreases in expression of CD16, CD18, and CD56. PMID:20883105

Lipid raft-associated β-adducin is required for PSGL-1-mediated neutrophil rolling on P-selectin.

PubMed

Xu, Tingshuang; Liu, Wenai; Yang, Chen; Ba, Xueqing; Wang, Xiaoguang; Jiang, Yong; Zeng, Xianlu

2015-02-01

Lipid rafts, a liquid-ordered plasma membrane microdomain, are related to cell-surface receptor function. PSGL-1, a major surface receptor protein for leukocyte, also acts as a signaling receptor in leukocyte rolling. To investigate the role of lipid raft in PSGL-1 signaling in human neutrophils, we quantitatively analyzed lipid raft proteome of human promyelocytic leukemia cell line HL-60 cells and identified a lipid raft-associated protein β-adducin. PSGL-1 ligation induced dissociation of the raft-associated protein β-adducin from lipid rafts and actin, as well as phosphorylation of β-adducin, indicating a transient uncoupling of lipid rafts from the actin cytoskeleton. Knockdown of β-adducin greatly attenuated HL-60 cells rolling on P-selectin. We also showed that Src kinase is crucial for PSGL-1 ligation-induced β-adducin phosphorylation and relocation. Taken together, these results show that β-adducin is a pivotal lipid raft-associated protein in PSGL-1-mediated neutrophil rolling on P-selectin. © Society for Leukocyte Biology.

Dipolar response of hydrated proteins

NASA Astrophysics Data System (ADS)

Matyushov, Dmitry V.

2012-02-01

The paper presents an analytical theory and numerical simulations of the dipolar response of hydrated proteins in solution. We calculate the effective dielectric constant representing the average dipole moment induced at the protein by a uniform external field. The dielectric constant shows a remarkable variation among the proteins, changing from 0.5 for ubiquitin to 640 for cytochrome c. The former value implies a negative dipolar susceptibility, that is a dia-electric dipolar response and negative dielectrophoresis. It means that ubiquitin, carrying an average dipole of ≃240 D, is expected to repel from the region of a stronger electric field. This outcome is the result of a negative cross-correlation between the protein and water dipoles, compensating for the positive variance of the intrinsic protein dipole in the overall dipolar susceptibility. In contrast to the neutral ubiquitin, charged proteins studied here show para-electric dipolar response and positive dielectrophoresis. The study suggests that the dipolar response of proteins in solution is strongly affected by the coupling of the protein surface charge to the hydration water. The protein-water dipolar cross-correlations are long-ranged, extending ˜2 nm from the protein surface into the bulk. A similar correlation length of about 1 nm is seen for the electrostatic potential produced by the hydration water inside the protein. The analysis of numerical simulations suggests that the polarization of the protein-water interface is highly heterogeneous and does not follow the standard dielectric results for cavities carved in dielectrics. The polarization of the water shell gains in importance, relative to the intrinsic protein dipole, at high frequencies, above the protein Debye peak. The induced interfacial dipole can be either parallel or antiparallel to the protein dipole, depending on the distribution of the protein surface charge. As a result, the high-frequency absorption of the protein solution can be either higher or lower than the absorption of water. Both scenarios have been experimentally observed in the THz window of radiation.

Dipolar response of hydrated proteins.

PubMed

Matyushov, Dmitry V

2012-02-28

The paper presents an analytical theory and numerical simulations of the dipolar response of hydrated proteins in solution. We calculate the effective dielectric constant representing the average dipole moment induced at the protein by a uniform external field. The dielectric constant shows a remarkable variation among the proteins, changing from 0.5 for ubiquitin to 640 for cytochrome c. The former value implies a negative dipolar susceptibility, that is a dia-electric dipolar response and negative dielectrophoresis. It means that ubiquitin, carrying an average dipole of ≃240 D, is expected to repel from the region of a stronger electric field. This outcome is the result of a negative cross-correlation between the protein and water dipoles, compensating for the positive variance of the intrinsic protein dipole in the overall dipolar susceptibility. In contrast to the neutral ubiquitin, charged proteins studied here show para-electric dipolar response and positive dielectrophoresis. The study suggests that the dipolar response of proteins in solution is strongly affected by the coupling of the protein surface charge to the hydration water. The protein-water dipolar cross-correlations are long-ranged, extending ~2 nm from the protein surface into the bulk. A similar correlation length of about 1 nm is seen for the electrostatic potential produced by the hydration water inside the protein. The analysis of numerical simulations suggests that the polarization of the protein-water interface is highly heterogeneous and does not follow the standard dielectric results for cavities carved in dielectrics. The polarization of the water shell gains in importance, relative to the intrinsic protein dipole, at high frequencies, above the protein Debye peak. The induced interfacial dipole can be either parallel or antiparallel to the protein dipole, depending on the distribution of the protein surface charge. As a result, the high-frequency absorption of the protein solution can be either higher or lower than the absorption of water. Both scenarios have been experimentally observed in the THz window of radiation.

Elucidation of metal-ion accumulation induced by hydrogen bonds on protein surfaces by using porous lysozyme crystals containing Rh(III) ions as the model surfaces.

PubMed

Ueno, Takafumi; Abe, Satoshi; Koshiyama, Tomomi; Ohki, Takahiro; Hikage, Tatsuo; Watanabe, Yoshihito

2010-03-01

Metal-ion accumulation on protein surfaces is a crucial step in the initiation of small-metal clusters and the formation of inorganic materials in nature. This event is expected to control the nucleation, growth, and position of the materials. There remain many unknowns, as to how proteins affect the initial process at the atomic level, although multistep assembly processes of the materials formation by both native and model systems have been clarified at the macroscopic level. Herein the cooperative effects of amino acids and hydrogen bonds promoting metal accumulation reactions are clarified by using porous hen egg white lysozyme (HEWL) crystals containing Rh(III) ions, as model protein surfaces for the reactions. The experimental results reveal noteworthy implications for initiation of metal accumulation, which involve highly cooperative dynamics of amino acids and hydrogen bonds: i) Disruption of hydrogen bonds can induce conformational changes of amino-acid residues to capture Rh(III) ions. ii) Water molecules pre-organized by hydrogen bonds can stabilize Rh(III) coordination as aqua ligands. iii) Water molecules participating in hydrogen bonds with amino-acid residues can be replaced by Rh(III) ions to form polynuclear structures with the residues. iv) Rh(III) aqua complexes are retained on amino-acid residues through stabilizing hydrogen bonds even at low pH (approximately 2). These metal-protein interactions including hydrogen bonds may promote native metal accumulation reactions and also may be useful in the preparation of new inorganic materials that incorporate proteins.

Molecular dynamics simulations of the adsorption of bone morphogenetic protein-2 on surfaces with medical relevance.

PubMed

Utesch, Tillmann; Daminelli, Grazia; Mroginski, Maria Andrea

2011-11-01

Bone morphogenetic protein-2 (BMP-2) plays a crucial role in osteoblast differentiation and proliferation. Its effective therapeutic use for ectopic bone and cartilage regeneration depends, among other factors, on the interaction with the carrier at the implant site. In this study, we used classical molecular dynamics (MD) and a hybrid approach of steered molecular dynamics (SMD) combined with MD simulations to investigate the initial stages of the adsorption of BMP-2 when approaching two implant surfaces, hydrophobic graphite and hydrophilic titanium dioxide rutile. Surface adsorption was evaluated for six different orientations of the protein, two end-on and four side-on, in explicit water environment. On graphite, we observed a weak but stable adsorption. Depending on the initial orientation, hydrophobic patches as well as flexible loops of the protein were involved in the interaction with graphite. On the contrary, BMP-2 adsorbed only loosely to hydrophilic titanium dioxide. Despite a favorable interaction energy between protein and the TiO(2) surface, the rapid formation of a two-layer water structure prevented the direct interaction between protein and titanium dioxide. The first water adlayer had a strong repulsive effect on the protein, while the second attracted the protein toward the surface. For both surfaces, hydrophobic graphite and hydrophilic titanium dioxide, denaturation of BMP-2 induced by adsorption was not observed on the nanosecond time scale.

GW domains of the Listeria monocytogenes invasion protein InlB are SH3-like and mediate binding to host ligands

PubMed Central

Marino, Michael; Banerjee, Manidipa; Jonquières, Renaud; Cossart, Pascale; Ghosh, Partho

2002-01-01

InlB, a surface-localized protein of Listeria monocytogenes, induces phagocytosis in non-phagocytic mammalian cells by activating Met, a receptor tyrosine kinase. InlB also binds glycosaminoglycans and the protein gC1q-R, two additional host ligands implicated in invasion. We present the structure of InlB, revealing a highly elongated molecule with leucine-rich repeats that bind Met at one end, and GW domains that dissociably bind the bacterial surface at the other. Surprisingly, the GW domains are seen to resemble SH3 domains. Despite this, GW domains are unlikely to act as functional mimics of SH3 domains since their potential proline-binding sites are blocked or destroyed. However, we do show that the GW domains, in addition to binding glycosaminoglycans, bind gC1q-R specifically, and that this binding requires release of InlB from the bacterial surface. Dissociable attachment to the bacterial surface via the GW domains may be responsible for restricting Met activation to a small, localized area of the host cell and for coupling InlB-induced host membrane dynamics with bacterial proximity during invasion. PMID:12411480

Molecular modelling study of changes induced by netropsin binding to nucleosome core particles.

PubMed Central

Pérez, J J; Portugal, J

1990-01-01

It is well known that certain sequence-dependent modulators in structure appear to determine the rotational positioning of DNA on the nucleosome core particle. That preference is rather weak and could be modified by some ligands as netropsin, a minor-groove binding antibiotic. We have undertaken a molecular modelling approach to calculate the relative energy of interaction between a DNA molecule and the protein core particle. The histones particle is considered as a distribution of positive charges on the protein surface that interacts with the DNA molecule. The molecular electrostatic potentials for the DNA, simulated as a discontinuous cylinder, were calculated using the values for all the base pairs. Computing these parameters, we calculated the relative energy of interaction and the more stable rotational setting of DNA. The binding of four molecules of netropsin to this model showed that a new minimum of energy is obtained when the DNA turns toward the protein surface by about 180 degrees, so a new energetically favoured structure appears where netropsin binding sites are located facing toward the histones surface. The effect of netropsin could be explained in terms of an induced change in the phasing of DNA on the core particle. The induced rotation is considered to optimize non-bonded contacts between the netropsin molecules and the DNA backbone. PMID:2165249

Denaturation of proteins near polar surfaces

NASA Astrophysics Data System (ADS)

Starzyk, Anna; Cieplak, Marek

2011-12-01

All-atom molecular dynamics simulations for proteins placed near a model mica surface indicate existence of two types of evolution. One type leads to the surface-induced unfolding and the other just to a deformation. The two behaviors are characterized by distinct properties of the radius of gyration and of a novel distortion parameter that distinguishes between elongated, globular, and planar shapes. They also differ in the nature of their single site diffusion and two-site distance fluctuations. The four proteins chosen for the studies, the tryptophan cage, protein G, hydrophobin and lyzozyme, are small to allow for a fair determination of the forces generated by the surface as the effects of finite cutoffs in the Coulombic interactions are thus minimized. When the net charge on the surface is set to zero artificially, infliction of deformation is seen to persists but no unfolding takes place. Unfolding may also be prevented by a cluster of disulfide bonds, as we observe in simulations of hydrophobin.

Surface-mediated molecular events in material-induced blood-plasma coagulation

NASA Astrophysics Data System (ADS)

Chatterjee, Kaushik

Coagulation and thrombosis persist as major impediments associated with the use of blood-contacting medical devices. We are investigating the molecular mechanism underlying material-induced blood-plasma coagulation focusing on the role of the surface as a step towards prospective development of improved hemocompatible biomaterials. A classic observation in hematology is that blood/blood-plasma in contact with clean glass surface clots faster than when in contact with many plastic surfaces. The traditional biochemical theory explaining the underlying molecular mechanism suggests that hydrophilic surfaces, like that of glass, are specific activators of the coagulation cascade because of the negatively-charged groups on the surface. Hydrophobic surfaces are poor procoagulants or essentially "benign" because they lack anionic groups. Further, these negatively-charged surfaces are believed to not only activate blood factor XII (FXII), the key protein in contact activation, but also play a cofactor role in the amplification and propagation reactions that ultimately lead to clot formation. In sharp contrast to the traditional theory, our investigations indicate a need for a paradigm shift in the proposed sequence of contact activation events to incorporate the role of protein adsorption at the material surfaces. These studies have lead to the central hypothesis for this work proposing that protein adsorption to hydrophobic surfaces attenuates the contact activation reactions so that poorly-adsorbent hydrophilic surfaces appear to be stronger procoagulants relative to hydrophobic surfaces. Our preliminary studies measuring the plasma coagulation response of activated FXII (FXIIa) on different model surfaces suggested that the material did not play a cofactor role in the processing of this enzyme dose through the coagulation pathway. Therefore, we focused our efforts on studying the mechanism of initial production of enzyme at the procoagulant surface. Calculations for the amounts of FXIIa generated at material surfaces in plasma using a mathematical model for measured coagulation responses indicate that the relative contributions of the individual pathways of enzyme generation are similar at both hydrophilic and hydrophobic surfaces, only the amounts of enzyme generated scale with surface energy and area of the activating surface. Further, from direct measurement of enzyme activation at test surfaces we observed that contact activation reactions are not specific to negatively-charged hydrophilic surfaces. Rather, the molecular interactions are attenuated at hydrophobic surfaces due to protein adsorption so that poorly-adsorbent hydrophilic surfaces exhibit an apparent specificity for contact activation reactions. Preliminary studies were preformed to assay the plasma coagulation response to low-fouling surfaces prepared by either grafting poly(ethylene glycol) chains or using zwitterions. Results indicate that poly(ethylene glycol)-modified surfaces are significantly weaker procoagulants than surfaces containing zwitterions underscoring a need to specifically evaluate the coagulation response despite similarities in observed protein adsorption to both surfaces. In summary, our studies demonstrate a need to incorporate protein-adsorption competition at procoagulant surfaces into the mechanism of contact activation to account for the observed moderation of FXII activation by blood proteins unrelated to the plasma coagulation cascade.

[Construction of Lactobacillus rhamnosus GG particles surface display system].

PubMed

Su, Runyu; Nie, Boyao; Yuan, Shengling; Tao, Haoxia; Liu, Chunjie; Yang, Bailiang; Wang, Yanchun

2017-01-25

To describe a novel particles surface display system which is consisted of gram-positive enhancer matrix (GEM) particles and anchor proteins for bacteria-like particles vaccines, we treated Lactobacillus rhamnosus GG bacteria with 10% heated-TCA for preparing GEM particles, and then identified the harvested GEM particles by electron microscopy, RT-PCR and SDS-PAGE. Meanwhile, Escherichia coli was induced to express hybrid proteins PA3-EGFP and P60-EGFP, and GEM particles were incubated with them. Then binding of anchor proteins were determined by Western blotting, transmission electron microscopy, fluorescence microscopy and spectrofluorometry. GEM particles preserved original size and shape, and proteins and DNA contents of GEM particles were released substantially. The two anchor proteins both had efficiently immobilized on the surface of GEM. GEM particles that were bounded by anchor proteins were brushy. The fluorescence of GEM particles anchoring PA3 was slightly brighter than P60, but the difference was not significant (P>0.05). GEM particles prepared from L. rhamnosus GG have a good binding efficiency with anchor proteins PA3-EGFP and P60-EGFP. Therefore, this novel foreign protein surface display system could be used for bacteria-like particle vaccines.

Effects of surfaces and leachables on the stability of biopharmaceuticals.

PubMed

Bee, Jared S; Randolph, Theodore W; Carpenter, John F; Bishop, Steven M; Dimitrova, Mariana N

2011-10-01

Therapeutic proteins are exposed to various potential contact surfaces, particles, and leachables during manufacturing, shipping, storage, and delivery. In this review, we present published examples of interfacial- or leachable-induced aggregation or particle formation, and discuss the mitigation strategies that were successfully utilized. Adsorption to interfaces or interactions with leachables and/or particles in some cases has been reported to cause protein aggregation or particle formation. Identification of the cause(s) of particle formation involving minute amounts of protein over extended periods of time can be challenging. Various formulation strategies such as addition of a nonionic surfactant (e.g., polysorbate) have been demonstrated to effectively mitigate adsorption-induced protein aggregation. However, not all stability problems associated with interfaces or leachables are best resolved by formulation optimization. Detectable leachables do not necessarily have any adverse impact on the protein but control of the leachable source is preferred when there is a concern. In other cases, preventing protein aggregation and particle formation may require manufacturing process and/or equipment changes, use of compatible materials at contact interfaces, and so on. This review summarizes approaches that have been used to minimize protein aggregation and particle formation during manufacturing and fill-finish operations, product storage and transportation, and delivery of protein therapeutics. Copyright © 2011 Wiley-Liss, Inc.

Photonics and microarray technology

NASA Astrophysics Data System (ADS)

Skovsen, E.; Duroux, M.; Neves-Petersen, M. T.; Duroux, L.; Petersen, S. B.

2007-05-01

Photonic induced immobilization of biosensor molecules is a novel technology that results in spatially oriented and spatially localized covalent coupling of a large variety of biomolecules onto thiol reactive surfaces, e.g. thiolated glass, quartz, gold or silicon. The reaction mechanism behind the reported new technology involves light-induced breakage of disulphide bridges in proteins upon UV illumination of nearby aromatic amino acids resulting in the formation of reactive molecules that will form covalent bonds with thiol reactive surfaces. This new technology has the potential of replacing present micro dispensing arraying technologies, where the size of the individual sensor spots are limited by the size of the dispensed droplets. Using light-induced immobilization the spatial resolution is defined by the area of the sensor surface that is illuminated by UV light and not by the physical size of the dispensed droplets of sensor molecules. This new technology allows for dense packing of different biomolecules on a surface, allowing the creation of multi-potent functionalized materials, such as biosensors with micrometer sized individual sensor spots. Thus, we have developed the necessary technology for preparing large protein arrays of enzymes and fragments of antibodies, with micrometer resolution, without the need for liquid micro dispensing.

Impact of silica nanoparticle surface chemistry on protein corona formation and consequential interactions with biological cells.

PubMed

Kurtz-Chalot, Andréa; Villiers, Christian; Pourchez, Jérémie; Boudard, Delphine; Martini, Matteo; Marche, Patrice N; Cottier, Michèle; Forest, Valérie

2017-06-01

Nanoparticles (NP) physico-chemical features greatly influence NP/cell interactions. NP surface functionalization is often used to improve NP biocompatibility or to enhance cellular uptake. But in biological media, the formation of a protein corona adds a level of complexity. The aim of this study was to investigate in vitro the influence of NP surface functionalization on their cellular uptake and the biological response induced. 50nm fluorescent silica NP were functionalized either with amine or carboxylic groups, in presence or in absence of polyethylene glycol (PEG). NP were incubated with macrophages, cellular uptake and cellular response were assessed in terms of cytotoxicity, pro-inflammatory response and oxidative stress. The NP protein corona was also characterized by protein mass spectroscopy. Results showed that NP uptake was enhanced in absence of PEG, while NP adsorption at the cell membrane was fostered by an initial positively charged NP surface. NP toxicity was not correlated with NP uptake. NP surface functionalization also influenced the formation of the protein corona as the profile of protein binding differed among the NP types. Copyright © 2017 Elsevier B.V. All rights reserved.

Immunity to East Coast fever in cattle induced by a polypeptide fragment of the major surface coat protein of Theileria parva sporozoites.

PubMed

Bishop, Richard; Nene, Vishvanath; Staeyert, Jan; Rowlands, John; Nyanjui, John; Osaso, Julius; Morzaria, Subhash; Musoke, Antony

2003-03-07

Full-length recombinant versions of p67, the 709 amino acid major surface protein of Theileria parva sporozoites, induce immunity to East Coast fever (ECF) in cattle. We show that a soluble Escherichia coli recombinant version of p67 (p67(635)), in which a prokaryotic signal peptide replaces the eukaryotic one, confers protection comparable to that induced by the full-length molecule, but is unstable. Peptides encoding 80 (p67C) and 205 (p67N) amino acid fragments of p67, containing epitopes recognised by sporozoite neutralising monoclonal antibodies, exhibit improved stability in E. coli. Antibodies raised against the central region of p67 (p67M) neutralise sporozoite infectivity in vitro. The p67C peptide induced immunity against ECF in cattle, at a level equivalent to p67(635), suggesting that a synthetic peptide vaccine might be achievable. Copyright 2002 Elsevier Science Ltd.

Protein crystal nucleation in pores.

PubMed

Nanev, Christo N; Saridakis, Emmanuel; Chayen, Naomi E

2017-01-16

The most powerful method for protein structure determination is X-ray crystallography which relies on the availability of high quality crystals. Obtaining protein crystals is a major bottleneck, and inducing their nucleation is of crucial importance in this field. An effective method to form crystals is to introduce nucleation-inducing heterologous materials into the crystallization solution. Porous materials are exceptionally effective at inducing nucleation. It is shown here that a combined diffusion-adsorption effect can increase protein concentration inside pores, which enables crystal nucleation even under conditions where heterogeneous nucleation on flat surfaces is absent. Provided the pore is sufficiently narrow, protein molecules approach its walls and adsorb more frequently than they can escape. The decrease in the nucleation energy barrier is calculated, exhibiting its quantitative dependence on the confinement space and the energy of interaction with the pore walls. These results provide a detailed explanation of the effectiveness of porous materials for nucleation of protein crystals, and will be useful for optimal design of such materials.

Quantification of the Influence of Protein-Protein Interactions on Adsorbed Protein Structure and Bioactivity

PubMed Central

Wei, Yang; Thyparambil, Aby A.; Latour, Robert A.

2013-01-01

While protein-surface interactions have been widely studied, relatively little is understood at this time regarding how protein-surface interaction effects are influenced by protein-protein interactions and how these effects combine with the internal stability of a protein to influence its adsorbed-state structure and bioactivity. The objectives of this study were to develop a method to study these combined effects under widely varying protein-protein interaction conditions using hen egg-white lysozyme (HEWL) adsorbed on silica glass, poly(methyl methacrylate), and polyethylene as our model systems. In order to vary protein-protein interaction effects over a wide range, HEWL was first adsorbed to each surface type under widely varying protein solution concentrations for 2 h to saturate the surface, followed by immersion in pure buffer solution for 15 h to equilibrate the adsorbed protein layers in the absence of additionally adsorbing protein. Periodic measurements were made at selected time points of the areal density of the adsorbed protein layer as an indicator of the level of protein-protein interaction effects within the layer, and these values were then correlated with measurements of the adsorbed protein’s secondary structure and bioactivity. The results from these studies indicate that protein-protein interaction effects help stabilize the structure of HEWL adsorbed on silica glass, have little influence on the structural behavior of HEWL on HDPE, and actually serve to destabilize HEWL’s structure on PMMA. The bioactivity of HEWL on silica glass and HDPE was found to decrease in direct proportion to the degree of adsorption-induce protein unfolding. A direct correlation between bioactivity and the conformational state of adsorbed HEWL was less apparent on PMMA, thus suggesting that other factors influenced HEWL’s bioactivity on this surface, such as the accessibility of HEWL’s bioactive site being blocked by neighboring proteins or the surface itself. The developed methods provide an effective means to characterize the influence of protein-protein interaction effects and provide new molecular-level insights into how protein-protein interaction effects combine with protein-surface interaction and internal protein stability effects to influence the structure and bioactivity of adsorbed protein. PMID:23751416

Physical characterization of MxiH and PrgI, the needle component of the type III secretion apparatus from Shigella and Salmonella.

PubMed

Darboe, Numukunda; Kenjale, Roma; Picking, Wendy L; Picking, William D; Middaugh, C Russell

2006-03-01

Shigella and Salmonella use similar type III secretion systems for delivering effector proteins into host cells. This secretion system consists of a base anchored in both bacterial membranes and an extracellular "needle" that forms a rod-like structure exposed on the pathogen surface. The needle is composed of multiple subunits of a single protein and makes direct contact with host cells to facilitate protein delivery. The proteins that make up the needle of Shigella and Salmonella are MxiH and PrgI, respectively. These proteins are attractive vaccine candidates because of their essential role in virulence and surface exposure. We therefore isolated, purified, and characterized the monomeric forms of MxiH and PrgI. Their far-UV circular dichroism spectra show structural similarities with hints of subtle differences in their secondary structure. Both proteins are highly helical and thermally unstable, with PrgI having a midpoint of thermal unfolding (Tm) near 37 degrees C and MxiH having a value near 42 degrees C. The two proteins also have comparable intrinsic stabilities as measured by chemically induced (urea) unfolding. MxiH, however, with a free energy of unfolding (DeltaG degrees 0,un) of 1.6 kcal/mol, is slightly more stable than PrgI (1.2 kcal/mol). The relatively low m-values obtained for the urea-induced unfolding of the proteins suggest that they undergo only a small change in solvent-accessible surface area. This argues that when MxiH and PrgI are incorporated into the needle complex, they obtain a more stable structural state through the introduction of protein-protein interactions.

Adsorption of soft and hard proteins onto OTCEs under the influence of an external electric field.

PubMed

Benavidez, Tomás E; Torrente, Daniel; Marucho, Marcelo; Garcia, Carlos D

2015-03-03

The adsorption behavior of hard and soft proteins under the effect of an external electric field was investigated by a combination of spectroscopic ellipsometry and molecular dynamics (MD) simulations. Optically transparent carbon electrodes (OTCE) were used as conductive, sorbent substrates. Lysozyme (LSZ) and ribonuclease A (RNase A) were selected as representative hard proteins, whereas myoglobin (Mb), α-lactalbumin (α-LAC), bovine serum albumin (BSA), glucose oxidase (GOx), and immunoglobulin G (IgG) were selected to represent soft proteins. In line with recent publications from our group, the experimental results revealed that while the adsorption of all investigated proteins can be enhanced by the potential applied to the electrode, the effect is more pronounced for hard proteins. In contrast with the incomplete monolayers formed at open-circuit potential, the application of +800 mV to the sorbent surface induced the formation of multiple layers of protein. These results suggest that this effect can be related to the intrinsic polarizability of the protein (induction of dipoles), the resulting surface accessible solvent area (SASA), and structural rearrangements induced upon the incorporation on the protein layer. The described experiments are critical to understand the relationship between the structure of proteins and their tendency to form (under electric stimulation) layers with thicknesses that greatly surpass those obtained at open-circuit conditions.

Adsorption of Soft and Hard Proteins onto OTCEs under the influence of an External Electric Field

PubMed Central

Benavidez, Tomás E.; Torrente, Daniel; Marucho, Marcelo; Garcia, Carlos D.

2015-01-01

The adsorption behavior of hard and soft proteins under the effect of an external electric field was investigated by a combination of spectroscopic ellipsometry and molecular dynamics (MD) simulations. Optically transparent carbon electrodes (OTCE) were used as conductive, sorbent substrates. Lysozyme (LSZ) and ribonuclease A (RNase A) were selected as representative hard proteins whereas myoglobin (Mb), α-lactalbumin (α-LAC), bovine serum albumin (BSA), glucose oxidase (GOx), and immunoglobulin G (IgG) were selected to represent soft proteins. In line with recent publications from our group, the experimental results revealed that while the adsorption of all investigated proteins can be enhanced by the potential applied to the electrode, the effect is more pronounced for hard proteins. In contrast with the incomplete monolayers formed at open-circuit potential, the application of +800mV to the sorbent surface induced the formation of multiple layers of protein. These results also suggest that this effect can be related to the intrinsic polarizability of the protein (induction of dipoles), the resulting surface accessible solvent area (SASA), and structural rearrangements induced upon the incorporation on the protein layer. The described experiments are critical to understand the relationship between the structure of proteins and their tendency to form (under electric stimulation) layers with thicknesses that greatly surpass those obtained at open-circuit conditions. PMID:25658387

Microbially induced separation of quartz from hematite using sulfate reducing bacteria.

PubMed

Prakasan, M R Sabari; Natarajan, K A

2010-07-01

Cells and metabolic products of Desulfovibrio desulfuricans were successfully used to separate quartz from hematite through environmentally benign microbially induced flotation. Bacterial metabolic products such as extracellular proteins and polysaccharides were isolated from both unadapted and mineral-adapted bacterial metabolite and their basic characteristics were studied in order to get insight into the changes brought about on bioreagents during adaptation. Interaction between bacterial cells and metabolites with minerals like hematite and quartz brought about significant surface-chemical changes on both the minerals. Quartz was rendered more hydrophobic, while hematite became more hydrophilic after biotreatment. The predominance of bacterial polysaccharides on interacted hematite and of proteins on quartz was responsible for the above surface-chemical changes, as attested through adsorption studies. Surface-chemical changes were also observed on bacterial cells after adaptation to the above minerals. Selective separation of quartz from hematite was achieved through interaction with quartz-adapted bacterial cells and metabolite. Mineral-specific proteins secreted by quartz-adapted cells were responsible for conferment of hydrophobicity on quartz resulting in enhanced separation from hematite through flotation. 2010 Elsevier B.V. All rights reserved.

A Multi-technique Characterization of Adsorbed Protein Films: Orientation and Structure by ToF-SIMS, NEXAFS, SFG, and XPS

NASA Astrophysics Data System (ADS)

Baio, Joseph E.

There are many techniques that allow surface scientists to study interfaces. However, few are routinely applied to probe biological surfaces. The work presented here demonstrates how detailed information about the conformation, orientation, chemical state, and molecular structure of biological molecules immobilized onto a surface can be assessed by electron spectroscopy, mass spectrometry, and nonlinear vibrational spectroscopy techniques. This investigation began with the development of simple model systems (small proteins, and peptides) and evolved into a study of more complex --- real world systems. Initially, two model systems based on the chemical and electrostatic immobilization of a small rigid protein (Protein G B1 domain, 6kDa) were built to develop the capabilities of time-of-flight secondary ion mass spectrometry (ToFSIMS), near edge X-ray absorption fine structure spectroscopy (NEXAFS) and sum frequency generation (SFG) spectroscopy as tools to probe the structure of surface immobilized proteins. X-ray photoelectron spectroscopy (XPS) was used to measure the amount of immobilized protein and ToF-SIMS sampled the amino acid composition of the exposed surface of the protein film. Within the ToF-SIMS spectra, an enrichment of secondary ions from amino acids located at opposite ends of the proteins were used to describe protein orientation. SFG spectral peaks characteristic of ordered alpha-helix and beta-sheet elements were observed for both systems and the phase of the peaks indicated a predominantly upright orientation for both the covalent and electrostatic configurations. Polarization dependence of the NEXAFS signal from the N 1s to pi* transition of the peptide bonds that make up the beta-sheets also indicated protein ordering at the surface. Building upon the Protein G B1 studies, the orientation and structure of a surface immobilized antibody (HuLys Fv: variant of humanized anti-lysozyme variable fragment, 26kDa) was characterized across two immobilization schemes. This protein contained both a hexahistidine tag and a cysteine residue, introduced at opposite ends of the HuLys Fv, for immobilization onto nitrilotriacetic acid (NTA) and maleimide oligo- (ethylene glycol) (MEG)-terminated substrates. The thiol group on the cysteine residue selectively binds to the MEG groups, while the his-tag selectively binds to the Ni-loaded NTA groups. XPS was used to monitor protein coverage on both surfaces by following the change in the nitrogen atomic %. The ToF-SIMS data provided a clear differentiation between the two samples due to the intensity differences of secondary ions originating from asymmetrically located amino acids in HuLys Fv. Indicating that the HuLys Fv fragment when adsorbed into the NTA and MEG substrates will be induced into two different orientations. On the NTA substrate the protein's binding site is accessible, while on the MEG substrate the binding site is oriented towards the surface. By taking advantage of the electron pathway through the heme group in cytochrome c (CytoC) electrochemists have built sensors based upon CytoC immobilized onto functionalized metal electrodes. When immobilized onto a charged surface, CytoC, with its distribution of lysine and glutamate residues around its surface, should orient and form a well-ordered protein film. Here a detailed examination of CytoC orientation when electrostatically immobilized onto both amine (NH 3+) and carboxyl (COO-) functionalized gold is presented. Again, protein coverage, on both surfaces, was monitored by the change in the atomic % N, as determined by XPS. ToF-SIMS data demonstrated a clear separation between the two samples based on the intensity differences of secondary ions stemming from amino acids located asymmetrically within CytoC, indicating opposite orientations of the protein on the two different surfaces. Spectral features within the in situ sum frequency generation vibrational spectra, acquired for the protein interacting with positively and negatively charged surfaces, indicates that these electrostatic interactions do induce the protein into a well ordered film.

The insulin response integrates increased TGF-β signaling through Akt-induced enhancement of cell surface delivery of TGF-β receptors

PubMed Central

Budi, Erine H.; Muthusamy, Baby Periyanayaki; Derynck, Rik

2015-01-01

Increased activity of transforming growth factor β (TGF-β), which binds to and stimulates cell surface receptors, contributes to cancer progression and fibrosis by driving epithelial cells toward a migratory mesenchymal phenotype and increasing the abundance of extracellular matrix proteins. The abundance of TGF-β receptors at the cell surface determines cellular responsiveness to TGF-β, which is often produced by the same cells that have the receptors, and thus serves as an autocrine signal. We found that Akt-mediated phosphorylation of AS160, a RabGAP [guanosine triphosphatase (GTPase)-activating protein] promoted the translocation of TGF-β receptors from intracellular stores to the plasma membrane of mouse embryonic fibroblasts (MEFs) and NMuMG epithelial cells. Consequently, insulin, which is commonly used to treat hyperglycemia and activates Akt signaling, increased the amount of TGF-β receptors at the cell surface, thereby enhancing TGF-β responsiveness. This insulin-induced increase in autocrine TGF-β signaling contributed to insulin-induced gene expression responses, attenuated the epithelial phenotype, and promoted the migration of NMuMG cells. Furthermore, the enhanced delivery of TGF-β receptors at the cell surface enabled insulin to increase TGF-β-induced gene responses. The enhancement of TGF-β responsiveness in response to Akt activation may help to explain the biological effects of insulin, the progression of cancers in which Akt is activated, and the increased incidence of fibroses in diabetes. PMID:26420907

The Role of Membrane Curvature in Nanoscale Topography-Induced Intracellular Signaling.

PubMed

Lou, Hsin-Ya; Zhao, Wenting; Zeng, Yongpeng; Cui, Bianxiao

2018-05-15

Over the past decade, there has been growing interest in developing biosensors and devices with nanoscale and vertical topography. Vertical nanostructures induce spontaneous cell engulfment, which enhances the cell-probe coupling efficiency and the sensitivity of biosensors. Although local membranes in contact with the nanostructures are found to be fully fluidic for lipid and membrane protein diffusions, cells appear to actively sense and respond to the surface topography presented by vertical nanostructures. For future development of biodevices, it is important to understand how cells interact with these nanostructures and how their presence modulates cellular function and activities. How cells recognize nanoscale surface topography has been an area of active research for two decades before the recent biosensor works. Extensive studies show that surface topographies in the range of tens to hundreds of nanometers can significantly affect cell functions, behaviors, and ultimately the cell fate. For example, titanium implants having rough surfaces are better for osteoblast attachment and host-implant integration than those with smooth surfaces. At the cellular level, nanoscale surface topography has been shown by a large number of studies to modulate cell attachment, activity, and differentiation. However, a mechanistic understanding of how cells interact and respond to nanoscale topographic features is still lacking. In this Account, we focus on some recent studies that support a new mechanism that local membrane curvature induced by nanoscale topography directly acts as a biochemical signal to induce intracellular signaling, which we refer to as the curvature hypothesis. The curvature hypothesis proposes that some intracellular proteins can recognize membrane curvatures of a certain range at the cell-to-material interface. These proteins then recruit and activate downstream components to modulate cell signaling and behavior. We discuss current technologies allowing the visualization of membrane deformation at the cell membrane-to-substrate interface with nanometer precision and demonstrate that vertical nanostructures induce local curvatures on the plasma membrane. These local curvatures enhance the process of clathrin-mediated endocytosis and affect actin dynamics. We also present evidence that vertical nanostructures can induce significant deformation of the nuclear membrane, which can affect chromatin distribution and gene expression. Finally, we provide a brief perspective on the curvature hypothesis and the challenges and opportunities for the design of nanotopography for manipulating cell behavior.

A novel carbohydrate-binding surface layer protein from the hyperthermophilic archaeon Pyrococcus horikoshii.

PubMed

Goda, Shuichiro; Koga, Tomoyuki; Yamashita, Kenichiro; Kuriura, Ryo; Ueda, Toshifumi

2018-04-08

In Archaea and Bacteria, surface layer (S-layer) proteins form the cell envelope and are involved in cell protection. In the present study, a putative S-layer protein was purified from the crude extract of Pyrococcus horikoshii using affinity chromatography. The S-layer gene was cloned and expressed in Escherichia coli. Isothermal titration calorimetry analyses showed that the S-layer protein bound N-acetylglucosamine and induced agglutination of the gram-positive bacterium Micrococcus lysodeikticus. The protein comprised a 21-mer structure, with a molecular mass of 1,340 kDa, as determined using small-angle X-ray scattering. This protein showed high thermal stability, with a midpoint of thermal denaturation of 79 °C in dynamic light scattering experiments. This is the first description of the carbohydrate-binding archaeal S-layer protein and its characteristics.

Identification and Characterization of Two Temperature-Induced Surface-Associated Proteins of Streptococcus suis with High Homologies to Members of the Arginine Deiminase System of Streptococcus pyogenes

PubMed Central

Winterhoff, Nora; Goethe, Ralph; Gruening, Petra; Rohde, Manfred; Kalisz, Henryk; Smith, Hilde E.; Valentin-Weigand, Peter

2002-01-01

The present study was performed to identify stress-induced putative virulence proteins of Streptococcus suis. For this, protein expression patterns of streptococci grown at 32, 37, and 42°C were compared by one- and two-dimensional gel electrophoresis. Temperature shifts from 32 and 37 to 42°C induced expression of two cell wall-associated proteins with apparent molecular masses of approximately 47 and 53 kDa. Amino-terminal sequence analysis of the two proteins indicated homologies of the 47-kDa protein with an ornithine carbamoyltransferase (OCT) from Streptococcus pyogenes and of the 53-kDa protein with the streptococcal acid glycoprotein (SAGP) from S. pyogenes, an arginine deiminase (AD) recently proposed as a putative virulence factor. Cloning and sequencing the genes encoding the putative OCT and AD of S. suis, octS and adiS, respectively, revealed that they had 81.2 (octS) and 80.2% (adiS) identity with the respective genes of S. pyogenes. Both genes belong to the AD system, also found in other bacteria. Southern hybridization analysis demonstrated the presence of the adiS gene in all 42 serotype 2 and 9 S. suis strains tested. In 9 of these 42 strains, selected randomly, we confirmed expression of the AdiS protein, homologous to SAGP, by immunoblot analysis using a specific antiserum against the SAGP of S. pyogenes. In all strains AD activity was detected. Furthermore, by immunoelectron microscopy using the anti-S. pyogenes SAGP antiserum we were able to demonstrate that the AdiS protein is expressed on the streptococcal surface in association with the capsular polysaccharides but is not coexpressed with them. PMID:12446626

Modeling Pressure-Driven Transport of Proteins through a Nanochannel

PubMed Central

Carr, Rogan; Comer, Jeffrey; Ginsberg, Mark D.; Aksimentiev, Aleksei

2012-01-01

Reducing the size of a nanofluidic channel not only creates new opportunities for high-precision manipulation of biological macromolecules, but also makes the performance of the entire nanofluidic system more susceptible to undesirable interactions between the transported biomolecules and the walls of the channel. In this manuscript, we report molecular dynamics simulations of a pressure-driven flow through a silica nanochannel that characterized, with atomic resolution, adsorption of a model protein to its surface. Although the simulated adsorption of the proteins was found to be nonspecific, it had a dramatic effect on the rate of the protein transport. To determine the relative strength of the protein–silica interactions in different adsorbed states, we simulated flow-induced desorption of the proteins from the silica surface. Our analysis of the protein conformations in the adsorbed states did not reveal any simple dependence of the adsorption strength on the size and composition of the protein–silica contact, suggesting that the heterogeneity of the silica surface may be a important factor. PMID:22611338

Direct observation of the transition from calcite to aragonite growth as induced by abalone shell proteins.

PubMed Central

Thompson, J B; Paloczi, G T; Kindt, J H; Michenfelder, M; Smith, B L; Stucky, G; Morse, D E; Hansma, P K

2000-01-01

The mixture of EDTA-soluble proteins found in abalone nacre are known to cause the nucleation and growth of aragonite on calcite seed crystals in supersaturated solutions of calcium carbonate. Past atomic force microscope studies of the interaction of these proteins with calcite crystals did not observe this transition because no information about the crystal polymorph on the surface was obtained. Here we have used the atomic force microscope to directly observe changes in the atomic lattice on a calcite seed crystal after the introduction of abalone shell proteins. The observed changes are consistent with a transition to (001) aragonite growth on a (1014) calcite surface. PMID:11106633

Aggregation and Particle Formation of Therapeutic Proteins in Contact With a Novel Fluoropolymer Surface Versus Siliconized Surfaces: Effects of Agitation in Vials and in Prefilled Syringes.

PubMed

Teska, Brandon M; Brake, Jeffrey M; Tronto, Gregory S; Carpenter, John F

2016-07-01

We examined the effects of an accelerated agitation protocol on 2 protein therapeutics, intravenous immunoglobulin (IVIG) and Avastin (bevacizumab), in contact with a novel fluoropolymer surface and more typical siliconized surfaces. The fluoropolymer surface provides "solid-phase" lubrication for the syringe plunger-obviating the need for silicone oil lubrication in prefilled syringes. We tested the 2 surfaces in a vial system and in prefilled glass syringes. We also examined the effects of 2 buffers, phosphate-buffered saline (PBS) and 0.2-M glycine, with and without the addition of polysorbate 20, on agitation-induced aggregation of IVIG. Aggregation was monitored by measuring subvisible particle formation and soluble protein loss. In both vials and syringes, protein particle formation was much lower during agitation with the fluoropolymer surface than with the siliconized surface. Also, particle formation was greater in PBS than in glycine buffer, an effect attributed to lower colloidal stability of IVIG in PBS. Polysorbate 20 in the formulation greatly inhibited protein particle formation. Overall, the fluoropolymer plunger surface in an unsiliconized glass barrel was demonstrated to be a viable solution for eliminating silicone oil droplets from prefilled syringe formulations and providing a consistent system for rationale formulation development and simplified particle analysis. Copyright © 2016 American Pharmacists Association®. Published by Elsevier Inc. All rights reserved.

Phosphatidylserine as an anchor for plasminogen and its plasminogen receptor, Histone H2B, to the macrophage surface

PubMed Central

DAS, R.; PLOW, E. F.

2013-01-01

Summary Background Plasminogen (Plg) binding to cell surface Plg receptors (Plg-Rs) on the surface of macrophages facilitates Plg activation and migration of these cells. Histone H2B (H2B) acts as a Plg-R and its cell surface expression is upregulated when monocytes are differentiated to macrophages via a pathway dependent on L-type Ca2+ channels and intracellular Ca2+. Objectives We sought to investigate the mechanism by which H2B, a protein without a transmembrane domain, is retained on themacrophage surface. Methods THP-1 monocytoid cells were induced to differentiate with interferon gamma + Vitamin D3 or to undergo apoptosis by treatment with camptothecin. Flow cytometry and cell surface biotinylation followed by Western blotting were used to measure the interrelationship between Plg binding, cell surface expression of H2B and outermembrane exposure of phosphatidylserine (PS). Results H2B interacted directly with PS via an electrostatic interaction. Anti-PS or PS binding proteins, annexin V and protein S, diminished H2B interaction with PS on the surface of differentiated or apoptotic cells and these same reagents inhibited Plg binding to these cells. L-type Ca2+ channels played a significant role in PS exposure, H2B surface expression and Plg binding induced either by differentiation or apoptosis. Conclusions These data suggest that H2B tethers to the surface of cells by interacting with PS on differentiated or apoptotic monocytoid cells. L-type Ca2+ channels regulate PS exposure on the surface of these cells. The exposed PS interacts directly with H2B and hence provides sites for Plg to bind to. PMID:21040449

Serological responses in humans to the smallpox vaccine LC16m8

PubMed Central

Johnson, Benjamin F.; Kanatani, Yasuhiro; Fujii, Tatsuya; Saito, Tomoya; Yokote, Hiroyuki

2011-01-01

In response to potential bioterrorism with smallpox, members of the Japanese Self-Defense Forces were vaccinated with vaccinia virus (VACV) strain LC16m8, an attenuated smallpox vaccine derived from VACV strain Lister. The serological response induced by LC16m8 to four virion-surface proteins and the intracellular mature virus (IMV) and extracellular enveloped virus (EEV) was investigated. LC16m8 induced antibody response against the IMV protein A27 and the EEV protein A56. LC16m8 also induced IMV-neutralizing antibodies, but unlike the VACV strain Lister, did not induce either EEV-neutralizing antibody or antibody to EEV protein B5, except after revaccination. Given that B5 is the only target for EEV-neutralizing antibody and that neutralization of both IMV and EEV give optimal protection against orthopoxvirus challenge, these data suggest that immunity induced by LC16m8 might be less potent than that deriving from strain Lister. This potential disadvantage should be balanced against the advantage of the greater safety of LC16m8. PMID:21715598

Physical characterization of MxiH and PrgI, the needle component of the type III secretion apparatus from Shigella and Salmonella

PubMed Central

Darboe, Numukunda; Kenjale, Roma; Picking, Wendy L.; Picking, William D.; Middaugh, C. Russell

2006-01-01

Shigella and Salmonella use similar type III secretion systems for delivering effector proteins into host cells. This secretion system consists of a base anchored in both bacterial membranes and an extracellular “needle” that forms a rod-like structure exposed on the pathogen surface. The needle is composed of multiple subunits of a single protein and makes direct contact with host cells to facilitate protein delivery. The proteins that make up the needle of Shigella and Salmonella are MxiH and PrgI, respectively. These proteins are attractive vaccine candidates because of their essential role in virulence and surface exposure. We therefore isolated, purified, and characterized the monomeric forms of MxiH and PrgI. Their far-UV circular dichroism spectra show structural similarities with hints of subtle differences in their secondary structure. Both proteins are highly helical and thermally unstable, with PrgI having a midpoint of thermal unfolding (Tm) near 37°C and MxiH having a value near 42°C. The two proteins also have comparable intrinsic stabilities as measured by chemically induced (urea) unfolding. MxiH, however, with a free energy of unfolding (ΔG°0,un) of 1.6 kcal/mol, is slightly more stable than PrgI (1.2 kcal/mol). The relatively low m-values obtained for the urea-induced unfolding of the proteins suggest that they undergo only a small change in solvent-accessible surface area. This argues that when MxiH and PrgI are incorporated into the needle complex, they obtain a more stable structural state through the introduction of protein–protein interactions. PMID:16501225

Surface-Induced Dissociation of Protein Complexes in a Hybrid Fourier Transform Ion Cyclotron Resonance Mass Spectrometer

DOE Office of Scientific and Technical Information (OSTI.GOV)

Yan, Jing; Zhou, Mowei; Gilbert, Joshua D.

Mass spectrometry continues to develop as a valuable tool in the analysis of proteins and protein complexes. In protein complex mass spectrometry studies, surface-induced dissociation (SID) has been successfully applied in quadrupole time-of-flight (Q-TOF) instruments. SID provides structural information on noncovalent protein complexes that is complementary to other techniques. However, the mass resolution of Q-TOF instruments can limit the information that can be obtained for protein complexes by SID. Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS) provides ultrahigh resolution and ultrahigh mass accuracy measurements. Here in this study, an SID device was designed and successfully installed in amore » hybrid FT-ICR instrument in place of the standard gas collision cell. The SID-FT-ICR platform has been tested with several protein complex systems (homooligomers, a heterooligomer, and a protein-ligand complex, ranging from 53 to 85 kDa), and the results are consistent with data previously acquired on Q-TOF platforms, matching predictions from known protein interface information. Lastly, SID fragments with the same m/z but different charge states are well-resolved based on distinct spacing between adjacent isotope peaks, and the addition of metal cations and ligands can also be isotopically resolved with the ultrahigh mass resolution available in FT-ICR.« less

Surface-Induced Dissociation of Protein Complexes in a Hybrid Fourier Transform Ion Cyclotron Resonance Mass Spectrometer

DOE Office of Scientific and Technical Information (OSTI.GOV)

Yan, Jing; Zhou, Mowei; Gilbert, Joshua D.

Mass spectrometry continues to develop as a valuable tool in the analysis of proteins and protein complexes. In protein complex mass spectrometry studies, surface-induced dissociation (SID) has been successfully applied in quadrupole time-of-flight (Q-TOF) instruments. SID provides structural information on non-covalent protein complexes that is complementary to other techniques. However, the mass resolution of Q-TOF instruments can limit the information that can be obtained for protein complexes by SID. Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS) provides ultrahigh resolution and ultrahigh mass accuracy measurements. In this study, an SID device was designed and successfully installed in a hybridmore » FT-ICR instrument in place of the standard gas collision cell. The SID-FT-ICR platform has been tested with several protein complex systems (homooligomers, a heterooligomer, and a protein-ligand complex, ranging from 53 kDa to 85 kDa), and the results are consistent with data previously acquired on Q-TOF platforms, matching predictions from known protein interface information. SID fragments with the same m/z but different charge states are well-resolved based on distinct spacing between adjacent isotope peaks, and the addition of metal cations and ligands can also be isotopically resolved with the ultrahigh mass resolution available in FT-ICR.« less

Surface-Induced Dissociation of Protein Complexes in a Hybrid Fourier Transform Ion Cyclotron Resonance Mass Spectrometer

DOE PAGES

Yan, Jing; Zhou, Mowei; Gilbert, Joshua D.; ...

2016-12-02

Mass spectrometry continues to develop as a valuable tool in the analysis of proteins and protein complexes. In protein complex mass spectrometry studies, surface-induced dissociation (SID) has been successfully applied in quadrupole time-of-flight (Q-TOF) instruments. SID provides structural information on noncovalent protein complexes that is complementary to other techniques. However, the mass resolution of Q-TOF instruments can limit the information that can be obtained for protein complexes by SID. Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS) provides ultrahigh resolution and ultrahigh mass accuracy measurements. Here in this study, an SID device was designed and successfully installed in amore » hybrid FT-ICR instrument in place of the standard gas collision cell. The SID-FT-ICR platform has been tested with several protein complex systems (homooligomers, a heterooligomer, and a protein-ligand complex, ranging from 53 to 85 kDa), and the results are consistent with data previously acquired on Q-TOF platforms, matching predictions from known protein interface information. Lastly, SID fragments with the same m/z but different charge states are well-resolved based on distinct spacing between adjacent isotope peaks, and the addition of metal cations and ligands can also be isotopically resolved with the ultrahigh mass resolution available in FT-ICR.« less

Insulin-induced exocytosis regulates the cell surface level of low-density lipoprotein-related protein-1 in Müller Glial cells.

PubMed

Actis Dato, Virginia; Grosso, Rubén A; Sánchez, María C; Fader, Claudio M; Chiabrando, Gustavo A

2018-05-15

Low-density lipoprotein (LDL) receptor-related protein-1 (LRP1) is expressed in retinal Müller glial cells (MGCs) and regulates intracellular translocation to the plasma membrane (PM) of the membrane proteins involved in cellular motility and activity. Different functions of MGCs may be influenced by insulin, including the removal of extracellular glutamate in the retina. In the present work, we investigated whether insulin promotes LRP1 translocation to the PM in the Müller glial-derived cell line MIO-M1 (human retinal Müller glial cell-derived cell line). We demonstrated that LRP1 is stored in small vesicles containing an approximate size of 100 nm (mean diameter range of 100-120 nm), which were positive for sortilin and VAMP2, and also incorporated GLUT4 when it was transiently transfected. Next, we observed that LRP1 translocation to the PM was promoted by insulin-regulated exocytosis through intracellular activation of the IR/PI 3 K/Akt axis and Rab-GTPase proteins such as Rab8A and Rab10. In addition, these Rab-GTPases regulated both the constitutive and insulin-induced LRP1 translocation to the PM. Finally, we found that dominant-negative Rab8A and Rab10 mutants impaired insulin-induced intracellular signaling of the IR/PI3K/Akt axis, suggesting that these GTPase proteins as well as the LRP1 level at the cell surface are involved in insulin-induced IR activation. © 2018 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.

Overexpression of human virus surface glycoprotein precursors induces cytosolic unfolded protein response in Saccharomyces cerevisiae

PubMed Central

2011-01-01

Background The expression of human virus surface proteins, as well as other mammalian glycoproteins, is much more efficient in cells of higher eukaryotes rather than yeasts. The limitations to high-level expression of active viral surface glycoproteins in yeast are not well understood. To identify possible bottlenecks we performed a detailed study on overexpression of recombinant mumps hemagglutinin-neuraminidase (MuHN) and measles hemagglutinin (MeH) in yeast Saccharomyces cerevisiae, combining the analysis of recombinant proteins with a proteomic approach. Results Overexpressed recombinant MuHN and MeH proteins were present in large aggregates, were inactive and totally insoluble under native conditions. Moreover, the majority of recombinant protein was found in immature form of non-glycosylated precursors. Fractionation of yeast lysates revealed that the core of viral surface protein aggregates consists of MuHN or MeH disulfide-linked multimers involving eukaryotic translation elongation factor 1A (eEF1A) and is closely associated with small heat shock proteins (sHsps) that can be removed only under denaturing conditions. Complexes of large Hsps seem to be bound to aggregate core peripherally as they can be easily removed at high salt concentrations. Proteomic analysis revealed that the accumulation of unglycosylated viral protein precursors results in specific cytosolic unfolded protein response (UPR-Cyto) in yeast cells, characterized by different action and regulation of small Hsps versus large chaperones of Hsp70, Hsp90 and Hsp110 families. In contrast to most environmental stresses, in the response to synthesis of recombinant MuHN and MeH, only the large Hsps were upregulated whereas sHsps were not. Interestingly, the amount of eEF1A was also increased during this stress response. Conclusions Inefficient translocation of MuHN and MeH precursors through ER membrane is a bottleneck for high-level expression in yeast. Overexpression of these recombinant proteins induces the UPR's cytosolic counterpart, the UPR-Cyto, which represent a subset of proteins involved in the heat-shock response. The involvement of eEF1A may explain the mechanism by which only large chaperones, but not small Hsps are upregulated during this stress response. Our study highlights important differences between viral surface protein expression in yeast and mammalian cells at the first stage of secretory pathway. PMID:21595909

The Density and Refractive Index of Adsorbing Protein Layers

PubMed Central

Vörös, Janos

2004-01-01

The structure of the adsorbing layers of native and denatured proteins (fibrinogen, γ-immunoglobulin, albumin, and lysozyme) was studied on hydrophilic TiO2 and hydrophobic Teflon-AF surfaces using the quartz crystal microbalance with dissipation and optical waveguide lightmode spectroscopy techniques. The density and the refractive index of the adsorbing protein layers could be determined from the complementary information provided by the two in situ instruments. The observed density and refractive index changes during the protein-adsorption process indicated the presence of conformational changes (e.g., partial unfolding) in general, especially upon contact with the hydrophobic surface. The structure of the formed layers was found to depend on the size of the proteins and on the experimental conditions. On the TiO2 surface smaller proteins formed a denser layer than larger ones and the layer of unfolded proteins was less dense than that adsorbed from the native conformation. The hydrophobic surface induced denaturation and resulted in the formation of thin compact protein films of albumin and lysozyme. A linear correlation was found between the quartz crystal microbalance measured dissipation factor and the total water content of the layer, suggesting the existence of a dissipative process that is related to the solvent molecules present inside the adsorbed protein layer. Our measurements indicated that water and solvent molecules not only influence the 3D structure of proteins in solution but also play a crucial role in their adsorption onto surfaces. PMID:15240488

Mechanism for CARMIL Protein Inhibition of Heterodimeric Actin-capping Protein*

PubMed Central

Kim, Taekyung; Ravilious, Geoffrey E.; Sept, David; Cooper, John A.

2012-01-01

Capping protein (CP) controls the polymerization of actin filaments by capping their barbed ends. In lamellipodia, CP dissociates from the actin cytoskeleton rapidly, suggesting the possible existence of an uncapping factor, for which the protein CARMIL (capping protein, Arp2/3 and myosin-I linker) is a candidate. CARMIL binds to CP via two motifs. One, the CP interaction (CPI) motif, is found in a number of unrelated proteins; the other motif is unique to CARMILs, the CARMIL-specific interaction motif. A 115-aa CARMIL fragment of CARMIL with both motifs, termed the CP-binding region (CBR), binds to CP with high affinity, inhibits capping, and causes uncapping. We wanted to understand the structural basis for this function. We used a collection of mutants affecting the actin-binding surface of CP to test the possibility of a steric-blocking model, which remained open because a region of CBR was not resolved in the CBR/CP co-crystal structure. The CP actin-binding mutants bound CBR normally. In addition, a CBR mutant with all residues of the unresolved region changed showed nearly normal binding to CP. Having ruled out a steric blocking model, we tested an allosteric model with molecular dynamics. We found that CBR binding induces changes in the conformation of the actin-binding surface of CP. In addition, ∼30-aa truncations on the actin-binding surface of CP decreased the affinity of CBR for CP. Thus, CARMIL promotes uncapping by binding to a freely accessible site on CP bound to a filament barbed end and inducing a change in the conformation of the actin-binding surface of CP. PMID:22411988

TiO2 Nanoparticle-Induced Oxidation of the Plasma Membrane: Importance of the Protein Corona.

PubMed

Runa, Sabiha; Lakadamyali, Melike; Kemp, Melissa L; Payne, Christine K

2017-09-21

Titanium dioxide (TiO 2 ) nanoparticles, used as pigments and photocatalysts, are widely present in modern society. Inhalation or ingestion of these nanoparticles can lead to cellular-level interactions. We examined the very first step in this cellular interaction, the effect of TiO 2 nanoparticles on the lipids of the plasma membrane. Within 12 h of TiO 2 nanoparticle exposure, the lipids of the plasma membrane were oxidized, determined with a malondialdehyde assay. Lipid peroxidation was inhibited by surface passivation of the TiO 2 nanoparticles, incubation with an antioxidant (Trolox), and the presence of serum proteins in solution. Subsequent experiments determined that serum proteins adsorbed on the surface of the TiO 2 nanoparticles, forming a protein corona, inhibit lipid peroxidation. Super-resolution fluorescence microscopy showed that these serum proteins were clustered on the nanoparticle surface. These protein clusters slow lipid peroxidation, but by 24 h, the level of lipid peroxidation is similar, independent of the protein corona or free serum proteins. Additionally, over 24 h, this corona of proteins was displaced from the nanoparticle surface by free proteins in solution. Overall, these experiments provide the first mechanistic investigation of plasma membrane oxidation by TiO 2 nanoparticles, in the absence of UV light and as a function of the protein corona, approximating a physiological environment.

Designing pH induced fold switch in proteins

NASA Astrophysics Data System (ADS)

Baruah, Anupaul; Biswas, Parbati

2015-05-01

This work investigates the computational design of a pH induced protein fold switch based on a self-consistent mean-field approach by identifying the ensemble averaged characteristics of sequences that encode a fold switch. The primary challenge to balance the alternative sets of interactions present in both target structures is overcome by simultaneously optimizing two foldability criteria corresponding to two target structures. The change in pH is modeled by altering the residual charge on the amino acids. The energy landscape of the fold switch protein is found to be double funneled. The fold switch sequences stabilize the interactions of the sites with similar relative surface accessibility in both target structures. Fold switch sequences have low sequence complexity and hence lower sequence entropy. The pH induced fold switch is mediated by attractive electrostatic interactions rather than hydrophobic-hydrophobic contacts. This study may provide valuable insights to the design of fold switch proteins.

Molecular Diversity of Macrophages in Allergic Reaction: Comparison between the Allergenic Modes; Th1- and -Th2-Derived Immune Conditions.

PubMed

Bagheri, Mozhdeh; Dong, Yupeng; Ono, Masao

2015-06-01

Activated macrophages have been classified into classical (M1) and alternative (M2) macrophages. We aimed to establish a method to yield enough number of macrophages to analyze their molecular, biological and immunological functions. We used drugs; adjuvant albumin from chicken egg whites--Imject Alum (OVA-Alum) and OVA Complete Freund Adjuvant (OVA-CFA), to induce macrophages to M2 and M1 respectively. We analyzed the phenotype of purified macrophages induced under these immune conditions, using flow cytometry (FACS) to detect cell-surface molecules and the enzyme-linked immunosorbent assay (ELISA) was used to detect cytokines. The cDNA microarray was employed to measure changes in expression level of cell surface protein between M1 and M2 macrophages. Phenotype analysis of purified macrophages, induced under these immune conditions, showed macrophages induced by OVA-Alum was almost M2 while the proportion of M1 macrophages induced by OVA-CFA was significantly higher. The results also showed higher expression level of macrophage galactose N- acetyl-galactosamine specific lectin-2 protein (MGL1/2-PE), a known M2 macrophage marker, on the surface of Alum-induced macrophages. On the basis of these preliminary data, ELISA results revealed that after macrophage stimulation with lipopolysaccharides (LPS), the level of interleukin (IL)-10 produced by Alum- induced macrophages was higher than the level of IL-10 produced by CFA-induced macrophages. In contrast, the level of tumor necrosis factor-alpha (TNF-α) produced by CFA-induced macrophages was higher than Alum-induced macrophages. The cDNA microarray confirmed previous results and suggest immunoglobulin-like type 2 receptor alpha (Pilra) as a new marker for M1, macrophage galactose N-acetylgalactosamine-specific lectin 2 (Mgl2) as M2 macrophages marker.

Water promotes the sealing of nanoscale packing defects in folding proteins.

PubMed

Fernández, Ariel

2014-05-21

A net dipole moment is shown to arise from a non-Debye component of water polarization created by nanoscale packing defects on the protein surface. Accordingly, the protein electrostatic field exerts a torque on the induced dipole, locally impeding the nucleation of ice at the protein-water interface. We evaluate the solvent orientation steering (SOS) as the reversible work needed to align the induced dipoles with the Debye electrostatic field and computed the SOS for the variable interface of a folding protein. The minimization of the SOS is shown to drive protein folding as evidenced by the entrainment of the total free energy by the SOS energy along trajectories that approach a Debye limit state where no torque arises. This result suggests that the minimization of anomalous water polarization at the interface promotes the sealing of packing defects, thereby maintaining structural integrity and committing the protein chain to fold.

Control of adhesion of human induced pluripotent stem cells to plasma-patterned polydimethylsiloxane coated with vitronectin and γ-globulin.

PubMed

Yamada, Ryotaro; Hattori, Koji; Tachikawa, Saoko; Tagaya, Motohiro; Sasaki, Toru; Sugiura, Shinji; Kanamori, Toshiyuki; Ohnuma, Kiyoshi

2014-09-01

Human induced pluripotent stem cells (hiPSCs) are a promising source of cells for medical applications. Recently, the development of polydimethylsiloxane (PDMS) microdevices to control the microenvironment of hiPSCs has been extensively studied. PDMS surfaces are often treated with low-pressure air plasma to facilitate protein adsorption and cell adhesion. However, undefined molecules present in the serum and extracellular matrix used to culture cells complicate the study of cell adhesion. Here, we studied the effects of vitronectin and γ-globulin on hiPSC adhesion to plasma-treated and untreated PDMS surfaces under defined culture conditions. We chose these proteins because they have opposite properties: vitronectin mediates hiPSC attachment to hydrophilic siliceous surfaces, whereas γ-globulin is adsorbed by hydrophobic surfaces and does not mediate cell adhesion. Immunostaining showed that, when applied separately, vitronectin and γ-globulin were adsorbed by both plasma-treated and untreated PDMS surfaces. In contrast, when PDMS surfaces were exposed to a mixture of the two proteins, vitronectin was preferentially adsorbed onto plasma-treated surfaces, whereas γ-globulin was adsorbed onto untreated surfaces. Human iPSCs adhered to the vitronectin-rich plasma-treated surfaces but not to the γ-globulin-rich untreated surfaces. On the basis of these results, we used perforated masks to prepare plasma-patterned PDMS substrates, which were then used to pattern hiPSCs. The patterned hiPSCs expressed undifferentiated-cell markers and did not escape from the patterned area for at least 7 days. The patterned PDMS could be stored for up to 6 days before hiPSCs were plated. We believe that our results will be useful for the development of hiPSC microdevices. Copyright © 2014 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

An investigation of vibration-induced protein desorption mechanism using a micromachined membrane and PZT plate.

PubMed

Yeh, Po Ying; Le, Yevgeniya; Kizhakkedathu, Jayachandran N; Chiao, Mu

2008-10-01

A micromachined vibrating membrane is used to remove adsorbed proteins on a surface. A lead zirconate titanate (PZT) composite (3 x 1 x 0.5 mm) is attached to a silicon membrane (2,000 x 500 x 3 microm) and vibrates in a flexural plate wave (FPW) mode with wavelength of 4,000/3 microm at a resonant frequency of 308 kHz. The surface charge on the membrane and fluid shear stress contribute in minimizing the protein adsorption on the SiO(2) surface. In vitro characterization shows that 57 +/- 10% of the adsorbed bovine serum albumin (BSA), 47 +/- 13% of the immunoglobulin G (IgG), and 55.3~59.2 +/- 8% of the proteins from blood plasma are effectively removed from the vibrating surface. A simulation study of the vibration-frequency spectrum and vibrating amplitude distribution matches well with the experimental data. Potentially, a microelectromechanical system (MEMS)-based vibrating membrane could be the tool to minimize biofouling of in vivo MEMS devices.

Protein-linked glycans in periodontal bacteria: prevalence and role at the immune interface.

PubMed

Settem, Rajendra P; Honma, Kiyonobu; Stafford, Graham P; Sharma, Ashu

2013-10-17

Protein modification with complex glycans is increasingly being recognized in many pathogenic and non-pathogenic bacteria, and is now thought to be central to the successful life-style of those species in their respective hosts. This review aims to convey current knowledge on the extent of protein glycosylation in periodontal pathogenic bacteria and its role in the modulation of the host immune responses. The available data show that surface glycans of periodontal bacteria orchestrate dendritic cell cytokine responses to drive T cell immunity in ways that facilitate bacterial persistence in the host and induce periodontal inflammation. In addition, surface glycans may help certain periodontal bacteria protect against serum complement attack or help them escape immune detection through glycomimicry. In this review we will focus mainly on the generalized surface-layer protein glycosylation system of the periodontal pathogen Tannerella forsythia in shaping innate and adaptive host immunity in the context of periodontal disease. In addition, we will also review the current state of knowledge of surface protein glycosylation and its potential for immune modulation in other periodontal pathogens.

Release of Membrane-Bound Vesicles and Inhibition of Tumor Cell Adhesion by the Peptide Neopetrosiamide A

PubMed Central

Austin, Pamela; Heller, Markus; Williams, David E.; McIntosh, Lawrence P.; Vogl, A. Wayne; Foster, Leonard J.; Andersen, Raymond J.; Roberge, Michel; Roskelley, Calvin D.

2010-01-01

Background Neopetrosiamide A (NeoA) is a 28-amino acid tricyclic peptide originally isolated from a marine sponge as a tumor cell invasion inhibitor whose mechanism of action is unknown. Methodology/Principal Findings We show that NeoA reversibly inhibits tumor cell adhesion, disassembles focal adhesions in pre-attached cells, and decreases the level of β1 integrin subunits on the cell surface. NeoA also induces the formation of dynamic, membrane-bound protrusions on the surface of treated cells and the release of membrane-bound vesicles into the culture medium. Proteomic analysis indicates that the vesicles contain EGF and transferrin receptors as well as a number of proteins involved in adhesion and migration including: β1 integrin and numerous α integrin subunits; actin and actin-binding proteins such as cofilin, moesin and myosin 1C; and membrane modulating eps15 homology domain (EHD) proteins. Surface labeling, trafficking inhibition, and real-time imaging experiments all suggest that β1 integrin-containing vesicles are released directly from NeoA-induced cell surface protrusions rather than from vesicles generated intracellularly. The biological activity of NeoA is dependent on its disulfide bond pattern and NMR spectroscopy indicates that the peptide is globular with a continuous ridge of hydrophobic groups flanked by charged amino acid residues that could facilitate a simultaneous interaction with lipids and proteins in the membrane. Conclusions/Significance NeoA is an anti-adhesive peptide that decreases cell surface integrin levels through a novel, yet to be elucidated, mechanism that involves the release of adhesion molecule-containing vesicles from the cell surface. PMID:20520768

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

DOE Office of Scientific and Technical Information (OSTI.GOV)

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 /sup 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 modulatedmore » 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/sup 0/C instead of 100/sup 0/C.« less

Surfactant protein D delays Fas- and TRAIL-mediated extrinsic pathway of apoptosis in T cells.

PubMed

Djiadeu, Pascal; Kotra, Lakshmi P; Sweezey, Neil; Palaniyar, Nades

2017-05-01

Only a few extracellular soluble proteins are known to modulate apoptosis. We considered that surfactant-associated protein D (SP-D), an innate immune collectin present on many mucosal surfaces, could regulate apoptosis. Although SP-D is known to be important for immune cell homeostasis, whether SP-D affects apoptosis is unknown. In this study we aimed to determine the effects of SP-D on Jurkat T cells and human T cells dying by apoptosis. Here we show that SP-D binds to Jurkat T cells and delays the progression of Fas (CD95)-Fas ligand and TRAIL-TRAIL receptor induced, but not TNF-TNF receptor-mediated apoptosis. SP-D exerts its effects by reducing the activation of initiator caspase-8 and executioner caspase-3. SP-D also delays the surface exposure of phosphatidylserine. The effect of SP-D was ablated by the presence of caspase-8 inhibitor, but not by intrinsic pathway inhibitors. The binding ability of SP-D to dying cells decreases during the early stages of apoptosis, suggesting the release of apoptotic cell surface targets during apoptosis. SP-D also delays FasL-induced death of primary human T cells. SP-D delaying the progression of the extrinsic pathway of apoptosis could have important implications in regulating immune cell homeostasis at mucosal surfaces.

Probing mass-transport and binding inhomogeneity in macromolecular interactions by molecular interferometric imaging

NASA Astrophysics Data System (ADS)

Zhao, Ming; Wang, Xuefeng; Nolte, David

2009-02-01

In solid-support immunoassays, the transport of target analyte in sample solution to capture molecules on the sensor surface controls the detected binding signal. Depletion of the target analyte in the sample solution adjacent to the sensor surface leads to deviations from ideal association, and causes inhomogeneity of surface binding as analyte concentration varies spatially across the sensor surface. In the field of label-free optical biosensing, studies of mass-transport-limited reaction kinetics have focused on the average response on the sensor surface, but have not addressed binding inhomogeneities caused by mass-transport limitations. In this paper, we employ Molecular Interferometric Imaging (MI2) to study mass-transport-induced inhomogeneity of analyte binding within a single protein spot. Rabbit IgG binding to immobilized protein A/G was imaged at various concentrations and under different flow rates. In the mass-transport-limited regime, enhanced binding at the edges of the protein spots was caused by depletion of analyte towards the center of the protein spots. The magnitude of the inhomogeneous response was a function of analyte reaction rate and sample flow rate.

Biomimetic surface coatings from modular amphiphilic proteins

NASA Astrophysics Data System (ADS)

Harden, James; Wan, Fan; Fischer, Stephen; Dick, Scott

2010-03-01

Recombinant DNA methods have been used to develop a library of diblock protein polymers for creating designer biofunctional interfaces. These proteins are composed of a surface-active, amphiphilic block joined to a disordered, water soluble block with an end terminal bioactive domain. The amphiphilic block has a strong affinity for many synthetic polymer surfaces, providing a facile means of imparting biological functionality to otherwise bio-neutral materials through physical self-assembly. We have incorporated a series of bioactive end domains into this diblock motif, including sequences that encode specific cell binding and signaling functions of extracellular matrix constituents (e.g. RGD and YIGSR). In this talk, we show that these diblock constructs self-assemble into biofunctional surface coatings on several model synthetic polymer materials. We demonstrate that surface adsorption of the proteins has minimal impacts on the presentation of the bioactive domains in the soluble block, and through the use of microscopic and cell proliferation assays, we show that the resulting biofunctional interfaces are capable of inducing appropriate cellular responses in a variety of human cell types.

Probing the interplay between amyloidogenic proteins and membranes using lipid monolayers and bilayers.

PubMed

Relini, Annalisa; Marano, Nadia; Gliozzi, Alessandra

2014-05-01

Many degenerative diseases such as Alzheimer's and Parkinson's involve proteins that have a tendency to misfold and aggregate eventually forming amyloid fibers. This review describes the use of monolayers, bilayers, supported membranes, and vesicles as model systems that have helped elucidate the mechanisms and consequences of the interactions between amyloidogenic proteins and membranes. These are twofold: membranes favor the formation of amyloid structures and these induce damage in those membranes. We describe studies that show how interfaces, especially charged ones, favor amyloidogenic protein aggregation by several means. First, surfaces increase the effective protein concentration reducing a three-dimensional system to a two-dimensional one. Second, charged surfaces allow electrostatic interactions with the protein. Anionic lipids as well as rafts, rich in cholesterol and gangliosides, prove to play an especially important role. Finally, these amphipathic systems also offer a hydrophobic environment favoring conformational changes, oligomerization, and eventual formation of mature fibers. In addition, we examine several models for membrane permeabilization: protein pores, leakage induced by extraction of lipids, chaotic pores, and membrane tension, presenting illustrative examples of experimental evidence in support of these models. The picture that emerges from recent work is one where more than one mechanism is in play. Which mechanism prevails depends on the protein, its aggregation state, and the lipid environment in which the interactions occur. © 2013.

Single molecule force measurements delineate salt, pH and surface effects on biopolymer adhesion

NASA Astrophysics Data System (ADS)

Pirzer, T.; Geisler, M.; Scheibel, T.; Hugel, T.

2009-06-01

In this paper we probe the influence of surface properties, pH and salt on the adhesion of recombinant spider silk proteins onto solid substrates with single molecule force spectroscopy. A single engineered spider silk protein (monomeric C16 or dimeric (QAQ)8NR3) is covalently bound with one end to an AFM tip, which assures long-time measurements for hours with one and the same protein. The tip with the protein is brought into contact with various substrates at various buffer conditions and then retracted to desorb the protein. We observe a linear dependence of the adhesion force on the concentration of three selected salts (NaCl, NaH2PO4 and NaI) and a Hofmeister series both for anions and cations. As expected, the more hydrophobic C16 shows a higher adhesion force than (QAQ)8NR3, and the adhesion force rises with the hydrophobicity of the substrate. Unexpected is the magnitude of the dependences—we never observe a change of more than 30%, suggesting a surprisingly well-regulated balance between dispersive forces, water-structure-induced forces as well as co-solute-induced forces in biopolymer adhesion.

Anomalous Dynamics of Water Confined in Protein-Protein and Protein-DNA Interfaces.

PubMed

Chong, Song-Ho; Ham, Sihyun

2016-10-06

Confined water often exhibits anomalous properties not observable in the bulk phase. Although water in hydrophobic confinement has been the focus of intense investigation, the behavior of water confined between hydrophilic surfaces, which are more frequently found in biological systems, has not been fully explored. Here, we investigate using molecular dynamics simulations dynamical properties of the water confined in hydrophilic protein-protein and protein-DNA interfaces. We find that the interfacial water exhibits glassy slow relaxations even at 300 K. In particular, the rotational dynamics show a logarithmic decay that was observed in glass-forming liquids at deeply supercooled states. We argue that such slow water dynamics are indeed induced by the hydrophilic binding surfaces, which is in opposition to the picture that the hydration water slaves protein motions. Our results will significantly impact the view on the role of water in biomolecular interactions.

Correlation of the protein structure and gelling properties in dried egg white products.

PubMed

Handa, A; Hayashi, K; Shidara, H; Kuroda, N

2001-08-01

The relationship between protein structure and aggregation, as well as heat-induced gelling properties, of seven dried egg white (DEW) products was investigated. Strong correlations were found between average molecular weight and hydrophobicity plus surface SH groups of DEW-soluble protein aggregate (SPA). This suggests that hydrophobic interactions and disulfide bond formation between protein molecules were involved in the aggregation. The average molecular weight of DEW products with alkaline pHs was relatively higher than those with neutral pHs and the same degree of protein unfolding, probably because of more disulfide bond formation between protein molecules. In addition, strong correlations were found between hydrophobicity, surface SH groups plus average molecular weight of DEW-SPA, and physical properties of the gels from DEW products. These data indicated that controlling the aggregation of DEW proteins in the dry state is crucial to controlling the gelling properties of DEW.

Influence of hesperidin on renal cell surface glycoprotein content, nucleic acids, lysosomal enzymes and macromolecules against 7, 12-dimethylbenz [a] anthracene induced experimental breast carcinoma.

PubMed

Nandakumar, Natarajan; Jayaprakash, Ramachandran; Balasubramanian, Maruthaiveeran Periyasamy

2012-01-01

Therapeutic substances may reduce the risk of developing cancer by modulating the factors responsible for carcinogenesis. To evaluate these hypotheses, the present study was designed to investigate the modulatory effect of bioflavonoid "Hesperidin" against DMBA induced experimental breast cancer with reference to renal cell surface glycoproteins, nucleic acids, protein content, lipid profile and lysosomal enzymes. The female sprague-dawley rats were orally administered with single dose of 7, 12-DMBA to induce breast cancer and were treated with hesperidin [30 mg/kg/body weight] for a consecutive 45 days. The results revealed that there was a significant elevation in the levels of glycoproteins, nucleic acids, lysosomal enzymes and also significant alterations in macromolecules in renal tissues of cancer bearing animals. Interestingly, the altered levels of these parameters were remarkably reverted back to near normal in hesperidin treatment. The histopathological analysis of liver and kidney tissues were well supported the biochemical alterations and inevitably proves the protective role of hesperidin. It is proposed that, the effect of hesperidin during DMBA induced breast cancer could be due to the intervention strategies of hesperidin in the protein, nucleic acid biosynthesis, membrane stabilizing potentials on lysosomal compartment and inhibitory effect on cell surface glycoproteins and bio-fuel such as lipids.

Polyclonal cell activity of a repeat peptide derived from the sequence of an 85-kilodalton surface protein of Trypanosoma cruzi trypomastigotes.

PubMed Central

Pestel, J; Defoort, J P; Gras-Masse, H; Afchain, D; Capron, A; Tartar, A; Ouaissi, A

1992-01-01

Some in vitro and in vivo biological activities of an octadecapeptide derived from an 85-kDa surface protein of Trypanosoma cruzi trypomastigote were studied. The peptide coupled to a carrier protein induced the proliferative response of lymph node cells from mice immunized with various antigens. Moreover, sera from mice immunized with the coupled peptide were found to contain antibodies against a number of self and nonself antigens: fibronectin, bovine serum albumin, myosin, tetanus toxoid, ovalbumin, keyhole limpet hemocyanin, and DNA. These results are discussed in the context of Chagas' disease immunopathology. PMID:1730508

Highly multiplexed and quantitative cell-surface protein profiling using genetically barcoded antibodies.

PubMed

Pollock, Samuel B; Hu, Amy; Mou, Yun; Martinko, Alexander J; Julien, Olivier; Hornsby, Michael; Ploder, Lynda; Adams, Jarrett J; Geng, Huimin; Müschen, Markus; Sidhu, Sachdev S; Moffat, Jason; Wells, James A

2018-03-13

Human cells express thousands of different surface proteins that can be used for cell classification, or to distinguish healthy and disease conditions. A method capable of profiling a substantial fraction of the surface proteome simultaneously and inexpensively would enable more accurate and complete classification of cell states. We present a highly multiplexed and quantitative surface proteomic method using genetically barcoded antibodies called phage-antibody next-generation sequencing (PhaNGS). Using 144 preselected antibodies displayed on filamentous phage (Fab-phage) against 44 receptor targets, we assess changes in B cell surface proteins after the development of drug resistance in a patient with acute lymphoblastic leukemia (ALL) and in adaptation to oncogene expression in a Myc-inducible Burkitt lymphoma model. We further show PhaNGS can be applied at the single-cell level. Our results reveal that a common set of proteins including FLT3, NCR3LG1, and ROR1 dominate the response to similar oncogenic perturbations in B cells. Linking high-affinity, selective, genetically encoded binders to NGS enables direct and highly multiplexed protein detection, comparable to RNA-sequencing for mRNA. PhaNGS has the potential to profile a substantial fraction of the surface proteome simultaneously and inexpensively to enable more accurate and complete classification of cell states. Copyright © 2018 the Author(s). Published by PNAS.

Appearance and distribution of surface proteins of the human erythrocyte membrane. An electron microscope and immunochemical labeling study

PubMed Central

Shotton, D.; Thompson, K.; Wofsy, L.; Branton, D.

1978-01-01

We have used freeze-etching, before and after immunoferritin labeling, to visualize spectrin molecules and other surface proteins of the human erythrocyte membrane. After intramembrane particle aggregation was induced, spectrin molecules, identified by labeling with ferritin-conjugated antispectrin, were clustered on the cytoplasmic surface of the membrane in patches directly underlying the particle clusters. This labeling pattern confirms the involvement of spectrin in such particle aggregates, as previously inferred from indirect evidence. Ferritin-conjugated antihapten molecules, directed against external and cytoplasmic surface proteins of the erythrocyte membrane which had been covalently labeled nonspecifically with the hapten p-diazoniumphenyl-beta-D-lactoside, were similarly found in direct association with such intramembrane particle aggregates. This indicates that when spectrin and the intramembrane particles are aggregated, all the major proteins of the erythrocyte membrane are constrained to coaggregate with them. Although giving no direct information concerning the freedom of translational movement of proteins in the unperturbed erythrocyte membrane, these experiments suggest that a close dynamic association may exist between the integral and peripheral protein components of the membrane, such that immobilization of one component can restrict the lateral mobility of others. PMID:10605454

Interactions of urea with native and unfolded proteins: a volumetric study.

PubMed

Son, Ikbae; Shek, Yuen Lai; Tikhomirova, Anna; Baltasar, Eduardo Hidalgo; Chalikian, Tigran V

2014-11-26

We describe a statistical thermodynamic approach to analyzing urea-dependent volumetric properties of proteins. We use this approach to analyze our urea-dependent data on the partial molar volume and adiabatic compressibility of lysozyme, apocytochrome c, ribonuclease A, and α-chymotrypsinogen A. The analysis produces the thermodynamic properties of elementary urea-protein association reactions while also yielding estimates of the effective solvent-accessible surface areas of the native and unfolded protein states. Lysozyme and apocytochrome c do not undergo urea-induced transitions. The former remains folded, while the latter is unfolded between 0 and 8 M urea. In contrast, ribonuclease A and α-chymotrypsinogen A exhibit urea-induced unfolding transitions. Thus, our data permit us to characterize urea-protein interactions in both the native and unfolded states. We interpreted the urea-dependent volumetric properties of the proteins in terms of the equilibrium constant, k, and changes in volume, ΔV0, and compressibility, ΔKT0, for a reaction in which urea binds to a protein with a concomitant release of two waters of hydration to the bulk. Comparison of the values of k, ΔV0, and ΔKT0 with the similar data obtained on small molecules mimicking protein groups reveals lack of cooperative effects involved in urea-protein interactions. In general, the volumetric approach, while providing a unique characterization of cosolvent-protein interactions, offers a practical way for evaluating the effective solvent accessible surface area of biologically significant fully or partially unfolded polypeptides.

Surface charge effects in protein adsorption on nanodiamonds

NASA Astrophysics Data System (ADS)

Aramesh, M.; Shimoni, O.; Ostrikov, K.; Prawer, S.; Cervenka, J.

2015-03-01

Understanding the interaction of proteins with charged diamond nanoparticles is of fundamental importance for diverse biomedical applications. Here we present a thorough study of protein binding, adsorption kinetics and structure on strongly positively (hydrogen-terminated) and negatively (oxygen-terminated) charged nanodiamond particles using a quartz crystal microbalance by dissipation and infrared spectroscopy. By using two model proteins (bovine serum albumin and lysozyme) of different properties (charge, molecular weight and rigidity), the main driving mechanism responsible for the protein binding to the charged nanoparticles was identified. Electrostatic interactions were found to dominate the protein adsorption dynamics, attachment and conformation. We developed a simple electrostatic model that can qualitatively explain the observed adsorption behaviour based on charge-induced pH modifications near the charged nanoparticle surfaces. Under neutral conditions, the local pH around the positively and negatively charged nanodiamonds becomes very high (11-12) and low (1-3) respectively, which has a profound impact on the protein charge, hydration and affinity to the nanodiamonds. Small proteins (lysozyme) were found to form multilayers with significant conformational changes to screen the surface charge, while larger proteins (albumin) formed monolayers with minor conformational changes. The findings of this study provide a step forward toward understanding and eventually predicting nanoparticle interactions with biofluids.Understanding the interaction of proteins with charged diamond nanoparticles is of fundamental importance for diverse biomedical applications. Here we present a thorough study of protein binding, adsorption kinetics and structure on strongly positively (hydrogen-terminated) and negatively (oxygen-terminated) charged nanodiamond particles using a quartz crystal microbalance by dissipation and infrared spectroscopy. By using two model proteins (bovine serum albumin and lysozyme) of different properties (charge, molecular weight and rigidity), the main driving mechanism responsible for the protein binding to the charged nanoparticles was identified. Electrostatic interactions were found to dominate the protein adsorption dynamics, attachment and conformation. We developed a simple electrostatic model that can qualitatively explain the observed adsorption behaviour based on charge-induced pH modifications near the charged nanoparticle surfaces. Under neutral conditions, the local pH around the positively and negatively charged nanodiamonds becomes very high (11-12) and low (1-3) respectively, which has a profound impact on the protein charge, hydration and affinity to the nanodiamonds. Small proteins (lysozyme) were found to form multilayers with significant conformational changes to screen the surface charge, while larger proteins (albumin) formed monolayers with minor conformational changes. The findings of this study provide a step forward toward understanding and eventually predicting nanoparticle interactions with biofluids. Electronic supplementary information (ESI) available: The FTIR spectrum of nanodiamonds, QCM-D profiles of 50 nm nanodiamond adsorption on silica surfaces, QCM-D profiles of protein desorption after rinsing with water (rinsing experiment) and the full FTIR spectrum of proteins before and after adsorption on ND particles. See DOI: 10.1039/c5nr00250h

Major surfome and secretome profile of Streptococcus agalactiae from Nile tilapia (Oreochromis niloticus): Insight into vaccine development.

PubMed

Li, Wei; Wang, Hai-Qing; He, Run-Zhen; Li, Yan-Wei; Su, You-Lu; Li, An-Xing

2016-08-01

Streptococcus agalactiae is a major piscine pathogen that is responsible for huge economic losses to the aquaculture industry. Safe recombinant vaccines, based on a small number of antigenic proteins, are emerging as the most attractive, cost-effective solution against S. agalactiae. The proteins of S. agalactiae exposed to the environment, including surface proteins and secretory proteins, are important targets for the immune system and they are likely to be good vaccine candidates. To obtain a precise profile of its surface proteins, S. agalactiae strain THN0901, which was isolated from tilapia (Oreochromis niloticus), was treated with proteinase K to cleave surface-exposed proteins, which were identified by liquid chromatography-tandem spectrometry (LC-MS/MS). Forty surface-associated proteins were identified, including ten proteins containing cell wall-anchoring motifs, eight lipoproteins, eleven membrane proteins, seven secretory proteins, three cytoplasmic proteins, and one unknown protein. In addition, culture supernatant proteins of S. agalactiae were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and all of the Coomassie-stained bands were subsequently identified by LC-MS/MS. A total of twenty-six extracellular proteins were identified, including eleven secretory proteins, seven cell wall proteins, three membrane proteins, two cytoplasmic proteins and three unknown proteins. Of these, six highly expressed surface-associated and secretory proteins are putative to be vaccine candidate of piscine S. agalactiae. Moreover, immunogenic secreted protein, a highly expressed protein screened from the secretome in the present study, was demonstrated to induce high antibody titer in tilapia, and it conferred protection against S. agalactiae, as evidenced by the relative percent survival (RPS) 48.61± 8.45%. The data reported here narrow the scope of screening protective antigens, and provide guidance in the development of a novel vaccine against piscine S. agalactiae. Copyright © 2016 Elsevier Ltd. All rights reserved.

Quantitative characterization of conformational-specific protein-DNA binding using a dual-spectral interferometric imaging biosensor

NASA Astrophysics Data System (ADS)

Zhang, Xirui; Daaboul, George G.; Spuhler, Philipp S.; Dröge, Peter; Ünlü, M. Selim

2016-03-01

DNA-binding proteins play crucial roles in the maintenance and functions of the genome and yet, their specific binding mechanisms are not fully understood. Recently, it was discovered that DNA-binding proteins recognize specific binding sites to carry out their functions through an indirect readout mechanism by recognizing and capturing DNA conformational flexibility and deformation. High-throughput DNA microarray-based methods that provide large-scale protein-DNA binding information have shown effective and comprehensive analysis of protein-DNA binding affinities, but do not provide information of DNA conformational changes in specific protein-DNA complexes. Building on the high-throughput capability of DNA microarrays, we demonstrate a quantitative approach that simultaneously measures the amount of protein binding to DNA and nanometer-scale DNA conformational change induced by protein binding in a microarray format. Both measurements rely on spectral interferometry on a layered substrate using a single optical instrument in two distinct modalities. In the first modality, we quantitate the amount of binding of protein to surface-immobilized DNA in each DNA spot using a label-free spectral reflectivity technique that accurately measures the surface densities of protein and DNA accumulated on the substrate. In the second modality, for each DNA spot, we simultaneously measure DNA conformational change using a fluorescence vertical sectioning technique that determines average axial height of fluorophores tagged to specific nucleotides of the surface-immobilized DNA. The approach presented in this paper, when combined with current high-throughput DNA microarray-based technologies, has the potential to serve as a rapid and simple method for quantitative and large-scale characterization of conformational specific protein-DNA interactions.DNA-binding proteins play crucial roles in the maintenance and functions of the genome and yet, their specific binding mechanisms are not fully understood. Recently, it was discovered that DNA-binding proteins recognize specific binding sites to carry out their functions through an indirect readout mechanism by recognizing and capturing DNA conformational flexibility and deformation. High-throughput DNA microarray-based methods that provide large-scale protein-DNA binding information have shown effective and comprehensive analysis of protein-DNA binding affinities, but do not provide information of DNA conformational changes in specific protein-DNA complexes. Building on the high-throughput capability of DNA microarrays, we demonstrate a quantitative approach that simultaneously measures the amount of protein binding to DNA and nanometer-scale DNA conformational change induced by protein binding in a microarray format. Both measurements rely on spectral interferometry on a layered substrate using a single optical instrument in two distinct modalities. In the first modality, we quantitate the amount of binding of protein to surface-immobilized DNA in each DNA spot using a label-free spectral reflectivity technique that accurately measures the surface densities of protein and DNA accumulated on the substrate. In the second modality, for each DNA spot, we simultaneously measure DNA conformational change using a fluorescence vertical sectioning technique that determines average axial height of fluorophores tagged to specific nucleotides of the surface-immobilized DNA. The approach presented in this paper, when combined with current high-throughput DNA microarray-based technologies, has the potential to serve as a rapid and simple method for quantitative and large-scale characterization of conformational specific protein-DNA interactions. Electronic supplementary information (ESI) available: DNA sequences and nomenclature (Table 1S); SDS-PAGE assay of IHF stock solution (Fig. 1S); determination of the concentration of IHF stock solution by Bradford assay (Fig. 2S); equilibrium binding isotherm fitting results of other DNA sequences (Table 2S); calculation of dissociation constants (Fig. 3S, 4S; Table 2S); geometric model for quantitation of DNA bending angle induced by specific IHF binding (Fig. 4S); customized flow cell assembly (Fig. 5S); real-time measurement of average fluorophore height change by SSFM (Fig. 6S); summary of binding parameters obtained from additive isotherm model fitting (Table 3S); average surface densities of 10 dsDNA spots and bound IHF at equilibrium (Table 4S); effects of surface densities on the binding and bending of dsDNA (Tables 5S, 6S and Fig. 7S-10S). See DOI: 10.1039/c5nr06785e

Relationship between Immunity to Borrelia burgdorferi Outer-surface Protein A (OspA) and Lyme Arthritis

PubMed Central

Drouin, Elise E.; Glickstein, Lisa J.

2011-01-01

Antibiotic-refractory Lyme arthritis may result from Borrelia burgdorferi–induced autoimmunity in affected joints. Such patients usually have certain HLA-DRB1 molecules that bind an epitope of B. burgdorferi outer-surface protein A (OspA163–175), and cellular and humoral immune responses to OspA are greater in patients with antibiotic-refractory arthritis than in those with antibiotic-responsive arthritis. Recent work in a mouse model suggests that, during B. burgdorferi infection, OspA in genetically susceptible individuals stimulates a particularly strong TH1 response, which may be one of several factors that can help set the stage for a putative autoimmune response in affected joints. However, vaccination with OspA did not induce arthritis in this mouse model, and case and control comparisons in human vaccine trials did not show an increased frequency of arthritis among OspA-vaccinated individuals. Thus, a vaccine-induced immune response to OspA does not replicate the sequence of events needed in the natural infection to induce antibiotic-refractory Lyme arthritis. PMID:21217173

Physicochemical properties of natural actomyosin from threadfin bream (Nemipterus spp.) induced by high hydrostatic pressure.

PubMed

Zhou, Aimei; Lin, Liying; Liang, Yan; Benjakul, Soottawat; Shi, Xiaoling; Liu, Xin

2014-08-01

Changes of physicochemical properties in natural actomyosin (NAM) from threadfin bream (Nemipterus spp.) induced by high hydrostatic pressure (200, 400, 600MPa for 10, 30, 50min) were studied. The increase in turbidity of NAM was coincidental with the decrease in protein solubility with increasing pressure and time, suggesting the formation of protein aggregates. SDS-PAGE showed that polymerisation and degradation of myosin heavy chain were induced by high pressure. Ca(2+)-ATPase activity of NAM treated by high pressure was lost, suggesting the denaturation of myosin and the dissociation of actomyosin complex. Surface hydrophobicity of NAM increased when the pressure and pressurization time increased, indicating that the exposed hydrophobic residues increased upon application of high pressure. Decrease in total sulfhydryl content and increase in surface-reactive sulfhydryl content of NAM samples were observed with the extension of pressurizing time, indicating the formation of disulphide bonds through oxidation of SH groups or disulphide interchanges. The above changes of physicochemical properties suggested conformational changes of NAM from muscle of threadfin bream induced by high hydrostatic pressure. Copyright © 2014 Elsevier Ltd. All rights reserved.

Nanoparticles as conjugated delivery agents for therapeutic applications

NASA Astrophysics Data System (ADS)

Muroski, Megan Elizabeth

This dissertation explores the use of nanoparticles as conjugated delivery agents. Chapter 1 is a general introduction. Chapter 2 discusses the delivery by a nanoparticle platform provides a method to manipulate gene activation, by taking advantage of the high surface area of a nanoparticle and the ability to selectively couple a desired biological moiety to the NP surface. The nanoparticle based transfection approach functions by controlled release of gene regulatory elements from a 6 nm AuNP (gold nanoparticle) surface. The endosomal release of the regulatory elements from the nanoparticle surface results in endogenous protein knockdown simultaneously with exogenous protein expression for the first 48 h. The use of fluorescent proteins as the endogenous and exogenous signals for protein expression enables the efficiency of co-delivery of siRNA (small interfering RNA) for GFP (green fluorescent protein) knockdown and a dsRed-express linearized plasmid for induction to be optically analyzed in CRL-2794, a human kidney cell line expressing an unstable green fluorescent protein. Delivery of the bimodal nanoparticle in cationic liposomes results in 20% GFP knockdown within 24 h of delivery and continues exhibiting knockdown for up to 48 h for the bimodal agent. Simultaneous dsRed expression is observed to initiate within the same time frame with expression levels reaching 34% after 25 days although cells have divided approximately 20 times, implying daughter cell transfection has occurred. Fluorescence cell sorting results in a stable colony, as demonstrated by Western blot analysis. The simultaneous delivery of siRNA and linearized plasmid DNA on the surface of a single nanocrystal provides a unique method for definitive genetic control within a single cell and leads to a very efficient cell transfection protocol. In Chapter 3, we wanted to understand the NP complex within the cell, and to look at the dynamics of release utilizing nanometal surface energy transfer as a molecular beacon. The development of non-viral transfection approaches using gold nanoparticles (AuNP) as a gene carrier allows the implementation of advanced biophysical tools to follow the transfection cycle by utilizing nanometal surface energy transfer (NSET) molecular beacon methods coupled to delivery of a gene that induces a fluorescent protein. The change in photoluminescence of an appended dye following gene release from the AuNP surface within endosomes can be tempo-rally and spatially followed. The ability to correlate the release events with the protein expression event by simultaneously monitoring fluorescent protein production provides insight into package uptake, nanoparticle disassembly, and final gene expression. Employing AuNP transfection constructs and then monitoring the stages of the transfection cycle via NSET, indicates delivery of the constructs leads to gene release from the AuNP surface within the endosome followed by slow cytosolic diffusion. The slow diffusion is the limiting step for transfection and impacts the protein yield due to competing degradation processes. Chapter 4 aims to improve the NP conjugate through the use of cell penetrating peptides (CPP) to Transfect Primary Cells. All future clinical applications of mesenchymal stem cell (MSC) therapies must allow the MSC to be harvested, transfected, and induced to express a desired protein or selection of proteins to have medical benefit. For the full potential of MSC cell therapy to be realized, it is desirable to be able to systematically alter the protein expression in harvested MSC cells with high fidelity in a single transfection event. We have developed a bimodal delivery platform based on the use of a solid gold core nanoparticle that has been surface modified to produce a chimera containing a protein transduction domain (PTD) sequence to enhance cellular uptake and a linearized expression vector to induce protein production. The transfection chimera is observed to be an efficient inducer of protein expression following a single treatment of femur bone marrow isolated rat MSCs. Use of the neutral penta-peptide, Ku70, designed from Bax-inhibiting peptides in a 500:1 ratio to the linearized gene yields >80% transfection efficiencies. Chapter 5 further develops this idea by using cell penetrating peptides. Research over the past decade has identified several of the key limiting features in multidrug resistance therapy applications, such as, cellular targeting, protection from multidrug resistant mediators and retention of intact and functional drugs. Cell penetrating peptides are able to overcome the difficulties of drug transport resulting in improved efficacy of delivery. Functionalizing the cell penetrating peptide onto the surface of a quantum dot, allows the capability of creating an individualized package for further downstream studies. Four distinct cell penetrating peptides, TAT, VP-22, Ku-70, and hCT (9-32), were utilized to study the different profiles in gliosarcoma lines (rat 9L) with varying resistances to one of the most prescribed drugs in treating glioblastoma in the clinic; BCNU. (Abstract shortened by UMI.)

Surface layer protein from Lactobacillus acidophilus NCFM inhibit intestinal pathogen-induced apoptosis in HT-29 cells.

PubMed

Meng, Jun; Zhang, Qiu-Xiang; Lu, Rong-Rong

2017-03-01

Intestinal pathogens have been proposed to adhere to epithelial cells and cause apoptosis. This study was to investigate the inhibitory effects of surface layer protein (SLP, 46kDa) from Lactobacillus acidophilus NCFM on Escherichia coli and Salmonella-induced apoptosis in HT-29 cells and the mechanism of the inhibition was also studied. The SLP could alleviate the chromatin condensation caused by intestinal pathogens as observed under fluorescent microscope. Flow cytometry analysis showed that the SLP decreased E. coli and Salmonella-induced apoptosis by 46% and 48%, respectively. The SLP could also inhibit the mitochondrial membrane potential reduction and Ca 2+ level increase in HT-29 cells. Furthermore, the activation of caspase-9 and caspase-3 induced by E. coli and Salmonella was significantly decreased by the addition of SLP. These results suggested that L. acidophilus NCFM SLP could protect HT-29 cells against intestinal pathogen-induced apoptosis through a mitochondria-mediated pathway. These findings may reveal a new method for the treatment of intestinal infection and provide a theoretical basis for the practical application of SLP in food, biological and pharmaceutical fields. Copyright © 2017 Elsevier B.V. All rights reserved.

Outer membrane protein A of Acinetobacter baumannii induces differentiation of CD4+ T cells toward a Th1 polarizing phenotype through the activation of dendritic cells.

PubMed

Lee, Jun Sik; Lee, Je Chul; Lee, Chang-Min; Jung, In Duk; Jeong, Young-Il; Seong, Eun-Young; Chung, Hae-Young; Park, Yeong-Min

2007-06-30

Acinetobacter baumannii is an increasing hospital-acquired pathogen that causes a various type of infections, but little is known about the protective immune response to this microorganism. Outer membrane protein A of A. baumannii (AbOmpA) is a major porin protein and plays an important role in pathogenesis. We analyzed interaction between AbOmpA and dendritic cells (DCs) to characterize the role of this protein in promoting innate and adaptive immune responses. AbOmpA functionally activates bone marrow-derived DCs by augmenting expression of the surface markers, CD40, CD54, B7 family (CD80 and CD86) and major histocompatibility complex class I and II. AbOmpA induces production of Th1-promoting interleukin-12 from DCs and augments the syngeneic and allogeneic immunostimulatory capacity of DCs. AbOmpA stimulates production of interferon-gamma from T cells in mixed lymphocyte reactions, which suggesting Th1-polarizing capacity. CD4(+) T cells stimulated by AbOmpA-stimulated DCs show a Th1-polarizing cytokine profile. The expression of surface markers on DCs is mediated by both mitogen-activated protein kinases and NF-kappaB pathways. Our findings suggest that AbOmpA induces maturation of DCs and drives Th1 polarization, which are important properties for determining the nature of immune response against A. baumannii.

Protein-Coupled Fluorescent Probe To Visualize Potassium Ion Transition on Cellular Membranes.

PubMed

Hirata, Tomoya; Terai, Takuya; Yamamura, Hisao; Shimonishi, Manabu; Komatsu, Toru; Hanaoka, Kenjiro; Ueno, Tasuku; Imaizumi, Yuji; Nagano, Tetsuo; Urano, Yasuteru

2016-03-01

K(+) is the most abundant metal ion in cells, and changes of [K(+)] around cell membranes play important roles in physiological events. However, there is no practical method to selectively visualize [K(+)] at the surface of cells. To address this issue, we have developed a protein-coupled fluorescent probe for K(+), TLSHalo. TLSHalo is responsive to [K(+)] in the physiological range, with good selectivity over Na(+) and retains its K(+)-sensing properties after covalent conjugation with HaloTag protein. By using cells expressing HaloTag on the plasma membrane, we successfully directed TLSHalo specifically to the outer surface of target cells. This enabled us to visualize localized extracellular [K(+)] change with TLSHalo under a fluorescence microscope in real time. To confirm the experimental value of this system, we used TLSHalo to monitor extracellular [K(+)] change induced by K(+) ionophores or by activation of a native Ca(2+)-dependent K(+) channel (BK channel). Further, we show that K(+) efflux via BK channel induced by electrical stimulation at the bottom surface of the cells can be visualized with TLSHalo by means of total internal reflection fluorescence microscope (TIRFM) imaging. Our methodology should be useful to analyze physiological K(+) dynamics with high spatiotemporal resolution.

Heme Oxygenase-1 Protects Corexit 9500A-Induced Respiratory Epithelial Injury across Species

PubMed Central

Oliva, Octavio M.; Karki, Suman; Surolia, Ranu; Wang, Zheng; Watson, R. Douglas; Thannickal, Victor J.; Powell, Mickie; Watts, Stephen; Kulkarni, Tejaswini; Batra, Hitesh; Bolisetty, Subhashini; Agarwal, Anupam; Antony, Veena B.

2015-01-01

The effects of Corexit 9500A (CE) on respiratory epithelial surfaces of terrestrial mammals and marine animals are largely unknown. This study investigated the role of CE-induced heme oxygenase-1 (HO-1), a cytoprotective enzyme with anti-apoptotic and antioxidant activity, in human bronchial airway epithelium and the gills of exposed aquatic animals. We evaluated CE-mediated alterations in human airway epithelial cells, mice lungs and gills from zebrafish and blue crabs. Our results demonstrated that CE induced an increase in gill epithelial edema and human epithelial monolayer permeability, suggesting an acute injury caused by CE exposure. CE induced the expression of HO-1 as well as C-reactive protein (CRP) and NADPH oxidase 4 (NOX4), which are associated with ROS production. Importantly, CE induced caspase-3 activation and subsequent apoptosis of epithelial cells. The expression of the intercellular junctional proteins, such as tight junction proteins occludin, zonula occludens (ZO-1), ZO-2 and adherens junctional proteins E-cadherin and Focal Adhesion Kinase (FAK), were remarkably inhibited by CE, suggesting that these proteins are involved in CE-induced increased permeability and subsequent apoptosis. The cytoskeletal protein F-actin was also disrupted by CE. Treatment with carbon monoxide releasing molecule-2 (CORM-2) significantly inhibited CE-induced ROS production, while the addition of HO-1 inhibitor, significantly increased CE-induced ROS production and apoptosis, suggesting a protective role of HO-1 or its reaction product, CO, in CE-induced apoptosis. Using HO-1 knockout mice, we further demonstrated that HO-1 protected against CE-induced inflammation and cellular apoptosis and corrected CE-mediated inhibition of E-cadherin and FAK. These observations suggest that CE activates CRP and NOX4-mediated ROS production, alters permeability by inhibition of junctional proteins, and leads to caspase-3 dependent apoptosis of epithelial cells, while HO-1 and its reaction products protect against oxidative stress and apoptosis. PMID:25835394

The simulation study of protein-protein interfaces based on the 4-helix bundle structure

NASA Astrophysics Data System (ADS)

Fukuda, Masaki; Komatsu, Yu; Morikawa, Ryota; Miyakawa, Takeshi; Takasu, Masako; Akanuma, Satoshi; Yamagishi, Akihiko

2013-02-01

Docking of two protein molecules is induced by intermolecular interactions. Our purposes in this study are: designing binding interfaces on the two proteins, which specifically interact to each other; and inducing intermolecular interactions between the two proteins by mixing them. A 4-helix bundle structure was chosen as a scaffold on which binding interfaces were created. Based on this scaffold, we designed binding interfaces involving charged and nonpolar amino acid residues. We performed molecular dynamics (MD) simulation to identify suitable amino acid residues for the interfaces. We chose YciF protein as the scaffold for the protein-protein docking simulation. We observed the structure of two YciF protein molecules (I and II), and we calculated the distance between centroids (center of gravity) of the interfaces' surface planes of the molecules I and II. We found that the docking of the two protein molecules can be controlled by the number of hydrophobic and charged amino acid residues involved in the interfaces. Existence of six hydrophobic and five charged amino acid residues within an interface were most suitable for the protein-protein docking.

Forced interaction of cell surface proteins with Derlin-1 in the endoplasmic reticulum is sufficient to induce their dislocation into the cytosol for degradation.

PubMed

Cho, Sunglim; Lee, Miriam; Jun, Youngsoo

2013-01-11

Aberrantly folded proteins in the endoplasmic reticulum (ER) are rapidly removed into the cytosol for degradation by the proteasome via an evolutionarily conserved process termed ER-associated protein degradation (ERAD). ERAD of a subset of proteins requires Derlin-1 for dislocation into the cytosol; however, the molecular function of Derlin-1 remains unclear. Human cytomegalovirus US11 exploits Derlin-1-dependent ERAD to degrade major histocompatibility complex class I (MHC-I) molecules for immune evasion. Because US11 binds to both MHC-I molecules and Derlin-1 via its luminal and transmembrane domains (TMDs), respectively, the major role of US11 has been proposed to simply be delivery of MHC-I molecules to Derlin-1. Here, we directly tested this proposal by generating a hybrid MHC-I molecule, which contains the US11 TMD, and thus can associate with Derlin-1 in the absence of US11. Intriguingly, this MHC-I hybrid was rapidly degraded in a Derlin-1- and proteasome-dependent manner. Similarly, the vesicular stomatitis virus G protein, otherwise expressed at the cell surface, was degraded via Derlin-1-dependent ERAD when its TMD was replaced with that of US11. Thus, forced interaction of cell surface proteins with Derlin-1 is sufficient to induce their degradation via ERAD. Taken together, these results suggest that the main role of US11 is to recruit MHC-I molecules to Derlin-1, which then mediates the dislocation of MHC-I molecules into the cytosol for degradation. Copyright © 2012 Elsevier Inc. All rights reserved.

Biocidal action of ozone-treated polystyrene surfaces on vegetative and sporulated bacteria

NASA Astrophysics Data System (ADS)

Mahfoudh, Ahlem; Barbeau, Jean; Moisan, Michel; Leduc, Annie; Séguin, Jacynthe

2010-03-01

Surfaces of materials can be modified to ensure specific interaction features with microorganisms. The current work discloses biocidal properties of polystyrene (PS) Petri-dish surfaces that have been exposed to a dry gaseous-ozone flow. Such treated PS surfaces are able to inactivate various species of vegetative and sporulated bacteria on a relatively short contact time. Denaturation of proteins seems likely based on a significant loss of enzymatic activity of the lysozyme protein. Characterization of these surfaces by atomic-force microscopy (AFM), Fourier-transform infra-red (FTIR) spectroscopy and X-ray photoelectron spectroscopy (XPS) reveals specific structural and chemical modifications as compared to untreated PS. Persistence of the biocidal properties of these treated surfaces is observed. This ozone-induced process is technically simple to achieve and does not require active precursors as in grafting.

Kinetics of protein physicochemical changes induced by heating in meat using mimetic models: (1) relative effects of heat and oxidants.

PubMed

Promeyrat, A; Daudin, J D; Gatellier, P

2013-05-01

Optimizing the nutritional quality of cooked meat needs a better understanding of the mechanisms responsible for protein changes induced by heating. The relative contributions of chemical and thermal effects on protein physicochemical changes were studied using meat models. Two models were tested: a basic model made of an aqueous suspension of myofibrillar proteins, and a complex model, in which oxidants were added in physiological concentrations. Various heating time-temperature combinations were applied to both models in the ranges 45-90 °C and 5-120 min. Protein oxidation was evaluated by carbonyl and free thiol contents. Conformational changes of proteins were assessed by measurements of surface hydrophobicity and aggregation. Carbonyl formation was weakly affected by the thermal process alone but exacerbated by oxidants. A synergistic effect of oxidants and heat treatments on protein oxidation was noted. Changes in protein hydrophobicity and aggregation were dominated by the thermal process. Copyright © 2012 Elsevier Ltd. All rights reserved.

Unique surface adsorption behaviors of serum proteins on chemically uniform and alternating surfaces

NASA Astrophysics Data System (ADS)

Song, Sheng

With increasing interests of studying proteins adsorption on the surfaces with nanoscale features in biomedical field, it is crucial to have fundamental understandings on how the proteins are adsorbed on such a surface and what factors contribute to the driving forces of adsorption. Besides, exploring more available nanoscale templates would greatly offer more possibilities one could design surface bio-detection methods with favorable protein-surface interactions. Thus, to fulfill the purpose, the work in this dissertation has been made into three major sections. First, to probe the intermediate states which possibly exist between stable and unstable phases described in mean-field theory diagram, a solvent vapor annealing method is chosen to slowly induce the copolymer polystyrene-block-polyvinylpyridine (PS-b-PVP)'s both blocks undergoing micro-phase separations from initial spherical nanodomains into terminal cylindrical nanodomains. During this process, real time atomic force microscopy (AFM) has been conducted to capture other six intermediate states with different morphologies on the polymeric film surfaces. Secondly, upon recognizing each intermediate state, the solution of immunoglobulin gamma (IgG) proteins has been deposited on the surface and been rinsed off with buffer solution before the protein-bounded surface is imaged by AFM. It has been found IgG showing a strong adsorption preference on PS over P4VP block. Among all the six intermediate states, the proteins are almost exclusively adsorbed on PS nanodomains regardless the concentration and deposition time. Thirdly, a trinodular shape protein fibrinogen (Fg) is selected for investigating how geometry and surface charge of proteins would interplay with cylindrical nanodomains on a surface developed from Polystyrene -block-Poly-(methyl methacrylate) PS-b-PMMA. Also, Fg adsorptions on chemically homogeneous surfaces are included here to have a better contrast of showing how much difference it can make by using it on a nanoscale surface. Interestingly, higher concentration of protein solution promotes the occurrences of single phase packed Fg on the PS domain. The densely packed network has formed where each Fg keeps its main body in PS domain and leaves its two alpha C chains on nearby PMMA domain. We believe this conformation and orientation would maximize both the hydrophobic and electrostatic interactions between Fg and the underlying surface.

Different protein of Echinococcus granulosus stimulates dendritic induced immune response.

PubMed

Wang, Yana; Wang, Qiang; Lv, Shiyu; Zhang, Shengxiang

2015-06-01

Cystic echinococcosis is a chronic infectious disease that results from a host/parasite interaction. Vaccination with ferritin derived from Echinococcus granulosus is a potential preventative treatment. To understand whether ferritin is capable of inducing a host immune response, we investigated the response of dendritic cells (DCs) to both recombinant ferritin protein and the hydatid fluid (HF) of E. granulosus. We evaluated the immunomodulatory potential of these antigens by performing, immunocytochemistry, electron microscopy and in vivo imaging of monocyte-derived murine DCs. During antigen stimulation of DCs, ferritin cause DCs maturation and induced higher levels of surface marker expression and activated T-cell proliferation and migration. On contrary, HF failed to induce surface marker expression and to stimulate T-cell proliferation. In response to HF, DCs produced interleukin-6 (IL-6), but no IL-12 and IL-10. DCs stimulated with ferritin produced high levels of cytokines. Overall, HF appears to induce host immunosuppression in order to ensure parasite survival via inhibits DC maturation and promotes Th2-dependent secretion of cytokines. Although ferritin also promoted DC maturation and cytokine release, it also activates CD4+T-cell proliferation, but regard of the mechanism of the Eg.ferritin induce host to eradicate E. granulosus were not clear.

Arf and Rho GAP adapter protein ARAP1 participates in the mobilization of TRAIL-R1/DR4 to the plasma membrane.

PubMed

Símová, Sárka; Klíma, Martin; Cermak, Lukas; Sourková, Vladimíra; Andera, Ladislav

2008-03-01

TRAIL, a ligand of the TNFalpha family, induces upon binding to its pro-death receptors TRAIL-R1/DR4 and TRAIL-R2/DR5 the apoptosis of cancer cells. Activated receptors incite the formation of the Death-Inducing Signaling Complex followed by the activation of the downstream apoptotic signaling. TRAIL-induced apoptosis is regulated at multiple levels, one of them being the presence and relative number of TRAIL pro- and anti-apoptotic receptors on the cytoplasmic membrane. In a yeast two-hybrid search for proteins that interact with the intracellular part (ICP) of DR4, we picked ARAP1, an adapter protein with ArfGAP and RhoGAP activities. In yeast, DR4(ICP) interacts with the alternatively spliced ARAP1 lacking 11 amino acids from the PH5 domain. Transfected ARAP1 co-precipitates with DR4 and co-localizes with it in the endoplasmic reticulum/Golgi, at the cytoplasmic membrane and in early endosomes of TRAIL-treated cells. ARAP1 knockdown significantly compromises the localization of DR4 at the cell surface of several tumor cell lines and slows down their TRAIL-induced death. ARAP1 overexpressed in HEL cells does not affect their TRAIL-induced apoptosis or the membrane localization of DR4, but it enhances the cell-surface presentation of phosphatidyl serine. Our data indicate that ARAP1 is likely involved in the regulation of the cell-specific trafficking of DR4 and might thus affect the efficacy of TRAIL-induced apoptosis.

Hepatitis C Virus E2 Protein Induces Upregulation of IL-8 Pathways and Production of Heat Shock Proteins in Human Thyroid Cells.

PubMed

Hammerstad, Sara Salehi; Stefan, Mihaela; Blackard, Jason; Owen, Randall P; Lee, Hanna J; Concepcion, Erlinda; Yi, Zhengzi; Zhang, Weijia; Tomer, Yaron

2017-02-01

Thyroiditis is one of the most common extrahepatic manifestations of hepatitis C virus (HCV) infection. By binding to surface cell receptor CD81, HCV envelope glycoprotein E2 mediates entry of HCV into cells. Studies have shown that different viral proteins may individually induce host responses to infection. We hypothesized that HCV E2 protein binding to CD81 expressed on thyroid cells activates a cascade of inflammatory responses that can trigger autoimmune thyroiditis in susceptible individuals. Human thyroid cell lines ML-1 and human thyrocytes in primary cell culture were treated with HCV recombinant E2 protein. The expression of major proinflammatory cytokines was measured at the messenger RNA and protein levels. Next-generation transcriptome analysis was used to identify early changes in gene expression in thyroid cells induced by E2. HCV envelope protein E2 induced strong inflammatory responses in human thyrocytes, resulting in production of interleukin (IL)-8, IL-6, and tumor necrosis factor-α. Furthermore, the E2 protein induced production of several heat shock proteins including HSP60, HSP70p12A, and HSP10, in human primary thyrocytes. In thyroid cell line ML-1, RNA sequencing identified upregulation of molecules involved in innate immune pathways with high levels of proinflammatory cytokines and chemokines and increased expression of costimulatory molecules, specifically CD40, known to be a major thyroid autoimmunity gene. Our data support a key role for HCV envelope protein E2 in triggering thyroid autoimmunity through activation of cytokine pathways by bystander mechanisms. Copyright © 2017 by the Endocrine Society

Hydrolysis-controlled protein adsorption and antifouling behaviors of mixed charged self-assembled monolayer: A molecular simulation study.

PubMed

Liu, Jie; Zhou, Jian

2016-08-01

Understanding the mechanism of the antimicrobial and antifouling properties of mixed charged materials is of great significance. The interactions between human gamma fibrinogen (γFg) and mixed carboxylic methyl ether-terminated (COOCH3-) and trimethylamino-terminated (N(CH3)3(+)-) SAMs and the influence of hydrolysis were studied by molecular simulations. After hydrolysis, the mixed SAMs exhibit behaviors from antimicrobial to antifouling, since the COOCH3-thiols were translated into carboxylic acid (COO(-)-) terminated thiols, which carried a net charge of -1 e. Simulation results showed that the main differences between COOCH3-/N(CH3)3(+)-SAM and COO(-)-/N(CH3)3(+)-SAM are the charged property and the hydration layer above the surface. γFg could stably adsorb on the positively-charged COOCH3-/N(CH3)3(+)-SAM. The adsorption behavior is mainly induced by the strong electrostatic attraction. There is a single hydration layer bound to the surface, which is related to the N(CH3)3(+) groups. The van der Waals repulsion between γFg and the single hydration layer are not strong enough to compensate the strong electrostatic attraction. After hydrolysis, the positively-charged SAM was transferred to a neutral mixed charged surface, the electrostatic attraction between γFg and the surface disappears. Meanwhile, the SAM surface is covered by double hydration layers, which is induced by the N(CH3)3(+) and COO(-) groups; water molecules around COO(-) groups are obviously denser than that around N(CH3)3(+) groups. With the combined contribution from double hydration layers and the vanishment of electrostatic attraction, γFg is forced to desorb from the surface. After hydrolysis, the internal structure of mixed SAM appears more ordered due to the electrostatic interactions between charged groups on the top of SAMs. The antimicrobial and antifouling materials are of great importance in many biological applications. The strong hydration property of surfaces and the interactions between proteins and surfaces play a key role in resisting protein adsorption. The mixed SAMs, constructed from a 1:1 combination of COOCH3- and N(CH3)3(+)-terminated thiols, can induce protein adsorption mainly through the electrostatic interaction. When the COOCH3-terminated thiols were hydrolyzed to negatively charged COO(-)-terminated thiols, the mixed-charged SAMs switched from antimicrobial to antifouling. Due to the strong hydration property of the mixed charged SAMs, the adsorbed γFg moved away from the surface. Understanding the interactions between protein and mixed-charged SAMs in the atomistic level is important for the practical design and development of new antimicrobial and antifouling materials. Copyright © 2016 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

Variable Lysozyme Transport Dynamics on Oxidatively Functionalized Polystyrene Films.

PubMed

Moringo, Nicholas A; Shen, Hao; Tauzin, Lawrence J; Wang, Wenxiao; Bishop, Logan D C; Landes, Christy F

2017-10-17

Tuning protein adsorption dynamics at polymeric interfaces is of great interest to many biomedical and material applications. Functionalization of polymer surfaces is a common method to introduce application-specific surface chemistries to a polymer interface. In this work, single-molecule fluorescence microscopy is utilized to determine the adsorption dynamics of lysozyme, a well-studied antibacterial protein, at the interface of polystyrene oxidized via UV exposure and oxygen plasma and functionalized by ligand grafting to produce varying degrees of surface hydrophilicity, surface roughness, and induced oxygen content. Single-molecule tracking indicates lysozyme loading capacities, and surface mobility at the polymer interface is hindered as a result of all functionalization techniques. Adsorption dynamics of lysozyme depend on the extent and the specificity of the oxygen functionalities introduced to the polystyrene surface. Hindered adsorption and mobility are dominated by hydrophobic effects attributed to water hydration layer formation at the functionalized polystyrene surfaces.

Ursolic Acid Inhibits Na+/K+-ATPase Activity and Prevents TNF-α-Induced Gene Expression by Blocking Amino Acid Transport and Cellular Protein Synthesis

PubMed Central

Yokomichi, Tomonobu; Morimoto, Kyoko; Oshima, Nana; Yamada, Yuriko; Fu, Liwei; Taketani, Shigeru; Ando, Masayoshi; Kataoka, Takao

2011-01-01

Pro-inflammatory cytokines, such as tumor necrosis factor (TNF)-α, induce the expression of a wide variety of genes, including intercellular adhesion molecule-1 (ICAM-1). Ursolic acid (3β-hydroxy-urs-12-en-28-oic acid) was identified to inhibit the cell-surface ICAM-1 expression induced by pro-inflammatory cytokines in human lung carcinoma A549 cells. Ursolic acid was found to inhibit the TNF-α-induced ICAM-1 protein expression almost completely, whereas the TNF-α-induced ICAM-1 mRNA expression and NF-κB signaling pathway were decreased only partially by ursolic acid. In line with these findings, ursolic acid prevented cellular protein synthesis as well as amino acid uptake, but did not obviously affect nucleoside uptake and the subsequent DNA/RNA syntheses. This inhibitory profile of ursolic acid was similar to that of the Na+/K+-ATPase inhibitor, ouabain, but not the translation inhibitor, cycloheximide. Consistent with this notion, ursolic acid was found to inhibit the catalytic activity of Na+/K+-ATPase. Thus, our present study reveals a novel molecular mechanism in which ursolic acid inhibits Na+/K+-ATPase activity and prevents the TNF-α-induced gene expression by blocking amino acid transport and cellular protein synthesis. PMID:24970122

Nucleation of Iron Oxide Nanoparticles Mediated by Mms6 Protein in Situ

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kashyap, Sanjay; Woehl, Taylor J; Liu, Xunpei

2014-09-23

Biomineralization proteins are widely used as templating agents in biomimetic synthesis of a variety of organic–inorganic nanostructures. However, the role of the protein in controlling the nucleation and growth of biomimetic particles is not well understood, because the mechanism of the bioinspired reaction is often deduced from ex situ analysis of the resultant nanoscale mineral phase. Here we report the direct visualization of biomimetic iron oxide nanoparticle nucleation mediated by an acidic bacterial recombinant protein, Mms6, during an in situ reaction induced by the controlled addition of sodium hydroxide to solution-phase Mms6 protein micelles incubated with ferric chloride. Using inmore » situ liquid cell scanning transmission electron microscopy we observe the liquid iron prenucleation phase and nascent amorphous nanoparticles forming preferentially on the surface of protein micelles. Our results provide insight into the early steps of protein-mediated biomimetic nucleation of iron oxide and point to the importance of an extended protein surface during nanoparticle formation.« less

Occludin confers adhesiveness when expressed in fibroblasts.

PubMed

Van Itallie, C M; Anderson, J M

1997-05-01

Occludin is an integral membrane protein specifically associated with tight junctions. Previous studies suggest it is likely to function in forming the intercellular seal. In the present study, we expressed occludin under an inducible promotor in occludin-null fibroblasts to determine whether this protein confers intercellular adhesion. When human occludin is stably expressed in NRK and Rat-1 fibroblasts, which lack endogenous occludin and tight junctions but do have well developed ZO-1-containing adherens-like junctions, occludin colocalizes with ZO-1 to points of cell-cell contact. In contrast, L-cell fibroblasts which lack cadherin-based adherens junctions, target neither ZO-1 nor occludin to sites of cell contact. Occludin-induced adhesion was next quantified using a suspended cell assay. In NRK and Rat-1 cells, occludin expression induces adhesion in the absence of calcium, thus independent of cadherin-cadherin contacts. In contrast, L-cells are nonadhesive in this assay and show no increase in adhesion after induction of occludin expression. Binding of an antibody to the first of the putative extracellular loops of occludin confirmed that this sequence was exposed on the cell surface, and synthetic peptides containing the amino acid sequence of this loop inhibit adhesion induced by occludin expression. These results suggest that the extracellular surface of occludin is directly involved in cell-cell adhesion and the ability to confer adhesiveness correlates with the ability to colocalize with its cytoplasmic binding protein, ZO-1.

Requirements for cell rounding and surface protein down-regulation by Ebola virus glycoprotein.

PubMed

Francica, Joseph R; Matukonis, Meghan K; Bates, Paul

2009-01-20

Ebola virus causes an acute hemorrhagic fever that is associated with high morbidity and mortality. The viral glycoprotein is thought to contribute to pathogenesis, though precise mechanisms are unknown. Cellular pathogenesis can be modeled in vitro by expression of the Ebola viral glycoprotein (GP) in cells, which causes dramatic morphological changes, including cell rounding and surface protein down-regulation. These effects are known to be dependent on the presence of a highly glycosylated region of the glycoprotein, the mucin domain. Here we show that the mucin domain from the highly pathogenic Zaire subtype of Ebola virus is sufficient to cause characteristic cytopathology when expressed in the context of a foreign glycoprotein. Similarly to full length Ebola GP, expression of the mucin domain causes rounding, detachment from the extracellular matrix, and the down-regulation of cell surface levels of beta1 integrin and major histocompatibility complex class 1. These effects were not seen when the mucin domain was expressed in the context of a glycophosphatidylinositol-anchored isoform of the foreign glycoprotein. In contrast to earlier analysis of full length Ebola glycoproteins, chimeras carrying the mucin domains from the Zaire and Reston strains appear to cause similar levels of down-modulation and cell detachment. Cytopathology associated with Ebola glycoprotein expression does not occur when GP expression is restricted to the endoplasmic reticulum. In contrast to a previously published report, our results demonstrate that GP-induced surface protein down-regulation is not mediated through a dynamin-dependent pathway. Overall, these results support a model in which the mucin domain of Ebola GP acts at the cell surface to induce protein down modulation and cytopathic effects.

Evidence for an intermediate in tau filament formation.

PubMed

Chirita, Carmen N; Kuret, Jeff

2004-02-17

Alzheimer's disease is defined in part by the intraneuronal accumulation of filaments comprised of the microtubule-associated protein tau. In vitro, fibrillization of full-length, unphosphorylated recombinant tau can be induced under near-physiological conditions by treatment with various agents, including anionic surfactants. Here we examine the pathway through which anionic surfactants promote tau fibrillization using a combination of electron microscopy and fluorescence spectroscopy. Protein and surfactant first interacted in solution to form micelles, which then provided negatively charged surfaces that accumulated tau aggregates. Surface aggregation of tau protein was followed by the time-dependent appearance of a thioflavin S reactive intermediate that accumulated over a period of hours. The intermediate was unstable in the absence of anionic surfaces, suggesting it was not filamentous. Fibrillization proceeded after intermediate formation with classic nucleation-dependent kinetics, consisting of lag phase followed by the exponential increase in filament lengths, followed by an equilibrium phase reached in approximately 24 h. The pathway did not require protein insertion into the micelle hydrophobic core or conformational change arising from mixed micelle formation, because anionic microspheres constructed from impermeable polystyrene were capable of qualitatively reproducing all aspects of the fibrillization reaction. It is proposed that the progression from amorphous aggregation through intermediate formation and fibrillization may underlie the activity of other inducers such as hyperphosphorylation and may be operative in vivo.

Electrosynthesized MIPs for transferrin: Plastibodies or nano-filters?

PubMed

Zhang, Xiaorong; Yarman, Aysu; Erdossy, Júlia; Katz, Sagie; Zebger, Ingo; Jetzschmann, Katharina J; Altintas, Zeynep; Wollenberger, Ulla; Gyurcsányi, Róbert E; Scheller, Frieder W

2018-05-15

Molecularly imprinted polymer (MIP) nanofilms for transferrin (Trf) have been synthesized on gold surfaces by electro-polymerizing the functional monomer scopoletin in the presence of the protein target or around pre-adsorbed Trf. As determined by atomic force microscopy (AFM) the film thickness was comparable with the molecular dimension of the target. The target (re)binding properties of the electro-synthesized MIP films was evaluated by cyclic voltammetry (CV) and square wave voltammetry (SWV) through the target-binding induced permeability changes of the MIP nanofilms to the ferricyanide redox marker, as well as by surface plasmon resonance (SPR) and surface enhanced infrared absorption spectroscopy (SEIRAS) of the immobilized protein molecules. For Trf a linear concentration dependence in the lower micromolar range and an imprinting factor of ~5 was obtained by SWV and SPR. Furthermore, non-target proteins including the iron-free apo-Trf were discriminated by pronounced size and shape specificity. Whilst it is generally assumed that the rebinding of the target or of cross-reacting proteins exclusively takes place at the polymer here we considered also the interaction of the protein molecules with the underlying gold transducers. We demonstrate by SWV that adsorption of proteins suppresses the signal of the redox marker even at the bare gold surface and by SEIRAS that the treatment of the MIP with proteinase K or NaOH only partially removes the target protein. Therefore, we conclude that when interpreting binding of proteins to directly MIP-covered gold electrodes the interactions between the protein and the gold surface should also be considered. Copyright © 2018 Elsevier B.V. All rights reserved.

Metrics that differentiate the origins of osmolyte effects on protein stability: a test of the surface tension proposal.

PubMed

Auton, Matthew; Ferreon, Allan Chris M; Bolen, D Wayne

2006-09-01

Osmolytes that are naturally selected to protect organisms against environmental stresses are known to confer stability to proteins via preferential exclusion from protein surfaces. Solvophobicity, surface tension, excluded volume, water structure changes and electrostatic repulsion are all examples of forces proposed to account for preferential exclusion and the ramifications exclusion has on protein properties. What has been lacking is a systematic way of determining which force(s) is(are) responsible for osmolyte effects. Here, we propose the use of two experimental metrics for assessing the abilities of various proposed forces to account for osmolyte-mediated effects on protein properties. Metric 1 requires prediction of the experimentally determined ability of the osmolyte to bring about folding/unfolding resulting from the application of the force in question (i.e. prediction of the m-value of the protein in osmolyte). Metric 2 requires prediction of the experimentally determined ability of the osmolyte to contract or expand the Stokes radius of the denatured state resulting from the application of the force. These metrics are applied to test separate claims that solvophobicity/solvophilicity and surface tension are driving forces for osmolyte-induced effects on protein stability. The results show clearly that solvophobic/solvophilic forces readily account for protein stability and denatured state dimensional effects, while surface tension alone fails to do so. The agreement between experimental and predicted m-values involves both positive and negative m-values for three different proteins, and as many as six different osmolytes, illustrating that the tests are robust and discriminating. The ability of the two metrics to distinguish which forces account for the effects of osmolytes on protein properties and which do not, provides a powerful means of investigating the origins of osmolyte-protein effects.

Liquid-Vapor Interfacial Properties of Aqueous Solutions of Guanidinium and Methyl Guanidinium Chloride: Influence of Molecular Orientation on Interface Fluctuations

PubMed Central

Ou, Shuching; Cui, Di; Patel, Sandeep

2014-01-01

The guanidinium cation (C(NH2)3+) is a highly stable cation in aqueous solution due to its efficient solvation by water molecules and resonance stabilization of the charge. Its salts increase the solubility of nonpolar molecules (”salting-in”) and decrease the ordering of water. It is one of the strongest denaturants used in biophysical studies of protein folding. We investigate the behavior of guanidinium and its derivative, methyl guanidinium (an amino acid analogue) at the air-water surface, using atomistic molecular dynamics (MD) simulations and calculation of potentials of mean force. Methyl guanidinium cation is less excluded from the air-water surface than guanidinium cation, but both cations show orientational dependence of surface affinity. Parallel orientations of the guanidinium ring (relative to the Gibbs dividing surface) show pronounced free energy minima in the interfacial region, while ring orientations perpendicular to the GDS exhibit no discernible surface stability. Calculations of surface fluctuations demonstrate that near the air-water surface, the parallel-oriented cations generate significantly greater interfacial fluctuations compared to other orientations, which induces more long-ranged perturbations and solvent density redistribution. Our results suggest a strong correlation with induced interfacial fluctuations and ion surface stability. These results have implications for interpreting molecular-level, mechanistic action of this osmolyte’s interaction with hydrophobic interfaces as they impact protein denaturation (solubilization). PMID:23937431

Functionalization of alkyne-terminated thermally hydrocarbonized porous silicon nanoparticles with targeting peptides and antifouling polymers: effect on the human plasma protein adsorption.

PubMed

Wang, Chang-Fang; Mäkilä, Ermei M; Bonduelle, Colin; Rytkönen, Jussi; Raula, Janne; Almeida, Sérgio; Närvänen, Ale; Salonen, Jarno J; Lecommandoux, Sebastien; Hirvonen, Jouni T; Santos, Hélder A

2015-01-28

Porous silicon (PSi) nanomaterials combine a high drug loading capacity and tunable surface chemistry with various surface modifications to meet the requirements for biomedical applications. In this work, alkyne-terminated thermally hydrocarbonized porous silicon (THCPSi) nanoparticles were fabricated and postmodified using five bioactive molecules (targeting peptides and antifouling polymers) via a single-step click chemistry to modulate the bioactivity of the THCPSi nanoparticles, such as enhancing the cellular uptake and reducing the plasma protein association. The size of the nanoparticles after modification was increased from 176 to 180-220 nm. Dextran 40 kDa modified THCPSi nanoparticles showed the highest stability in aqueous buffer. Both peptide- and polymer-functionalized THCPSi nanoparticles showed an extensive cellular uptake which was dependent on the functionalized moieties presented on the surface of the nanoparticles. The plasma protein adsorption study showed that the surface modification with different peptides or polymers induced different protein association profiles. Dextran 40 kDa functionalized THCPSi nanoparticles presented the least protein association. Overall, these results demonstrate that the "click" conjugation of the biomolecules onto the alkyne-terminated THCPSi nanoparticles is a versatile and simple approach to modulate the surface chemistry, which has high potential for biomedical applications.

Insulin-Like Growth Factor Binding Proteins Increase Intracellular Calcium Levels in Two Different Cell Lines

PubMed Central

Seurin, Danielle; Lombet, Alain; Babajko, Sylvie; Godeau, François; Ricort, Jean-Marc

2013-01-01

Background Insulin-like growth factor binding proteins (IGFBPs) are six related secreted proteins that share IGF-dependent and -independent functions. If the former functions begin to be well described, the latter are somewhat more difficult to investigate and to characterize. At the cellular level, IGFBPs were shown to modulate numerous processes including cell growth, differentiation and apoptosis. However, the molecular mechanisms implicated remain largely unknown. We previously demonstrated that IGFBP-3, but not IGFBP-1 or IGFBP-5, increase intracellular calcium concentration in MCF-7 cells (Ricort J-M et al. (2002) FEBS lett 527: 293–297). Methodology/Principal Findings We perform a global analysis in which we studied, by two different approaches, the binding of each IGFBP isoform (i.e., IGFBP-1 to -6) to the surface of two different cellular models, MCF-7 breast adenocarcinoma cells and C2 myoblast proliferative cells, as well as the IGFBP-induced increase of intracellular calcium concentration. Using both confocal fluorescence microscopy and flow cytometry analysis, we showed that all IGFBPs bind to MCF-7 cell surface. By contrast, only four IGFBPs can bind to C2 cell surface since neither IGFBP-2 nor IGFBP-4 were detected. Among the six IGFBPs tested, only IGFBP-1 did not increased intracellular calcium concentration whatever the cellular model studied. By contrast, IGFBP-2, -3, -4 and -6, in MCF-7 cells, and IGFBP-3, -5 and -6, in C2 proliferative cells, induce a rapid and transient increase in intracellular free calcium concentration. Moreover, IGFBP-2 and -3 (in MCF-7 cells) and IGFBP-5 (in C2 cells) increase intracellular free calcium concentration by a pertussis toxin sensitive signaling pathway. Conclusions Our results demonstrate that IGFBPs are able to bind to cell surface and increase intracellular calcium concentration. By characterizing the IGFBPs-induced cell responses and intracellular couplings, we highlight the cellular specificity and complexity of the IGF-independent actions of these IGF binding proteins. PMID:23527161

Curcumin attenuates cardiomyocyte hypertrophy induced by high glucose and insulin via the PPARγ/Akt/NO signaling pathway.

PubMed

Chen, Rongchun; Peng, Xiaofeng; Du, Weimin; Wu, Yang; Huang, Bo; Xue, Lai; Wu, Qin; Qiu, Hongmei; Jiang, Qingsong

2015-05-01

To investigate the potential effect of curcumin on cardiomyocyte hypertrophy and a possible mechanism involving the PPARγ/Akt/NO signaling pathway in diabetes. The cardiomyocyte hypertrophy induced by high glucose (25.5mmol/L) and insulin (0.1μmol/L) (HGI) and the antihypertrophic effect of curcumin were evaluated in primary culture by measuring the cell surface area, protein content and atrial natriuretic factor (ANF) mRNA expression. The mRNA and protein expressions were assayed by reverse transcription PCR and Western blotting, whereas the NO concentration and endothelial NO synthase (eNOS) activity were determined using nitrate reduction and ELISA methods, respectively. The cardiomyocyte hypertrophy induced by HGI was characterized by increasing ANF mRNA expression, total protein content, and cell surface area, with downregulated mRNA and protein expressions of both PPARγ and Akt, which paralleled the declining eNOS mRNA expression, eNOS content, and NO concentration. The effects of HGI were inhibited by curcumin (1, 3, 10μmol/L) in a concentration-dependent manner. GW9662 (10μmol/L), a selective PPARγ antagonist, could abolish the effects of curcumin. LY294002 (20μmol/L), an Akt blocker, and N(G)-nitro-l-arginine-methyl ester (100μmol/L), a NOS inhibitor, could also diminish the effects of curcumin. The results suggested that curcumin supplementation can improve HGI-induced cardiomyocytes hypertrophy in vitro through the activation of PPARγ/Akt/NO signaling pathway. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

Neutron reflectivity measurement of protein A-antibody complex at the solid-liquid interface.

PubMed

Mazzer, Alice R; Clifton, Luke A; Perevozchikova, Tatiana; Butler, Paul D; Roberts, Christopher J; Bracewell, Daniel G

2017-05-26

Chromatography is a ubiquitous unit operation in the purification of biopharmaceuticals yet few studies have addressed the biophysical characterisation of proteins at the solution-resin interface. Chromatography and other adsorption and desorption processes have been shown to induce protein aggregation which is undesirable in biopharmaceutical products. In order to advance understanding of how adsorption processes might impact protein stability, neutron reflectivity was used to characterise the structure of adsorbed immunoglobulin G (IgG) on model surfaces. In the first model system, IgG was adsorbed directly to silica and demonstrated a side-on orientation with high surface contact. A maximum dimension of 60Å in the surface normal direction and high density surface coverage were observed under pH 4.1 conditions. In chromatography buffers, pH was found to influence IgG packing density and orientation at the solid-liquid interface. In the second model system, which was designed to mimic an affinity chromatography surface, protein A was attached to a silica surface to produce a configuration representative of a porous glass chromatography resin. Interfacial structure was probed during sequential stages from ligand attachment, through to IgG binding and elution. Adsorbed IgG structures extended up to 250Å away from the surface and showed dependence on surface blocking strategies. The data was suggestive of two IgG molecules bound to protein A with a somewhat skewed orientation and close proximity to the silica surface. The findings provide insight into the orientation of adsorbed antibody structures under conditions encountered during chromatographic separations. Copyright © 2017 The Author(s). Published by Elsevier B.V. All rights reserved.

Induction of neutralising antibodies by virus-like particles harbouring surface proteins from highly pathogenic H5N1 and H7N1 influenza viruses

PubMed Central

Szécsi, Judit; Boson, Bertrand; Johnsson, Per; Dupeyrot-Lacas, Pia; Matrosovich, Mikhail; Klenk, Hans-Dieter; Klatzmann, David; Volchkov, Viktor; Cosset, François-Loïc

2006-01-01

There is an urgent need to develop novel approaches to vaccination against the emerging, highly pathogenic avian influenza viruses. Here, we engineered influenza viral-like particles (Flu-VLPs) derived from retroviral core particles that mimic the properties of the viral surface of two highly pathogenic influenza viruses of either H7N1 or H5N1 antigenic subtype. We demonstrate that, upon recovery of viral RNAs from a field strain, one can easily generate expression vectors that encode the HA, NA and M2 surface proteins of either virus and prepare high-titre Flu-VLPs. We characterise these Flu-VLPs incorporating the HA, NA and M2 proteins and we show that they induce high-titre neutralising antibodies in mice. PMID:16948862

Monoclonal Antibody and an Antibody-Toxin Conjugate to a Cell Surface Proteoglycan of Melanoma Cells Suppress in vivo Tumor Growth

NASA Astrophysics Data System (ADS)

Bumol, T. F.; Wang, Q. C.; Reisfeld, R. A.; Kaplan, N. O.

1983-01-01

A monoclonal antibody directed against a cell surface chondroitin sulfate proteoglycan of human melanoma cells, 9.2.27, and its diphtheria toxin A chain (DTA) conjugate were investigated for their effects on in vitro protein synthesis and in vivo tumor growth of human melanoma cells. The 9.2.27 IgG and its DTA conjugate display similar serological activities against melanoma target cells but only the conjugate can induce consistent in vitro inhibition of protein synthesis and toxicity in M21 melanoma cells. However, both 9.2.27 IgG and its DTA conjugate effect significant suppression of M21 tumor growth in vivo in an immunotherapy model of a rapidly growing tumor in athymic nu/nu mice, suggesting that other host mechanisms may mediate monoclonal antibody-induced tumor suppression.

Pigment Epithelial-derived Factor (PEDF)-triggered Lung Cancer Cell Apoptosis Relies on p53 Protein-driven Fas Ligand (Fas-L) Up-regulation and Fas Protein Cell Surface Translocation*

PubMed Central

Li, Lei; Yao, Ya-Chao; Fang, Shu-Huan; Ma, Cai-Qi; Cen, Yi; Xu, Zu-Min; Dai, Zhi-Yu; Li, Cen; Li, Shuai; Zhang, Ting; Hong, Hong-Hai; Qi, Wei-Wei; Zhou, Ti; Li, Chao-Yang; Yang, Xia; Gao, Guo-Quan

2014-01-01

Pigment epithelium-derived factor (PEDF), a potent antiangiogenesis agent, has recently attracted attention for targeting tumor cells in several types of tumors. However, less is known about the apoptosis-inducing effect of PEDF on human lung cancer cells and the underlying molecular events. Here we report that PEDF has a growth-suppressive and proapoptotic effect on lung cancer xenografts. Accordingly, in vitro, PEDF apparently induced apoptosis in A549 and Calu-3 cells, predominantly via the Fas-L/Fas death signaling pathway. Interestingly, A549 and Calu-3 cells are insensitive to the Fas-L/Fas apoptosis pathway because of the low level of cell surface Fas. Our results revealed that, in addition to the enhancement of Fas-L expression, PEDF increased the sensitivity of A549 and Calu-3 cells to Fas-L-mediated apoptosis by triggering the translocation of Fas protein to the plasma membrane in a p53- and FAP-1-dependent manner. Similarly, the up-regulation of Fas-L by PEDF was also mediated by p53. Furthermore, peroxisome proliferator-activated receptor γ was determined to be the upstream regulator of p53. Together, these findings uncover a novel mechanism of tumor cell apoptosis induced by PEDF and provide a potential therapeutic strategy for tumors that are insensitive to Fas-L/Fas-dependent apoptosis because of a low level of cell surface Fas. PMID:25225287

Cell wall proteins of Sporothrix schenckii as immunoprotective agents.

PubMed

Alba-Fierro, Carlos A; Pérez-Torres, Armando; López-Romero, Everardo; Cuéllar-Cruz, Mayra; Ruiz-Baca, Estela

2014-01-01

Sporothrix schenckii is the etiological agent of sporotrichosis, an endemic subcutaneous mycosis in Latin America. Cell wall (CW) proteins located on the cell surface are inducers of cellular and humoral immune responses, potential candidates for diagnosis purposes and to generate vaccines to prevent fungal infections. This mini-review emphasizes the potential use of S. schenckii CW proteins as protective and therapeutic immune response inducers against sporotrichosis. A number of pathogenic fungi display CW components that have been characterized as inducers of protective cellular and humoral immune responses against the whole pathogen from which they were originally purified. The isolation and characterization of immunodominant protein components of the CW of S. schenckii have become relevant because of their potential in the development of protective and therapeutic immune responses against sporotrichosis. This manuscript is part of the series of works presented at the "V International Workshop: Molecular genetic approaches to the study of human pathogenic fungi" (Oaxaca, Mexico, 2012). Copyright © 2013 Revista Iberoamericana de Micología. Published by Elsevier Espana. All rights reserved.

The 170-kDa glucose-regulated stress protein is an endoplasmic reticulum protein that binds immunoglobulin.

PubMed Central

Lin, H Y; Masso-Welch, P; Di, Y P; Cai, J W; Shen, J W; Subjeck, J R

1993-01-01

Anoxia, glucose starvation, calcium ionophore A23187, EDTA, glucosamine, and several other conditions that adversely affect the function of the endoplasmic reticulum (ER) induce the synthesis of the glucose-regulated class of stress proteins (GRPs). The primary GRPs induced by these stresses migrate at 78 and 94 kDa (GRP78 and GRP94). In addition, another protein of approximately 150-170 kDa (GRP170) has been previously observed and is coordinately induced with GRP78 and GRP94. To characterize this novel stress protein, we have prepared an antisera against purified GRP170. Immunofluorescence, Endoglycosidase H sensitivity, and protease resistance of this protein in microsomes indicates that GRP170 is an ER lumenal glycoprotein retained in a pre-Golgi compartment. Immunoprecipitation of GRP170 with our antibody coprecipitates the GRP78 (also referred to as the B cell immunoglobulin-binding protein) and GRP94 members of this stress protein family in Chinese hamster ovary cells under stress conditions. ATP depletion, by immunoprecipitation in the presence of apyrase, does not affect the interaction between GRP78 and GRP170 but results in the coprecipitation of an unidentified 60-kDa protein. In addition, GRP170 is found to be coprecipitated with immunoglobulin (Ig) in four different B cell hybridomas expressing surface IgM, cytoplasmic Ig light chain only, cytoplasmic Ig heavy chain only, or an antigen specific secreted IgG. In addition, in IgM surface expressing WEHI-231 B cells, anti-IgM coprecipitates GRP78, GRP94, as well as GRP170; antibodies against GRP170 and GRP94 reciprocally coprecipitate GRP94/GRP170 as well as GRP78. Results suggest that this 170-kDa GRP is a retained ER lumenal glycoprotein that is constitutively present and that may play a role in immunoglobulin folding and assembly in conjunction or consecutively with GRP78 and GRP94. Images PMID:8305733

Dynamic interactions between a membrane binding protein and lipids induce fluctuating diffusivity

PubMed Central

Yamamoto, Eiji; Akimoto, Takuma; Kalli, Antreas C.; Yasuoka, Kenji; Sansom, Mark S. P.

2017-01-01

Pleckstrin homology (PH) domains are membrane-binding lipid recognition proteins that interact with phosphatidylinositol phosphate (PIP) molecules in eukaryotic cell membranes. Diffusion of PH domains plays a critical role in biological reactions on membrane surfaces. Although diffusivity can be estimated by long-time measurements, it lacks information on the short-time diffusive nature. We reveal two diffusive properties of a PH domain bound to the surface of a PIP-containing membrane using molecular dynamics simulations. One is fractional Brownian motion, attributed to the motion of the lipids with which the PH domain interacts. The other is temporally fluctuating diffusivity; that is, the short-time diffusivity of the bound protein changes substantially with time. Moreover, the diffusivity for short-time measurements is intrinsically different from that for long-time measurements. This fluctuating diffusivity results from dynamic changes in interactions between the PH domain and PIP molecules. Our results provide evidence that the complexity of protein-lipid interactions plays a crucial role in the diffusion of proteins on biological membrane surfaces. Changes in the diffusivity of PH domains and related membrane-bound proteins may in turn contribute to the formation/dissolution of protein complexes in membranes. PMID:28116358

Dysfunction of annexin A2 contributes to hyperglycaemia-induced loss of human endothelial cell surface fibrinolytic activity.

PubMed

Dai, Haibin; Yu, Zhanyang; Fan, Xiang; Liu, Ning; Yan, Min; Chen, Zhong; Lo, Eng H; Hajjar, Katherine A; Wang, Xiaoying

2013-06-01

Hyperglycaemia impairs fibrinolytic activity on the surface of endothelial cells, but the underlying mechanisms are not fully understood. In this study, we tested the hypothesis that hyperglycaemia causes dysfunction of the endothelial membrane protein annexin A2, thereby leading to an overall reduction of fibrinolytic activity. Hyperglycaemia for 7 days significantly reduced cell surface fibrinolytic activity in human brain microvascular endothelial cells (HBMEC). Hyperglycaemia also decreased tissue type plasminogen activator (t-PA), plasminogen, and annexin A2 mRNA and protein expression, while increasing plasminogen activator inhibitor-1 (PAI-1). No changes in p11 mRNA or protein expression were detected. Hyperglycaemia significantly increased AGE-modified forms of total cellular and membrane annexin A2. The hyperglycemia-associated reduction in fibrinolytic activity was fully restored upon incubation with recombinant annexin A2 (rA2), but not AGE-modified annexin A2 or exogenous t-PA. Hyperglycaemia decreased t-PA, upregulated PAI-1 and induced AGE-related disruption of annexin A2 function, all of which contributed to the overall reduction in endothelial cell surface fibrinolytic activity. Further investigations to elucidate the underlying molecular mechanisms and pathophysiological implications of A2 derivatisation might ultimately lead to a better understanding of mechanisms of impaired vascular fibrinolysis, and to development of new interventional strategies for the thrombotic vascular complications in diabetes.

Dysfunction of annexin A2 contributes to hyperglycaemia-induced loss of human endothelial cell surface fibrinolytic activity

PubMed Central

Dai, Haibin; Yu, Zhanyang; Fan, Xiang; Liu, Ning; Yan, Min; Chen, Zhong; Lo, Eng H.; Hajjar, Katherine A.; Wang, Xiaoying

2014-01-01

Summary Hyperglycaemia impairs fibrinolytic activity on the surface of endothelial cells, but the underlying mechanisms are not fully understood. In this study, we tested the hypothesis that hyperglycaemia causes dysfunction of the endothelial membrane protein annexin A2, thereby leading to an overall reduction of fibrinolytic activity. Hyperglycaemia for 7 days significantly reduced cell surface fibrinolytic activity in human brain microvascular endothelial cells (HBMEC). Hyperglycaemia also decreased tissue type plasminogen activator (t-PA), plasminogen, and annexin A2 mRNA and protein expression, while increasing plasminogen activator inhibitor-1 (PAI-1). No changes in p11 mRNA or protein expression were detected. Hyperglycaemia significantly increased AGE-modified forms of total cellular and membrane annexin A2. The hyperglycemia-associated reduction in fibrinolytic activity was fully restored upon incubation with recombinant annexin A2 (rA2), but not AGE-modified annexin A2 or exogenous t-PA. Hyperglycaemia decreased t-PA, upregulated PAI-1 and induced AGE-related disruption of annexin A2 function, all of which contributed to the overall reduction in endothelial cell surface fibrinolytic activity. Further investigations to elucidate the underlying molecular mechanisms and pathophysiological implications of A2 derivatisation might ultimately lead to a better understanding of mechanisms of impaired vascular fibrinolysis, and to development of new interventional strategies for the thrombotic vascular complications in diabetes. PMID:23572070

Enhanced osteogenic differentiation of MC3T3-E1 cells on grid-topographic surface and evidence for involvement of YAP mediator.

PubMed

Zhang, Yingying; Gong, He; Sun, Yan; Huang, Yan; Fan, Yubo

2016-05-01

Numerous studies have shown that surface topography can promote cell-substrate associations and deeply influence cell fate. The intracellular mechanism or how micro- or nano-patterned extracellular signal is ultimately linked to activity of nuclear transcription factors remains unknown. It has been reported that Yes-associated protein (YAP) can respond to extracellular matrix microenvironment signals, thus regulates stem cell differentiation process. We propose that YAP may play a role in mediating the topography induced cell differentiation. To this end, we fabricated polydimethylsiloxane (PDMS) micropatterns with grid topology (GT) (3 μm pattern width, 2 μm pattern interval length, 7 μm pattern height); nonpatterned PDMS substrates were used as the planar controls. The MC3T3-E1 cells were then cultured on these surfaces, respectively, in osteogenic inducing medium. Cell differentiation in terms of osteogenesis related gene expression, protein levels, alkaline phosphatase activity and extracellular matrix mineralization was assessed. It was shown that the cells on GT surfaces had stronger osteogenesis capacity. In addition, expression level of YAP was increased when MC3T3-E1 cells grew on GT substrates, which was similar to the levels of osteogenic differentiation markers. It was also shown that YAP knockdown attenuated GT substrates-induced MC3T3-E1 differentiation, which reduced the osteogenic differentiation effect of the GT substrates. Collectively, our findings indicate that GT substrates-induced MC3T3-E1 differentiation may be associated with YAP. This paper provides new target points for transcriptional mechanism research of microenvironment induced cell differentiation and a useful approach to obtain more biofunctionalization scaffolds for tissue engineering. © 2016 Wiley Periodicals, Inc.

Prediction of allosteric sites on protein surfaces with an elastic-network-model-based thermodynamic method.

PubMed

Su, Ji Guo; Qi, Li Sheng; Li, Chun Hua; Zhu, Yan Ying; Du, Hui Jing; Hou, Yan Xue; Hao, Rui; Wang, Ji Hua

2014-08-01

Allostery is a rapid and efficient way in many biological processes to regulate protein functions, where binding of an effector at the allosteric site alters the activity and function at a distant active site. Allosteric regulation of protein biological functions provides a promising strategy for novel drug design. However, how to effectively identify the allosteric sites remains one of the major challenges for allosteric drug design. In the present work, a thermodynamic method based on the elastic network model was proposed to predict the allosteric sites on the protein surface. In our method, the thermodynamic coupling between the allosteric and active sites was considered, and then the allosteric sites were identified as those where the binding of an effector molecule induces a large change in the binding free energy of the protein with its ligand. Using the proposed method, two proteins, i.e., the 70 kD heat shock protein (Hsp70) and GluA2 alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptor, were studied and the allosteric sites on the protein surface were successfully identified. The predicted results are consistent with the available experimental data, which indicates that our method is a simple yet effective approach for the identification of allosteric sites on proteins.

Surface charge effects in protein adsorption on nanodiamonds.

PubMed

Aramesh, M; Shimoni, O; Ostrikov, K; Prawer, S; Cervenka, J

2015-03-19

Understanding the interaction of proteins with charged diamond nanoparticles is of fundamental importance for diverse biomedical applications. Here we present a thorough study of protein binding, adsorption kinetics and structure on strongly positively (hydrogen-terminated) and negatively (oxygen-terminated) charged nanodiamond particles using a quartz crystal microbalance by dissipation and infrared spectroscopy. By using two model proteins (bovine serum albumin and lysozyme) of different properties (charge, molecular weight and rigidity), the main driving mechanism responsible for the protein binding to the charged nanoparticles was identified. Electrostatic interactions were found to dominate the protein adsorption dynamics, attachment and conformation. We developed a simple electrostatic model that can qualitatively explain the observed adsorption behaviour based on charge-induced pH modifications near the charged nanoparticle surfaces. Under neutral conditions, the local pH around the positively and negatively charged nanodiamonds becomes very high (11-12) and low (1-3) respectively, which has a profound impact on the protein charge, hydration and affinity to the nanodiamonds. Small proteins (lysozyme) were found to form multilayers with significant conformational changes to screen the surface charge, while larger proteins (albumin) formed monolayers with minor conformational changes. The findings of this study provide a step forward toward understanding and eventually predicting nanoparticle interactions with biofluids.

Prediction of allosteric sites on protein surfaces with an elastic-network-model-based thermodynamic method

NASA Astrophysics Data System (ADS)

Su, Ji Guo; Qi, Li Sheng; Li, Chun Hua; Zhu, Yan Ying; Du, Hui Jing; Hou, Yan Xue; Hao, Rui; Wang, Ji Hua

2014-08-01

Allostery is a rapid and efficient way in many biological processes to regulate protein functions, where binding of an effector at the allosteric site alters the activity and function at a distant active site. Allosteric regulation of protein biological functions provides a promising strategy for novel drug design. However, how to effectively identify the allosteric sites remains one of the major challenges for allosteric drug design. In the present work, a thermodynamic method based on the elastic network model was proposed to predict the allosteric sites on the protein surface. In our method, the thermodynamic coupling between the allosteric and active sites was considered, and then the allosteric sites were identified as those where the binding of an effector molecule induces a large change in the binding free energy of the protein with its ligand. Using the proposed method, two proteins, i.e., the 70 kD heat shock protein (Hsp70) and GluA2 alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptor, were studied and the allosteric sites on the protein surface were successfully identified. The predicted results are consistent with the available experimental data, which indicates that our method is a simple yet effective approach for the identification of allosteric sites on proteins.

Ectodomain shedding of TNF receptor 1 induced by protein synthesis inhibitors regulates TNF-{alpha}-mediated activation of NF-{kappa}B and caspase-8

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ogura, Hirotsugu; Tsukumo, Yoshinori; Department of Bioengineering, Tokyo Institute of Technology, 4259 Nagatsuta-cho, Midori-ku, Yokohama 226-8501

2008-04-01

The transcription factor nuclear factor {kappa}B (NF-{kappa}B) plays a major role in the inducible resistance to death receptor-mediated apoptosis. It has been established that the protein synthesis inhibitor cycloheximide (CHX) sensitizes many types of cells to tumor necrosis factor (TNF)-{alpha}-induced apoptosis, mainly due to its ability to block de novo synthesis of cellular FLICE-inhibitory protein (c-FLIP). Nevertheless, we have surprisingly found that CHX, as well as its structural analogue acetoxycycloheximide (Ac-CHX), prevents TNF-{alpha}-mediated activation of NF-{kappa}B and caspase-8 in human lung carcinoma A549 cells. Both CHX and Ac-CHX reduced the expression of cell surface TNF receptor 1 (TNF-R1) in amore » dose-dependent manner, while Ac-CHX was approximately 100-fold more effective than CHX. Consistent with this observation, Ac-CHX induced the proteolytic cleavage of TNF-R1 and its release into the culture medium. CHX and Ac-CHX profoundly decreased constitutive and inducible expression of c-FLIP, whereas these compounds potentiated TNF-{alpha}-induced caspase-8 activation only when metalloprotease inhibitors were present. Thus, our results indicate that ectodomain shedding of TNF-R1 induced by protein synthesis inhibitors regulates TNF-{alpha}-mediated activation of NF-{kappa}B and caspase-8.« less

The role of HBV-induced autophagy in HBV replication and HBV related-HCC.

PubMed

Xie, Mingjie; Yang, Zhenggang; Liu, Yanning; Zheng, Min

2018-04-27

Hepatitis B virus (HBV) is infecting about 364 million people around the world. It can cause various diseases, such as chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma (HCC). However, the present anti-viral treatment in clinics is limited; studies for new therapies are highly desired. Autophagy is a crucial and major catabolic process in the maintenance of normal intracellular homeostasis in host cells. Host cells use this unique process to degrade and recycle long-lived proteins, damaged organelles, and various pathogens for keeping the normal physiological functions. Recently, published studies indicated that HBV can induce autophagy in host cells; this autophagic response is involved in viral replication and pathogenesis. Several viral proteins, such as surface and X proteins, are assumed to be responsible for inducing autophagy in HBV infection. This review briefly summarizes some important mechanisms involved in HBV-induced autophagy and provides a novel perspective on therapies of HBV infection and HBV-related HCC. Copyright © 2017. Published by Elsevier Inc.

HLA-DR allele reading register shifting is associated with immunity induced by SERA peptide analogues.

PubMed

Salazar, Luz Mary; Bermúdez, Adriana; Patarroyo, Manuel E

2008-07-18

SERA protein is a leading candidate molecule to be included in an antimalarial vaccine. Conserved high activity binding peptides (HABP) binding to red blood cells (RBC) have been identified in this protein. One of them (6762) localising in the 18-kDa C-terminal fragment was used to induce protective immunity with negative result. Critical RBC binding residues (assessed by glycine-analogue scanning) were replaced by others having the same mass, volume and surface but different polarity, rendering some of them immunogenic as assessed by antibody production against the parasite or its proteins and protection-inducing against challenge with a highly infectious Aotus monkey-adapted Plasmodium falciparum strain. A shift in binding to purified HLA-DR allelic molecules from the same haplotype and in their reading register was found, suggesting that modified molecules had adopted a different (1)H NMR 3D structure allowing a better fit into the MHCII-pept-TCR complex, thereby representing a new mechanism for inducing immune protection.

A Comparison of Blood Factor XII Autoactivation in Buffer, Protein Cocktail, Serum, and Plasma Solutions

PubMed Central

Golas, Avantika; Yeh, Chyi-Huey Josh; Pitakjakpipop, Harit; Siedlecki, Christopher A.; Vogler, Erwin A.

2012-01-01

Activation of blood plasma coagulation in vitro by contact with material surfaces is demonstrably dependent on plasma-volume-to-activator-surface-area ratio. The only plausible explanation consistent with current understanding of coagulation-cascade biochemistry is that procoagulant stimulus arising from the activation complex of the intrinsic pathway is dependent on activator surface area. And yet, it is herein shown that activation of the blood zymogen factor XII (Hageman factor, FXII) dissolved in buffer, protein cocktail, heat-denatured serum, and FXI deficient plasma does not exhibit activator surface-area dependence. Instead, a highly-variable burst of procoagulant-enzyme yield is measured that exhibits no measurable kinetics, sensitivity to mixing, or solution-temperature dependence. Thus, FXII activation in both buffer and protein-containing solutions does not exhibit characteristics of a biochemical reaction but rather appears to be a “mechanochemical” reaction induced by FXII molecule interactions with hydrophilic activator particles that do not formally adsorb blood proteins from solution. Results of this study strongly suggest that activator surface-area dependence observed in contact activation of plasma coagulation does not solely arise at the FXII activation step of the intrinsic pathway. PMID:23117212

Self-similar assemblies of globular whey proteins at the air-water interface: effect of the structure.

PubMed

Mahmoudi, Najet; Gaillard, Cédric; Boué, François; Axelos, Monique A V; Riaublanc, Alain

2010-05-01

We investigated the structure of heat-induced assemblies of whey globular proteins using small angle neutron scattering (SANS), static and dynamic light scattering (SLS and DLS), and cryogenic transmission electron microscopy (Cryo-TEM). Whey protein molecules self-assemble in fractal aggregates with a structure density depending on the electrostatic interactions. We determined the static and dynamic properties of interfacial layer formed by the protein assemblies, upon adsorption and spreading at the air-water interface using surface film balance and interfacial dilatational rheology. Upon spreading, all whey protein systems show a power-law scaling behavior of the surface pressure versus concentration in the semi-dilute surface concentration regime, with an exponent ranging from 5.5 to 9 depending on the electrostatic interactions and the aggregation state. The dilatational modulus derived from surface pressure isotherms shows a main peak at 6-8 mN/m, generally considered to be the onset of a conformational change in the monolayer, and a second peak or a shoulder at 15 mN/m. Long-time adsorption kinetics give similar results for both the native whey proteins and the corresponding self-similar assemblies, with a systematic effect of the ionic strength. Copyright 2010 Elsevier Inc. All rights reserved.

B61 is a ligand for the ECK receptor protein-tyrosine kinase.

PubMed

Bartley, T D; Hunt, R W; Welcher, A A; Boyle, W J; Parker, V P; Lindberg, R A; Lu, H S; Colombero, A M; Elliott, R L; Guthrie, B A

1994-04-07

A protein ligand for the ECK receptor protein-tyrosine kinase has been isolated by using the extracellular domain (ECK-X) of the receptor as an affinity reagent. Initially, concentrated cell culture supernatants were screened for receptor binding activity using immobilized ECK-X in a surface plasmon resonance detection system. Subsequently, supernatants from selected cell lines were fractionated directly by receptor affinity chromatography, resulting in the single-step purification of B61, a protein previously identified as the product of an early response gene induced by tumour necrosis factor-alpha. We report here that recombinant B61 induces autophosphorylation of ECK in intact cells, consistent with B61 being an authentic ligand for ECK. ECK is a member of a large orphan receptor protein-tyrosine kinase family headed by EPH, and we suggest that ligands for other members of this family will be related to B61, and can be isolated in the same way.

Hemocompatibility of poly(vinylidene fluoride) membrane grafted with network-like and brush-like antifouling layer controlled via plasma-induced surface PEGylation.

PubMed

Chang, Yung; Shih, Yu-Ju; Ko, Chao-Yin; Jhong, Jheng-Fong; Liu, Ying-Ling; Wei, Ta-Chin

2011-05-03

In this work, the hemocompatibility of PEGylated poly(vinylidene fluoride) (PVDF) microporous membranes with varying grafting coverage and structures via plasma-induced surface PEGylation was studied. Network-like and brush-like PEGylated layers on PVDF membrane surfaces were achieved by low-pressure and atmospheric plasma treatment. The chemical composition, physical morphology, grafting structure, surface hydrophilicity, and hydration capability of prepared membranes were determined to illustrate the correlations between grafting qualities and hemocompatibility of PEGylated PVDF membranes in contact with human blood. Plasma protein adsorption onto different PEGylated PVDF membranes from single-protein solutions and the complex medium of 100% human plasma were measured by enzyme-linked immunosorbent assay (ELISA) with monoclonal antibodies. Hemocompatibility of the PEGylated membranes was evaluated by the antifouling property of platelet adhesion observed by scanning electron microscopy (SEM) and the anticoagulant activity of the blood coagulant determined by testing plasma-clotting time. The control of grafting structures of PEGylated layers highly regulates the PVDF membrane to resist the adsorption of plasma proteins, the adhesion of platelets, and the coagulation of human plasma. It was found that PVDF membranes grafted with brush-like PEGylated layers presented higher hydration capability with binding water molecules than with network-like PEGylated layers to improve the hemocompatible character of plasma protein and blood platelet resistance in human blood. This work suggests that the hemocompatible nature of grafted PEGylated polymers by controlling grafting structures gives them great potential in the molecular design of antithrombogenic membranes for use in human blood.

Simultaneous surface display and cargo loading of encapsulin nanocompartments and their use for rational vaccine design.

PubMed

Lagoutte, Priscillia; Mignon, Charlotte; Stadthagen, Gustavo; Potisopon, Supanee; Donnat, Stéphanie; Mast, Jan; Lugari, Adrien; Werle, Bettina

2018-05-11

In the past decades protein nanoparticles have successfully been used for vaccine applications. Their particulate nature and dense repetitive subunit organization makes them perfect carriers for antigen surface display and confers high immunogenicity. Nanoparticles have emerged as excellent candidates for vectorization of biological and immunostimulating molecules. Nanoparticles and biomolecular nanostructures such as ferritins or virus like particles have been used as diagnostic and therapeutic delivery systems, in vaccine development, as nanoreactors, etc. Recently, a new class of bacterial protein compartment has been discovered referred to as encapsulin nanocompartment. These compartments have been used for targeted diagnostics, as therapeutic delivery systems and as nanoreactors. Their biological origin makes them conveniently biocompatible and allows genetic functionalization. The aim of our study was to implement encapsulin nanocompartements for simultaneous epitope surface display and heterologous protein loading for rational vaccine design. For this proof-of-concept-study, we produced Thermotoga maritima encapsulin nanoparticles in E. coli. We demonstrated the ability of simultaneous display in our system by inserting Matrix protein 2 ectodomain (M2e) of influenza A virus at the nanoparticle surface and by packaging of a fluorescent reporter protein (GFP) into the internal cavity. Characterization of the nanoparticles by electronic microscopy confirmed homogenously shaped particles of 24 nm diameter in average. The results further show that engineering of the particle surface improved the loading capacity of the heterologous reporter protein suggesting that surface display may induce a critical elastic deformation resulting in improved stiffness. In Balb/c mice, nanoparticle immunization elicited antibody responses against both the surface epitope and the loaded cargo protein. These results confirm the potential of encapsulin nanocompartments for customized vaccine design and antigen delivery. Copyright © 2018 Elsevier Ltd. All rights reserved.

Antagonistic Regulation of Cystic Fibrosis Transmembrane Conductance Regulator Cell Surface Expression by Protein Kinases WNK4 and Spleen Tyrosine Kinase ▿

PubMed Central

Mendes, Ana Isabel; Matos, Paulo; Moniz, Sónia; Luz, Simão; Amaral, Margarida D.; Farinha, Carlos M.; Jordan, Peter

2011-01-01

Members of the WNK (with-no-lysine [K]) subfamily of protein kinases regulate various ion channels involved in sodium, potassium, and chloride homeostasis by either inducing their phosphorylation or regulating the number of channel proteins expressed at the cell surface. Here, we describe findings demonstrating that the cell surface expression of the cystic fibrosis transmembrane conductance regulator (CFTR) is also regulated by WNK4 in mammalian cells. This effect of WNK4 is independent of the presence of kinase and involves interaction with and inhibition of spleen tyrosine kinase (Syk), which phosphorylates Tyr512 in the first nucleotide-binding domain 1 (NBD1) of CFTR. Transfection of catalytically active Syk into CFTR-expressing baby hamster kidney cells reduces the cell surface expression of CFTR, whereas that of WNK4 promotes it. This is shown by biotinylation of cell surface proteins, immunofluorescence microscopy, and functional efflux assays. Mutation of Tyr512 to either glutamic acid or phenylalanine is sufficient to alter CFTR surface levels. In human airway epithelial cells, downregulation of endogenous Syk and WNK4 confirms their roles as physiologic regulators of CFTR surface expression. Together, our results show that Tyr512 phosphorylation is a novel signal regulating the prevalence of CFTR at the cell surface and that WNK4 and Syk perform an antagonistic role in this process. PMID:21807898

Quantitatively identifying the roles of interfacial water and solid surface in governing peptide adsorption.

PubMed

Xu, Zhijun; Yang, Xiao; Wei, Qichao; Zhao, Weilong; Cui, Beiliang; Yang, Xiaoning; Sahai, Nita

2018-06-11

Understanding the molecular mechanism of protein adsorption on solids is critical to their applications in materials synthesis and tissue engineering. Though the water phase at the surface/water interface has been recognized as three types: free water in the bulk region, intermediate water phase and surface-bound water layers adjacent to the surface, the roles of the water and surface in determining the protein adsorption are not clearly identified, particularly at the quantitative level. Herein, we provide a methodology involving the combination of microsecond strengthen sampling simulation and force integration to quantitatively characterize the water-induced contribution and the peptide-surface interactions into the adsorption free energy. Using hydroxyapatite and graphene surfaces as examples, we demonstrate how the distinct interfacial features dominate the delicate force balance between these two thermodynamics parameters, leading to surface preference/resistance to peptide adsorption. Specifically, the water layer provides sustained repelling force against peptide adsorption, as indicated by a monotonic increase in the water-induced free energy profile, whereas the contribution to the free energy from the surface effect is thermodynamically favorable, thus acting as the dominant driving force for peptide adsorptions. More importantly, the revealed adsorption mechanism is critically dictated by the distribution of water phase at the solid/water interface, which plays a crucial role in establishing the force balance between the interactions of the peptide with the water layer and the surface. For the HAP surface, the charged peptide exhibits strong binding affinity to the surface, which is ascribed to the controlling contribution of peptide-surface interaction in the intermediate water phase and the surface-bound water layers are observed as the origin of bioresistance of solid surfaces towards the adsorption of charge-neutral peptides. The preferred peptide adsorption on the graphene, however, is dominated by the surface-induced component at the water layers adjacent to the surface. Our results further elucidate that the intermediate water phase significantly shortens the effective range of the surface dispersion force to guide the diffusion of the peptide to the interface, in sharp contrast to the observation in interfacial systems involving the strong water-surface interaction.

A Study on the Effect of Surface Lysine to Arginine Mutagenesis on Protein Stability and Structure Using Green Fluorescent Protein

PubMed Central

Sokalingam, Sriram; Raghunathan, Govindan; Soundrarajan, Nagasundarapandian; Lee, Sun-Gu

2012-01-01

Two positively charged basic amino acids, arginine and lysine, are mostly exposed to protein surface, and play important roles in protein stability by forming electrostatic interactions. In particular, the guanidinium group of arginine allows interactions in three possible directions, which enables arginine to form a larger number of electrostatic interactions compared to lysine. The higher pKa of the basic residue in arginine may also generate more stable ionic interactions than lysine. This paper reports an investigation whether the advantageous properties of arginine over lysine can be utilized to enhance protein stability. A variant of green fluorescent protein (GFP) was created by mutating the maximum possible number of lysine residues on the surface to arginines while retaining the activity. When the stability of the variant was examined under a range of denaturing conditions, the variant was relatively more stable compared to control GFP in the presence of chemical denaturants such as urea, alkaline pH and ionic detergents, but the thermal stability of the protein was not changed. The modeled structure of the variant indicated putative new salt bridges and hydrogen bond interactions that help improve the rigidity of the protein against different chemical denaturants. Structural analyses of the electrostatic interactions also confirmed that the geometric properties of the guanidinium group in arginine had such effects. On the other hand, the altered electrostatic interactions induced by the mutagenesis of surface lysines to arginines adversely affected protein folding, which decreased the productivity of the functional form of the variant. These results suggest that the surface lysine mutagenesis to arginines can be considered one of the parameters in protein stability engineering. PMID:22792305

Searching for protein binding sites from Molecular Dynamics simulations and paramagnetic fragment-based NMR studies.

PubMed

Bernini, Andrea; Henrici De Angelis, Lucia; Morandi, Edoardo; Spiga, Ottavia; Santucci, Annalisa; Assfalg, Michael; Molinari, Henriette; Pillozzi, Serena; Arcangeli, Annarosa; Niccolai, Neri

2014-03-01

Hotspot delineation on protein surfaces represents a fundamental step for targeting protein-protein interfaces. Disruptors of protein-protein interactions can be designed provided that the sterical features of binding pockets, including the transient ones, can be defined. Molecular Dynamics, MD, simulations have been used as a reliable framework for identifying transient pocket openings on the protein surface. Accessible surface area and intramolecular H-bond involvement of protein backbone amides are proposed as descriptors for characterizing binding pocket occurrence and evolution along MD trajectories. TEMPOL induced paramagnetic perturbations on (1)H-(15)N HSQC signals of protein backbone amides have been analyzed as a fragment-based search for surface hotspots, in order to validate MD predicted pockets. This procedure has been applied to CXCL12, a small chemokine responsible for tumor progression and proliferation. From combined analysis of MD data and paramagnetic profiles, two CXCL12 sites suitable for the binding of small molecules were identified. One of these sites is the already well characterized CXCL12 region involved in the binding to CXCR4 receptor. The other one is a transient pocket predicted by Molecular Dynamics simulations, which could not be observed from static analysis of CXCL12 PDB structures. The present results indicate how TEMPOL, instrumental in identifying this transient pocket, can be a powerful tool to delineate minor conformations which can be highly relevant in dynamic discovery of antitumoral drugs. Copyright © 2013 Elsevier B.V. All rights reserved.

Influence of xanthan gum on the structural characteristics of myofibrillar proteins treated by high pressure.

PubMed

Villamonte, Gina; Jury, Vanessa; Jung, Stéphanie; de Lamballerie, Marie

2015-03-01

The effects of xanthan gum on the structural modifications of myofibrillar proteins (0.3 M NaCl, pH 6) induced by high pressure (200, 400, and 600 MPa, 6 min) were investigated. The changes in the secondary and tertiary structures of myofibrillar proteins were analyzed by circular dichroism. The protein denaturation was also evaluated by differential scanning calorimetry. Likewise, the protein surface hydrophobicity and the solubility of myofibrillar proteins were measured. High pressure (600 MPa) induced the loss of α-helix structures and an increase of β-sheet structures. However, the presence of xanthan gum hindered the former mechanism of protein denaturation by high pressure. In fact, changes in the secondary (600 MPa) and the tertiary structure fingerprint of high-pressure-treated myofibrillar proteins (400 to 600 MPa) were observed in the presence of xanthan gum. These modifications were confirmed by the thermal analysis, the thermal transitions of high-pressure (400 to 600 MPa)-treated myofibrillar proteins were modified in systems containing xanthan gum. As consequence, the high-pressure-treated myofibrillar proteins with xanthan gum showed increased solubility from 400 MPa, in contrast to high-pressure treatment (600 MPa) without xanthan gum. Moreover, the surface hydrophobicity of high-pressure-treated myofibrillar proteins was enhanced in the presence of xanthan gum. These effects could be due to the unfolding of myofibrillar proteins at high-pressure levels, which exposed sites that most likely interacted with the anionic polysaccharide. This study suggests that the role of food additives could be considered for the development of meat products produced by high-pressure processing. © 2015 Institute of Food Technologists®

DOE Office of Scientific and Technical Information (OSTI.GOV)

Seong, Yeon-Jae; Hafis Clinic, Seoul; Sung, Pil Soo

Cellular prion protein (PrP{sup C}) is widely expressed in various cell types, including cells of the immune system. However, the specific roles of PrP{sup C} in the immune system have not been clearly elucidated. In the present study, we investigated the effects of a soluble form of recombinant PrP{sup C} protein on human natural killer (NK) cells. Recombinant soluble PrP{sup C} protein was generated by fusion of human PrP{sup C} with the Fc portion of human IgG{sub 1} (PrP{sup C}-Fc). PrP{sup C}-Fc binds to the surface of human NK cells, particularly to CD56{sup dim} NK cells. PrP{sup C}-Fc induced themore » production of cytokines and chemokines and the degranulation of granzyme B from NK cells. In addition, PrP{sup C}-Fc facilitated the IL-15-induced proliferation of NK cells. PrP{sup C}-Fc induced phosphorylation of ERK-1/2 and JNK in NK cells, and inhibitors of the ERK or the JNK pathways abrogated PrP{sup C}-Fc-induced cytokine production in NK cells. In conclusion, the soluble form of recombinant PrP{sup C}-Fc protein activates human NK cells via the ERK and JNK signaling pathways. - Highlights: • Recombinant soluble PrP{sup C} (PrP{sup C}-Fc) was generated by fusion of human PrP{sup C} with IgG1 Fc portion. • PrP{sup C}-Fc protein induces the production of cytokines and degranulation from human NK cells. • PrP{sup C}-Fc protein enhances the IL-15-induced proliferation of human NK cells. • PrP{sup C}-Fc protein activates human NK cells via the ERK and JNK signaling pathways.« less

Drug-Induced Trafficking of P-Glycoprotein in Human Brain Capillary Endothelial Cells as Demonstrated by Exposure to Mitomycin C

PubMed Central

Noack, Andreas; Noack, Sandra; Hoffmann, Andrea; Maalouf, Katia; Buettner, Manuela; Couraud, Pierre-Olivier; Romero, Ignacio A.; Weksler, Babette; Alms, Dana; Römermann, Kerstin; Naim, Hassan Y.; Löscher, Wolfgang

2014-01-01

P-glycoprotein (Pgp; ABCB1/MDR1) is a major efflux transporter at the blood-brain barrier (BBB), restricting the penetration of various compounds. In other tissues, trafficking of Pgp from subcellular stores to the cell surface has been demonstrated and may constitute a rapid way of the cell to respond to toxic compounds by functional membrane insertion of the transporter. It is not known whether drug-induced Pgp trafficking also occurs in brain capillary endothelial cells that form the BBB. In this study, trafficking of Pgp was investigated in human brain capillary endothelial cells (hCMEC/D3) that were stably transfected with a doxycycline-inducible MDR1-EGFP fusion plasmid. In the presence of doxycycline, these cells exhibited a 15-fold increase in Pgp-EGFP fusion protein expression, which was associated with an increased efflux of the Pgp substrate rhodamine 123 (Rho123). The chemotherapeutic agent mitomycin C (MMC) was used to study drug-induced trafficking of Pgp. Confocal fluorescence microscopy of single hCMEC/D3-MDR1-EGFP cells revealed that Pgp redistribution from intracellular pools to the cell surface occurred within 2 h of MMC exposure. Pgp-EGFP exhibited a punctuate pattern at the cell surface compatible with concentrated regions of the fusion protein in membrane microdomains, i.e., lipid rafts, which was confirmed by Western blot analysis of biotinylated cell surface proteins in Lubrol-resistant membranes. MMC exposure also increased the functionality of Pgp as assessed in three functional assays with Pgp substrates (Rho123, eFluxx-ID Gold, calcein-AM). However, this increase occurred with some delay after the increased Pgp expression and coincided with the release of Pgp from the Lubrol-resistant membrane complexes. Disrupting rafts by depleting the membrane of cholesterol increased the functionality of Pgp. Our data present the first direct evidence of drug-induced Pgp trafficking at the human BBB and indicate that Pgp has to be released from lipid rafts to gain its full functionality. PMID:24505408

Drug-induced trafficking of p-glycoprotein in human brain capillary endothelial cells as demonstrated by exposure to mitomycin C.

PubMed

Noack, Andreas; Noack, Sandra; Hoffmann, Andrea; Maalouf, Katia; Buettner, Manuela; Couraud, Pierre-Olivier; Romero, Ignacio A; Weksler, Babette; Alms, Dana; Römermann, Kerstin; Naim, Hassan Y; Löscher, Wolfgang

2014-01-01

P-glycoprotein (Pgp; ABCB1/MDR1) is a major efflux transporter at the blood-brain barrier (BBB), restricting the penetration of various compounds. In other tissues, trafficking of Pgp from subcellular stores to the cell surface has been demonstrated and may constitute a rapid way of the cell to respond to toxic compounds by functional membrane insertion of the transporter. It is not known whether drug-induced Pgp trafficking also occurs in brain capillary endothelial cells that form the BBB. In this study, trafficking of Pgp was investigated in human brain capillary endothelial cells (hCMEC/D3) that were stably transfected with a doxycycline-inducible MDR1-EGFP fusion plasmid. In the presence of doxycycline, these cells exhibited a 15-fold increase in Pgp-EGFP fusion protein expression, which was associated with an increased efflux of the Pgp substrate rhodamine 123 (Rho123). The chemotherapeutic agent mitomycin C (MMC) was used to study drug-induced trafficking of Pgp. Confocal fluorescence microscopy of single hCMEC/D3-MDR1-EGFP cells revealed that Pgp redistribution from intracellular pools to the cell surface occurred within 2 h of MMC exposure. Pgp-EGFP exhibited a punctuate pattern at the cell surface compatible with concentrated regions of the fusion protein in membrane microdomains, i.e., lipid rafts, which was confirmed by Western blot analysis of biotinylated cell surface proteins in Lubrol-resistant membranes. MMC exposure also increased the functionality of Pgp as assessed in three functional assays with Pgp substrates (Rho123, eFluxx-ID Gold, calcein-AM). However, this increase occurred with some delay after the increased Pgp expression and coincided with the release of Pgp from the Lubrol-resistant membrane complexes. Disrupting rafts by depleting the membrane of cholesterol increased the functionality of Pgp. Our data present the first direct evidence of drug-induced Pgp trafficking at the human BBB and indicate that Pgp has to be released from lipid rafts to gain its full functionality.

Rational design of a conformation-switchable Ca2+- and Tb3+-binding protein without the use of multiple coupled metal-binding sites.

PubMed

Li, Shunyi; Yang, Wei; Maniccia, Anna W; Barrow, Doyle; Tjong, Harianto; Zhou, Huan-Xiang; Yang, Jenny J

2008-10-01

Ca2+, as a messenger of signal transduction, regulates numerous target molecules via Ca2+-induced conformational changes. Investigation into the determinants for Ca2+-induced conformational change is often impeded by cooperativity between multiple metal-binding sites or protein oligomerization in naturally occurring proteins. To dissect the relative contributions of key determinants for Ca2+-dependent conformational changes, we report the design of a single-site Ca2+-binding protein (CD2.trigger) created by altering charged residues at an electrostatically sensitive location on the surface of the host protein rat Cluster of Differentiation 2 (CD2).CD2.trigger binds to Tb3+ and Ca2+ with dissociation constants of 0.3 +/- 0.1 and 90 +/- 25 microM, respectively. This protein is largely unfolded in the absence of metal ions at physiological pH, but Tb3+ or Ca2+ binding results in folding of the native-like conformation. Neutralization of the charged coordination residues, either by mutation or protonation, similarly induces folding of the protein. The control of a major conformational change by a single Ca2+ ion, achieved on a protein designed without reliance on sequence similarity to known Ca2+-dependent proteins and coupled metal-binding sites, represents an important step in the design of trigger proteins.

PPE57 induces activation of macrophages and drives Th1-type immune responses through TLR2.

PubMed

Xu, Ying; Yang, Enzhuo; Huang, Qi; Ni, Wenwen; Kong, Cong; Liu, Guoyuan; Li, Guanghua; Su, Haibo; Wang, Honghai

2015-06-01

Proline-glutamic acid (PE) and proline-proline-glutamic acid (PPE) are related proteins exclusive to Mycobacteria that play diverse roles in modulating critical innate immune pathways. In this study, we observed that the PPE57 protein is associated with the cell wall and is exposed on the cell surface. PPE57 enhances Mycobacterium spp. entering into macrophages and plays a role in macrophage phagocytosis. To explore the underlying mechanism, we demonstrated that PPE57 is able to recognise Toll-like receptor 2 (TLR2) and further induce macrophage activation by augmenting the expression of several cell surface molecules (CD40, CD80, CD86 and MHC class II) and pro-inflammatory cytokines (TNF-α, IL-6 and IL-12p40) within macrophages. These molecules are involved in the mitogen-activated protein kinase (MAPK) and nuclear factor κB (NF-κB) signalling pathways. We demonstrated that PPE57 effectively polarises T cells to secrete interferon (IFN)-γ and IL-2 and to up-regulate CXCR3 expression in vivo and in vitro, suggesting that this protein may contribute to Th1 polarisation during the immune response. Moreover, recombinant Bacillus Calmette-Guérin (BCG) over-expressing PPE57 could provide better protective efficacy against Mycobacterium tuberculosis challenge compared with BCG. Taken together, our data provides several pieces of evidence that PPE57 may regulate innate and adaptive immunity by interacting with TLR2. These findings indicate that PPE57 protein is a potential antigen for the rational design of an efficient vaccine against M. tuberculosis. PPE57 is located on the cell surface and enhances mycobacterium entry into macrophage. PPE57 interacts directly with TLR2 on macrophages. PPE57 plays a key role in the activation of macrophages in a TLR2-dependent manner. PPE57 induces a Th1 immune response via TLR2-mediated macrophage functions. Recombinant BCG over-expressing PPE57 could improve protective efficacy against M. tuberculosis.

Chemical Functionalization of Germanium with Dextran Brushes for Immobilization of Proteins Revealed by Attenuated Total Reflection Fourier Transform Infrared Difference Spectroscopy.

PubMed

Schartner, Jonas; Hoeck, Nina; Güldenhaupt, Jörn; Mavarani, Laven; Nabers, Andreas; Gerwert, Klaus; Kötting, Carsten

2015-07-21

Protein immobilization studied by attenuated total reflection Fourier transform infrared (ATR-FT-IR) difference spectroscopy is an emerging field enabling the study of proteins at atomic detail. Gold or glass surfaces are frequently used for protein immobilization. Here, we present an alternative method for protein immobilization on germanium. Because of its high refractive index and broad spectral window germanium is the best material for ATR-FT-IR spectroscopy of thin layers. So far, this technique was mainly used for protein monolayers, which lead to a limited signal-to-noise ratio. Further, undesired protein-protein interactions can occur in a dense layer. Here, the germanium surface was functionalized with thiols and stepwise a dextran brush was generated. Each step was monitored by ATR-FT-IR spectroscopy. We compared a 70 kDa dextran with a 500 kDa dextran regarding the binding properties. All surfaces were characterized by atomic force microscopy, revealing thicknesses between 40 and 110 nm. To analyze the capability of our system we utilized N-Ras on mono-NTA (nitrilotriacetic acid) functionalized dextran, and the amount of immobilized Ras corresponded to several monolayers. The protein stability and loading capacity was further improved by means of tris-NTA for immobilization. Small-molecule-induced changes were revealed with an over 3 times higher signal-to-noise ratio compared to monolayers. This improvement may allow the observation of very small and so far hidden changes in proteins upon stimulus. Furthermore, we immobilized green fluorescent protein (GFP) and mCherry simultaneously enabling an analysis of the surface by fluorescence microscopy. The absence of a Förster resonance energy transfer (FRET) signal demonstrated a large protein-protein distance, indicating an even distribution of the protein within the dextran.

The Role of Protein-Mineral Interactions for Protein Adsorption or Fragmentation

NASA Astrophysics Data System (ADS)

Chacon, S. S.; Reardon, P.; Washton, N.; Kleber, M.

2014-12-01

Soil exo-enzymes (EE) are proteins with the capability to catalyze the depolymerization of soil organic matter (SOM). SOM must be disassembled by EEs in order to be transported through the microbial cell wall and become metabolized. One factor determining an EE's functionality is their affinity to mineral surfaces found in the soil. Our goal was to establish the range of protein modifications, either chemical or structural, as the protein becomes associated with mineral surfaces. We hypothesized that pedogenic oxides would generate more extensive chemical alterations to the protein structure than phyllosilicates. A well-characterized protein proxy (Gb1, IEP 4.0, 6.2 kDA) was adsorbed onto functionally different mineral surfaces (goethite, montmorillonite, kaolinite and birnesite) at pH 5 and pH 7. We used 1H 15N Heteronuclear Single Quantum Coherence Nuclear Magnetic Resonance Spectroscopy (HSQC NMR) to observe structural modifications in the unadsorbed Gb1 that was allowed to equilibrate during the adsorption process for kaolinite, goethite and birnessite. Solid state NMR was used to observe the structural modifications of Gb1 while adsorbed onto kaolinite and montmorillonite. Preliminary results in the HSQC NMR spectra observed no changes in the native conformation of Gb1 when allowed to interact with goethite and kaolinite while birnessite induced strong structural modification of Gb1 at an acidic pH. Our results suggest that not all mineral surfaces in soil act as sorbents for EEs and changes in their catalytic activity upon adsorption to minerals surfaces may not just be an indication of conformational changes but of fragmentation of the protein itself.

Time-resolved spectroscopy of self-assembly of CCMV protein capsids

NASA Astrophysics Data System (ADS)

Moore, Jelyn; Aronzon, Dina; Manoharan, V. N.

2008-10-01

In order to gain a deeper understanding of the process a virus undergoes to assemble; the purpose of this study to time resolve the self-assembly of a virus. Cowpea Chlorotic Mottle virus (CCMV), an icosahedral type virus, can assemble without its genetic code (RNA) depending on its chemical and physical surroundings. The surface plasmon resonance (SPR) of colloidal gold particles is known to display a shift when the gold interacts with the proteins of a virus. Surface plasmon resonance is the free electron oscillation occurring at the surface of the gold particle resulting in a characteristic peak location at maximal absorbance and peak width. The shift results from the change in the refractive index of the particles as induced by the presence of the proteins. We hope to detect this shift through total internal reflection microscopy (TIRM). The accomplishments of this research are the completion of the TIR setup and the purification of the virus and its proteins.

The human peripheral subunit-binding domain folds rapidly while overcoming repulsive Coulomb forces

PubMed Central

Arbely, Eyal; Neuweiler, Hannes; Sharpe, Timothy D; Johnson, Christopher M; Fersht, Alan R

2010-01-01

Peripheral subunit binding domains (PSBDs) are integral parts of large multienzyme complexes involved in carbohydrate metabolism. PSBDs facilitate shuttling of prosthetic groups between different catalytic subunits. Their protein surface is characterized by a high density of positive charges required for binding to subunits within the complex. Here, we investigated folding thermodynamics and kinetics of the human PSBD (HSBD) using circular dichroism and tryptophan fluorescence experiments. HSBD was only marginally stable under physiological solvent conditions but folded within microseconds via a barrier-limited apparent two-state transition, analogous to its bacterial homologues. The high positive surface-charge density of HSBD leads to repulsive Coulomb forces that modulate protein stability and folding kinetics, and appear to even induce native-state movement. The electrostatic strain was alleviated at high solution-ionic-strength by Debye-Hückel screening. Differences in ionic-strength dependent characteristics among PSBD homologues could be explained by differences in their surface charge distributions. The findings highlight the trade-off between protein function and stability during protein evolution. PMID:20662005

Angiotensin II reduces the surface abundance of KV 1.5 channels in arterial myocytes to stimulate vasoconstriction.

PubMed

Kidd, Michael W; Bulley, Simon; Jaggar, Jonathan H

2017-03-01

Several different voltage-dependent K + (K V ) channel isoforms are expressed in arterial smooth muscle cells (myocytes). Vasoconstrictors inhibit K V currents, but the isoform selectivity and mechanisms involved are unclear. We show that angiotensin II (Ang II), a vasoconstrictor, stimulates degradation of K V 1.5, but not K V 2.1, channels through a protein kinase C- and lysosome-dependent mechanism, reducing abundance at the surface of mesenteric artery myocytes. The Ang II-induced decrease in cell surface K V 1.5 channels reduces whole-cell K V 1.5 currents and attenuates K V 1.5 function in pressurized arteries. We describe a mechanism by which Ang II stimulates protein kinase C-dependent K V 1.5 channel degradation, reducing the abundance of functional channels at the myocyte surface. Smooth muscle cells (myocytes) of resistance-size arteries express several different voltage-dependent K + (K V ) channels, including K V 1.5 and K V 2.1, which regulate contractility. Myocyte K V currents are inhibited by vasoconstrictors, including angiotensin II (Ang II), but the mechanisms involved are unclear. Here, we tested the hypothesis that Ang II inhibits K V currents by reducing the plasma membrane abundance of K V channels in myocytes. Angiotensin II (applied for 2 h) reduced surface and total K V 1.5 protein in rat mesenteric arteries. In contrast, Ang II did not alter total or surface K V 2.1, or K V 1.5 or K V 2.1 cellular distribution, measured as the percentage of total protein at the surface. Bisindolylmaleimide (BIM; a protein kinase C blocker), a protein kinase C inhibitory peptide or bafilomycin A (a lysosomal degradation inhibitor) each blocked the Ang II-induced decrease in total and surface K V 1.5. Immunofluorescence also suggested that Ang II reduced surface K V 1.5 protein in isolated myocytes; an effect inhibited by BIM. Arteries were exposed to Ang II or Ang II plus BIM (for 2 h), after which these agents were removed and contractility measurements performed or myocytes isolated for patch-clamp electrophysiology. Angiotensin II reduced both whole-cell K V currents and currents inhibited by Psora-4, a K V 1.5 channel blocker. Angiotensin II also reduced vasoconstriction stimulated by Psora-4 or 4-aminopyridine, another K V channel inhibitor. These data indicate that Ang II activates protein kinase C, which stimulates K V 1.5 channel degradation, leading to a decrease in surface K V 1.5, a reduction in whole-cell K V 1.5 currents and a loss of functional K V 1.5 channels in myocytes of pressurized arteries. © 2016 The Authors. The Journal of Physiology © 2016 The Physiological Society.

Enhanced human bone marrow mesenchymal stem cell functions on cathodic arc plasma-treated titanium

PubMed Central

Zhu, Wei; Teel, George; O’Brien, Christopher M; Zhuang, Taisen; Keidar, Michael; Zhang, Lijie Grace

2015-01-01

Surface modification of titanium for use in orthopedics has been explored for years; however, an ideal method of integrating titanium with native bone is still required to this day. Since human bone cells directly interact with nanostructured extracellular matrices, one of the most promising methods of improving titanium’s osseointegration involves inducing bio-mimetic nanotopography to enhance cell–implant interaction. In this regard, we explored an approach to functionalize the surface of titanium by depositing a thin film of textured titanium nanoparticles via a cathodic arc discharge plasma. The aim is to improve human bone marrow mesenchymal stem cell (MSC) attachment and differentiation and to reduce deleterious effects of more complex surface modification methods. Surface functionalization was analyzed by scanning electron microscopy, atomic force microscopy, contact angle testing, and specific protein adsorption. Scanning electron microscopy and atomic force microscopy examination demonstrate the deposition of titanium nanoparticles and the surface roughness change after coating. The specific fibronectin adsorption was enhanced on the modified titanium surface that associates with the improved hydrophilicity. MSC adhesion and proliferation were significantly promoted on the nanocoated surface. More importantly, compared to bare titanium, greater production of total protein, deposition of calcium mineral, and synthesis of alkaline phosphatase were observed from MSCs on nanocoated titanium after 21 days. The method described herein presents a promising alternative method for inducing more cell favorable nanosurface for improved orthopedic applications. PMID:26677327

Enhanced human bone marrow mesenchymal stem cell functions on cathodic arc plasma-treated titanium.

PubMed

Zhu, Wei; Teel, George; O'Brien, Christopher M; Zhuang, Taisen; Keidar, Michael; Zhang, Lijie Grace

2015-01-01

Surface modification of titanium for use in orthopedics has been explored for years; however, an ideal method of integrating titanium with native bone is still required to this day. Since human bone cells directly interact with nanostructured extracellular matrices, one of the most promising methods of improving titanium's osseointegration involves inducing bio-mimetic nanotopography to enhance cell-implant interaction. In this regard, we explored an approach to functionalize the surface of titanium by depositing a thin film of textured titanium nanoparticles via a cathodic arc discharge plasma. The aim is to improve human bone marrow mesenchymal stem cell (MSC) attachment and differentiation and to reduce deleterious effects of more complex surface modification methods. Surface functionalization was analyzed by scanning electron microscopy, atomic force microscopy, contact angle testing, and specific protein adsorption. Scanning electron microscopy and atomic force microscopy examination demonstrate the deposition of titanium nanoparticles and the surface roughness change after coating. The specific fibronectin adsorption was enhanced on the modified titanium surface that associates with the improved hydrophilicity. MSC adhesion and proliferation were significantly promoted on the nanocoated surface. More importantly, compared to bare titanium, greater production of total protein, deposition of calcium mineral, and synthesis of alkaline phosphatase were observed from MSCs on nanocoated titanium after 21 days. The method described herein presents a promising alternative method for inducing more cell favorable nanosurface for improved orthopedic applications.

Effect of Ethanol on Differential Protein Production and Expression of Potential Virulence Functions in the Opportunistic Pathogen Acinetobacter baumannii

PubMed Central

Nwugo, Chika C.; Arivett, Brock A.; Zimbler, Daniel L.; Gaddy, Jennifer A.; Richards, Ashley M.; Actis, Luis A.

2012-01-01

Acinetobacter baumannii persists in the medical environment and causes severe human nosocomial infections. Previous studies showed that low-level ethanol exposure increases the virulence of A. baumannii ATCC 17978. To better understand the mechanisms involved in this response, 2-D gel electrophoresis combined with mass spectrometry was used to investigate differential protein production in bacteria cultured in the presence or absence of ethanol. This approach showed that the presence of ethanol significantly induces and represses the production of 22 and 12 proteins, respectively. Although over 25% of the ethanol-induced proteins were stress-response related, the overall bacterial viability was uncompromised when cultured under these conditions. Production of proteins involved in lipid and carbohydrate anabolism was increased in the presence of ethanol, a response that correlates with increased carbohydrate biofilm content, enhanced biofilm formation on abiotic surfaces and decrease bacterial motility on semi-solid surfaces. The presence of ethanol also induced the acidification of bacterial cultures and the production of indole-3-acetic acid (IAA), a ubiquitous plant hormone that signals bacterial stress-tolerance and promotes plant-bacteria interactions. These responses could be responsible for the significantly enhanced virulence of A. baumannii ATCC 17978 cells cultured in the presence of ethanol when tested with the Galleria mellonella experimental infection model. Taken together, these observations provide new insights into the effect of ethanol in bacterial virulence. This alcohol predisposes the human host to infections by A. baumannii and could favor the survival and adaptation of this pathogen to medical settings and adverse host environments. PMID:23284824

Effect of ethanol on differential protein production and expression of potential virulence functions in the opportunistic pathogen Acinetobacter baumannii.

PubMed

Nwugo, Chika C; Arivett, Brock A; Zimbler, Daniel L; Gaddy, Jennifer A; Richards, Ashley M; Actis, Luis A

2012-01-01

Acinetobacter baumannii persists in the medical environment and causes severe human nosocomial infections. Previous studies showed that low-level ethanol exposure increases the virulence of A. baumannii ATCC 17978. To better understand the mechanisms involved in this response, 2-D gel electrophoresis combined with mass spectrometry was used to investigate differential protein production in bacteria cultured in the presence or absence of ethanol. This approach showed that the presence of ethanol significantly induces and represses the production of 22 and 12 proteins, respectively. Although over 25% of the ethanol-induced proteins were stress-response related, the overall bacterial viability was uncompromised when cultured under these conditions. Production of proteins involved in lipid and carbohydrate anabolism was increased in the presence of ethanol, a response that correlates with increased carbohydrate biofilm content, enhanced biofilm formation on abiotic surfaces and decrease bacterial motility on semi-solid surfaces. The presence of ethanol also induced the acidification of bacterial cultures and the production of indole-3-acetic acid (IAA), a ubiquitous plant hormone that signals bacterial stress-tolerance and promotes plant-bacteria interactions. These responses could be responsible for the significantly enhanced virulence of A. baumannii ATCC 17978 cells cultured in the presence of ethanol when tested with the Galleria mellonella experimental infection model. Taken together, these observations provide new insights into the effect of ethanol in bacterial virulence. This alcohol predisposes the human host to infections by A. baumannii and could favor the survival and adaptation of this pathogen to medical settings and adverse host environments.

Effects of protein inter-layers on cell-diamond FET characteristics.

PubMed

Rezek, Bohuslav; Krátká, Marie; Kromka, Alexander; Kalbacova, Marie

2010-12-15

Diamond is recognized as an attractive material for merging solid-state and biological systems. The advantage of diamond field-effect transistors (FET) is that they are chemically resistant, bio-compatible, and can operate without gate oxides. Solution-gated FETs based on H-terminated nanocrystalline diamond films exhibiting surface conductivity are employed here for studying effects of fetal bovine serum (FBS) proteins and osteoblastic SAOS-2 cells on diamond electronic properties. FBS proteins adsorbed on the diamond FETs permanently decrease diamond conductivity as reflected by the -45 mV shift of the FET transfer characteristics. Cell cultivation for 2 days results in a further shift by another -78 mV. We attribute it to a change of diamond material properties rather than purely to the field-effect. Increase in gate leakage currents (by a factor of 4) indicates that the FBS proteins also decrease the diamond-electrolyte electronic barrier induced by C-H surface dipoles. We propose a model where the proteins replace ions in the very vicinity of the H-terminated diamond surface. Copyright © 2010 Elsevier B.V. All rights reserved.

Oral vaccine of Lactococcus lactis harbouring pandemic H1N1 2009 haemagglutinin1 and nisP anchor fusion protein elevates anti-HA1 sIgA levels in mice.

PubMed

Joan, Stella Siaw Xiu; Pui-Fong, Jee; Song, Adelene Ai-Lian; Chang, Li-Yen; Yusoff, Khatijah; AbuBakar, Sazaly; Rahim, Raha Abdul

2016-05-01

An oral lactococcal-based vaccine which haboured the haemagglutinin1 (HA1) antigen fused to nisP anchor protein for the purpose of surface displaying the HA1 antigen was developed against H1N1 virus. Recombinant L. lactis strains expressed HA1-nisP fusion proteins when induced with nisin, as confirmed through western blotting. However, immunofluorescense did not detect any surface-displayed proteins, suggesting that the protein was either unsuccessfully translocated or improperly displayed. Despite this, oral administration of recombinant L. lactis strains to BALB/c mice revealed that significant levels of anti-HA1 sIgA antibodies were detected in mice fecal suspension samples of mice group NZ9000 (pNZ:HN) when compared to the negative control NZ9000 (pNZ8048) group. Specific anti-HA1 sIgA antibodies were locally produced and live recombinant lactococcal vaccine was able to elicit humoral response of BALB/c mice despite unsuccessful surface display of the HA1 epitope.

DNA secondary structures are associated with recombination in major Plasmodium falciparum variable surface antigen gene families

PubMed Central

Sander, Adam F.; Lavstsen, Thomas; Rask, Thomas S.; Lisby, Michael; Salanti, Ali; Fordyce, Sarah L.; Jespersen, Jakob S.; Carter, Richard; Deitsch, Kirk W.; Theander, Thor G.; Pedersen, Anders Gorm; Arnot, David E.

2014-01-01

Many bacterial, viral and parasitic pathogens undergo antigenic variation to counter host immune defense mechanisms. In Plasmodium falciparum, the most lethal of human malaria parasites, switching of var gene expression results in alternating expression of the adhesion proteins of the Plasmodium falciparum-erythrocyte membrane protein 1 class on the infected erythrocyte surface. Recombination clearly generates var diversity, but the nature and control of the genetic exchanges involved remain unclear. By experimental and bioinformatic identification of recombination events and genome-wide recombination hotspots in var genes, we show that during the parasite’s sexual stages, ectopic recombination between isogenous var paralogs occurs near low folding free energy DNA 50-mers and that these sequences are heavily concentrated at the boundaries of regions encoding individual Plasmodium falciparum-erythrocyte membrane protein 1 structural domains. The recombinogenic potential of these 50-mers is not parasite-specific because these sequences also induce recombination when transferred to the yeast Saccharomyces cerevisiae. Genetic cross data suggest that DNA secondary structures (DSS) act as inducers of recombination during DNA replication in P. falciparum sexual stages, and that these DSS-regulated genetic exchanges generate functional and diverse P. falciparum adhesion antigens. DSS-induced recombination may represent a common mechanism for optimizing the evolvability of virulence gene families in pathogens. PMID:24253306

Influence of Ficoll on urea induced denaturation of fibrinogen

NASA Astrophysics Data System (ADS)

Sankaranarayanan, Kamatchi; Meenakshisundaram, N.

2016-03-01

Ficoll is a neutral, highly branched polymer used as a molecular crowder in the study of proteins. Ficoll is also part of Ficoll-Paque used in biology laboratories to separate blood to its components (erythrocytes, leukocytes etc.,). Role of Ficoll in the urea induced denaturation of protein Fibrinogen (Fg) has been analyzed using fluorescence, circular dichroism, molecular docking and interfacial studies. Fluorescence studies show that Ficoll prevents quenching of Fg in the presence of urea. From the circular dichroism spectra, Fg shows conformational transition to random coil with urea of 6 M concentration. Ficoll helps to shift this denaturation concentration to 8 M and thus constraints by shielding Fg during the process. Molecular docking studies indicate that Ficoll interacts favorably with the protein than urea. The surface tension and shear viscosity analysis shows clearly that the protein is shielded by Ficoll.

Anisotropy of the Coulomb Interaction between Folded Proteins: Consequences for Mesoscopic Aggregation of Lysozyme

PubMed Central

Chan, Ho Yin; Lankevich, Vladimir; Vekilov, Peter G.; Lubchenko, Vassiliy

2012-01-01

Toward quantitative description of protein aggregation, we develop a computationally efficient method to evaluate the potential of mean force between two folded protein molecules that allows for complete sampling of their mutual orientation. Our model is valid at moderate ionic strengths and accounts for the actual charge distribution on the surface of the molecules, the dielectric discontinuity at the protein-solvent interface, and the possibility of protonation or deprotonation of surface residues induced by the electric field due to the other protein molecule. We apply the model to the protein lysozyme, whose solutions exhibit both mesoscopic clusters of protein-rich liquid and liquid-liquid separation; the former requires that protein form complexes with typical lifetimes of approximately milliseconds. We find the electrostatic repulsion is typically lower than the prediction of the Derjaguin-Landau-Verwey-Overbeek theory. The Coulomb interaction in the lowest-energy docking configuration is nonrepulsive, despite the high positive charge on the molecules. Typical docking configurations barely involve protonation or deprotonation of surface residues. The obtained potential of mean force between folded lysozyme molecules is consistent with the location of the liquid-liquid coexistence, but produces dimers that are too short-lived for clusters to exist, suggesting lysozyme undergoes conformational changes during cluster formation. PMID:22768950

Aggregation of a Monoclonal Antibody Induced by Adsorption to Stainless Steel

PubMed Central

Bee, Jared S.; Davis, Michele; Freund, Erwin; Carpenter, John F.; Randolph, Theodore W.

2014-01-01

Stainless steel is a ubiquitous surface in therapeutic protein production equipment and is also present as the needle in some pre-filled syringe biopharmaceutical products. Stainless steel microparticles can cause of aggregation of a monoclonal antibody (mAb). The initial rate of mAb aggregation was second-order in steel surface area and zero-order in mAb concentration, generally consistent with a bimolecular surface aggregation being the rate-limiting step. Polysorbate 20 (PS20) suppressed the aggregation yet was unable to desorb the firmly bound first layer of protein that adsorbs to the stainless steel surface. Also, there was no exchange of mAb from the first adsorbed layer to the bulk phase, suggesting that the aggregation process actually occurs on subsequent adsorption layers. No oxidized Met residues were detected in the mass spectrum of a digest of a highly aggregated mAb, although there was five-fold increase in carbonyl groups due to protein oxidation. PMID:19725039

Bovine seminal PDC-109 protein: an overview of biochemical and functional properties.

PubMed

Srivastava, N; Jerome, A; Srivastava, S K; Ghosh, S K; Kumar, Amit

2013-04-01

Although long-term storage of bovine semen is desirable for wider use, successful cryopreservation depends on several factors, including various proteins present in seminal plasma. One such group of proteins, viz. bovine seminal plasma (BSP) proteins represents the major protein fraction in bovine seminal plasma. They constitute three major heparin-binding (HB-) acidic proteins secreted by seminal vesicles, viz. BSP-A1/-A2 (PDC-109), BSP-A3 and BSP-30-kDa. By purification studies it was deduced that PDC-109 is a polypeptide of 109 amino acids and contains two tandem repeating fibronectin type-II (Fn-II) domains, preceded by a 23 residue N-terminal domain. Though BSP-A1 and BSP-A2 are biochemically similar they differ only in glycosylation and their mixture is called PDC-109 or gonadostatins. PDC-109 exists as a polydisperse, multimeric self-associated molecule and possesses multifunctional properties, viz. binding to the surface of plasma membrane of spermatozoa causing conformational change in the sperm surface proteins and enhances motility. Besides binding, PDC-109 protein provokes cholesterol efflux from sperm membrane and promotes sperm reservoir by interacting with oviductal membrane. Interaction of sperm with PDC-109 protein induces sperm capacitation and acrosome reaction. However, prolonged exposure of spermatozoa with free floating PDC-109 protein as during processing for preservation, increases cholesterol efflux from spermatozoa. The efflux of sperm membrane cholesterol and disturbance in cholesterol:phospholipids ratio causes destabilization of plasma membrane thereby inducing cryoinjury to the sperm. In this review, the biochemical, functional properties of PDC-109 protein and its role during semen cryopreservation is summarized. Copyright © 2013 Elsevier B.V. All rights reserved.

FROM FOLDING THEORIES TO FOLDING PROTEINS: A Review and Assessment of Simulation Studies of Protein Folding and Unfolding

NASA Astrophysics Data System (ADS)

Shea, Joan-Emma; Brooks, Charles L., III

2001-10-01

Beginning with simplified lattice and continuum "minimalist" models and progressing to detailed atomic models, simulation studies have augmented and directed development of the modern landscape perspective of protein folding. In this review we discuss aspects of detailed atomic simulation methods applied to studies of protein folding free energy surfaces, using biased-sampling free energy methods and temperature-induced protein unfolding. We review studies from each on systems of particular experimental interest and assess the strengths and weaknesses of each approach in the context of "exact" results for both free energies and kinetics of a minimalist model for a beta-barrel protein. We illustrate in detail how each approach is implemented and discuss analysis methods that have been developed as components of these studies. We describe key insights into the relationship between protein topology and the folding mechanism emerging from folding free energy surface calculations. We further describe the determination of detailed "pathways" and models of folding transition states that have resulted from unfolding studies. Our assessment of the two methods suggests that both can provide, often complementary, details of folding mechanism and thermodynamics, but this success relies on (a) adequate sampling of diverse conformational regions for the biased-sampling free energy approach and (b) many trajectories at multiple temperatures for unfolding studies. Furthermore, we find that temperature-induced unfolding provides representatives of folding trajectories only when the topology and sequence (energy) provide a relatively funneled landscape and "off-pathway" intermediates do not exist.

Vimentin Is Involved in Peptidylarginine Deiminase 2-Induced Apoptosis of Activated Jurkat Cells

PubMed Central

Hsu, Pei-Chen; Liao, Ya-Fan; Lin, Chin-Li; Lin, Wen-Hao; Liu, Guang-Yaw; Hung, Hui-Chih

2014-01-01

Peptidylarginine deiminase type 2 (PADI2) deiminates (or citrullinates) arginine residues in protein to citrulline residues in a Ca2+-dependent manner, and is found in lymphocytes and macrophages. Vimentin is an intermediate filament protein and a well-known substrate of PADI2. Citrullinated vimentin is found in ionomycin-induced macrophage apoptosis. Citrullinated vimentin is the target of anti-Sa antibodies, which are specific to rheumatoid arthritis, and play a critical role in the pathogenesis of the disease. To investigate the role of PADI2 in apoptosis, we generated a Jurkat cell line that overexpressed the PADI2 transgene from a tetracycline-inducible promoter, and used a combination of 12-O-tetradecanoylphorbol-13-acetate and ionomycin to activate Jurkat cells. We found that PADI2 overexpression reduced the cell viability of activated Jurkat cells in a dose- and time-dependent manner. The PADI2-overexpressed and -activated Jurkat cells presented typical manifestations of apoptosis, and exhibited greater levels of citrullinated proteins, including citrullinated vimentin. Vimentin overexpression rescued a portion of the cells from apoptosis. In conclusion, PADI2 overexpression induces apoptosis in activated Jurkat cells. Vimentin is involved in PADI2-induced apoptosis. Moreover, PADI2-overexpressed Jurkat cells secreted greater levels of vimentin after activation, and expressed more vimentin on their cell surfaces when undergoing apoptosis. Through artificially highlighting PADI2 and vimentin, we demonstrated that PADI2 and vimentin participate in the apoptotic mechanisms of activated T lymphocytes. The secretion and surface expression of vimentin are possible ways of autoantigen presentation to the immune system. PMID:24850148

Hepatitis C virus core protein subverts the antiviral activities of human Kupffer cells.

PubMed

Tu, Zhengkun; Pierce, Robert H; Kurtis, Jonathan; Kuroki, Yoshio; Crispe, I Nicholas; Orloff, Mark S

2010-01-01

Kupffer cells (KC) are important innate immune cells of the liver, functioning as scavenging sinusoidal phagocytes and transducers of pattern recognition signals, including those of toll-like receptors (TLRs). The hepatitis C virus core protein (HCVc) engages TLR2 on peripheral blood monocytes and induces production of multiple inflammatory cytokines. We examined the effects of HCVc on human primary KC functions. KC were isolated from living donor allografts and stimulated with HCVc and/or ligands for TLRs. KC were examined for production of cytokines, expression of programmed death-ligand 1 (PD-L1), secretion of type 1 interferons (IFNs), and expression of the apoptosis-inducing protein tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL). HCVc acts as a ligand for TLR2 on human KC, inducing them to secrete interleukin (IL)-1beta, TNF-alpha, and IL-10 and up-regulate cell surface PD-L1. HCVc blocked TLR3-mediated secretion of IFN-alpha, IFN-beta, and cell surface expression of the cytotoxic molecule TRAIL. Inhibition of phosphoinositide 3 kinase with LY294002 blocked the up-regulation of PD-L1 by TLR ligands and the TLR3-specific induction of TRAIL and type 1 IFNs. KC are intravascular macrophages that are continuously exposed to, and tolerant of, bacterial TLR ligands, which are delivered via the portal circulation. By mimicking a bacterial TLR2 ligand and effectively blocking the TLR3-mediated, double-stranded RNA-induced antiviral response, HCVc might appear to exploit this unique aspect of immunity in the liver. Copyright 2010 AGA Institute. Published by Elsevier Inc. All rights reserved.

Quantification of Protein-Induced Membrane Remodeling Kinetics In Vitro with Lipid Multilayer Gratings

PubMed Central

Lowry, Troy W.; Hariri, Hanaa; Prommapan, Plengchart; Kusi-Appiah, Aubrey; Vafai, Nicholas; Bienkiewicz, Ewa A.; Van Winkle, David H.; Stagg, Scott M.

2016-01-01

The dynamic self-organization of lipids in biological systems is a highly regulated process that enables the compartmentalization of living systems at micro- and nanoscopic scales. Consequently, quantitative methods for assaying the kinetics of supramolecular remodeling such as vesicle formation from planar lipid bilayers or multilayers are needed to understand cellular self-organization. Here, a new nanotechnology-based method for quantitative measurements of lipid–protein interactions is presented and its suitability for quantifying the membrane binding, inflation, and budding activity of the membrane-remodeling protein Sar1 is demonstrated. Lipid multilayer gratings are printed onto surfaces using nanointaglio and exposed to Sar1, resulting in the inflation of lipid multilayers into unilamellar structures, which can be observed in a label-free manner by monitoring the diffracted light. Local variations in lipid multilayer volume on the surface is used to vary substrate availability in a microarray format. A quantitative model is developed that allows quantification of binding affinity (KD) and kinetics (kon and koff). Importantly, this assay is uniquely capable of quantifying membrane remodeling. Upon Sar1-induced inflation of single bilayers from surface supported multilayers, the semicylindrical grating lines are observed to remodel into semispherical buds when a critical radius of curvature is reached. PMID:26649649

The effect of milk processing on the microstructure of the milk fat globule and rennet induced gel observed using confocal laser scanning microscopy.

PubMed

Ong, L; Dagastine, R R; Kentish, S E; Gras, S L

2010-04-01

Confocal laser scanning microscopy (CLSM) was successfully used to observe the effect of milk processing on the size and the morphology of the milk fat globule in raw milk, raw ultrafiltered milk, and standardized and pasteurized milk prepared for cheese manufacture (cheese-milk) and commercial pasteurized and homogenized milk. Fat globule size distributions for the milk preparations were analyzed using both image analysis and light scattering and both measurements produced similar data trends. Changes to the native milk fat globule membrane (MFGM) were tracked using a MFGM specific fluorescent stain that allowed MFGM proteins and adsorbed proteins to be differentiated on the fat globule surface. Sodium dodecyl sulfate polyacrylamide gel electrophoresis confirmed the identity of native MFGM proteins isolated from the surface of fat globules within raw, UF retentate, and cheese-milk preparations, whereas only casein was detected on the surface of fat globules in homogenized milk. The microstructure, porosity, and gel strength of the rennet induced gel made from raw milk and cheese-milk was also found to be comparable and significantly different to that made from homogenized milk. Our results highlight the potential use of CLSM as a tool to observe the structural details of the fat globule and associated membrane close to its native environment.

The biomolecular corona is retained during nanoparticle uptake and protects the cells from the damage induced by cationic nanoparticles until degraded in the lysosomes.

PubMed

Wang, Fengjuan; Yu, Lu; Monopoli, Marco P; Sandin, Peter; Mahon, Eugene; Salvati, Anna; Dawson, Kenneth A

2013-11-01

Nanoparticles have unique capacities of interacting with the cellular machinery and entering cells. To be able to exploit this potential, it is essential to understand what controls the interactions at the interface between nanoparticles and cells: it is now established that nanoparticles in biological media are covered by proteins and other biomolecules forming a "corona" on the nanoparticle surface, which confers a new identity to the nanoparticles. By labelling the proteins of the serum, using positively-charged polystyrene, we now show that this adsorbed layer is strong enough to be retained on the nanoparticles as they enter cells and is trafficked to the lysosomes on the nanoparticles. There, the corona is degraded and this is followed by lysosomal damage, leading to cytosolic release of lysosomal content, and ultimately apoptosis. Thus the corona protects the cells from the damage induced by the bare nanoparticle surface until enzymatically cleared in the lysosomes. This study investigates the effects of protein corona that normally forms on the surface of nanoparticles during in vivo use, describing the steps of intracellular processing of such particles, to enhance our understanding of how these particles interact with the cellular machinery. Copyright © 2013 Elsevier Inc. All rights reserved.

Shigella Outer Membrane Protein PSSP-1 Is Broadly Protective against Shigella Infection

PubMed Central

Rho, Semi; Kim, Su Hee; Kim, Heejoo; Song, Hyo Jin; Kim, Eun Jin; Kim, Ryang Yeo; Kim, Eun Hye; Sinha, Anuradha; Dey, Ayan; Yang, Jae Seung; Song, Man Ki; Nandy, Ranjan Kumar; Czerkinsky, Cecil

2015-01-01

In developing countries, Shigella is a primary cause of diarrhea in infants and young children. Although antibiotic therapy is an effective treatment for shigellosis, therapeutic options are narrowing due to the emergence of antibiotic resistance. Thus, preventive vaccination could become the most efficacious approach for controlling shigellosis. We have identified several conserved protein antigens that are shared by multiple Shigella serotypes and species. Among these, one antigen induced cross-protection against experimental shigellosis, and we have named it pan-Shigella surface protein 1 (PSSP-1). PSSP-1-induced protection requires a mucosal administration route and coadministration of an adjuvant. When PSSP-1 was administered intranasally, it induced cross-protection against Shigella flexneri serotypes 2a, 5a, and 6, Shigella boydii, Shigella sonnei, and Shigella dysenteriae serotype 1. Intradermally administered PSSP-1 induced strong serum antibody responses but failed to induce protection in the mouse lung pneumonia model. In contrast, intranasal administration elicited efficient local and systemic antibody responses and production of interleukin 17A and gamma interferon. Interestingly, blood samples from patients with recent-onset shigellosis showed variable but significant mucosal antibody responses to other conserved Shigella protein antigens but not to PSSP-1. We suggest that PSSP-1 is a promising antigen for a broadly protective vaccine against Shigella. PMID:25651919

Reusable hydroxyapatite nanocrystal sensors for protein adsorption.

PubMed

Tagaya, Motohiro; Ikoma, Toshiyuki; Hanagata, Nobutaka; Chakarov, Dinko; Kasemo, Bengt; Tanaka, Junzo

2010-08-01

The repeatability of the adsorption and removal of fibrinogen and fetal bovine serum on hydroxyapatite (HAp) nanocrystal sensors was investigated by Fourier transform infrared (FTIR) spectroscopy and quartz crystal microbalance with dissipation (QCM-D) monitoring technique. The HAp nanocrystals were coated on a gold-coated quartz sensor by electrophoretic deposition. Proteins adsorbed on the HAp sensors were removed by (i) ammonia/hydrogen peroxide mixture (APM), (ii) ultraviolet light (UV), (iii) UV/APM, (iv) APM/UV and (v) sodium dodecyl sulfate (SDS) treatments. FTIR spectra of the reused surfaces revealed that the APM and SDS treatments left peptide fragments or the proteins adsorbed on the surfaces, whereas the other methods successfully removed the proteins. The QCM-D measurements indicated that in the removal treatments, fibrinogen was slowly adsorbed in the first cycle because of the change in surface wettability revealed by contact angle measurements. The SDS treatment was not effective in removing proteins. The APM or UV treatment decreased the frequency shifts for the reused HAp sensors. The UV/APM treatment did not induce the frequency shifts but decreased the dissipation shifts. Therefore, we conclude that the APM/UV treatment is the most useful method for reproducing protein adsorption behavior on HAp sensors.

Reusable hydroxyapatite nanocrystal sensors for protein adsorption

NASA Astrophysics Data System (ADS)

Tagaya, Motohiro; Ikoma, Toshiyuki; Hanagata, Nobutaka; Chakarov, Dinko; Kasemo, Bengt; Tanaka, Junzo

2010-08-01

The repeatability of the adsorption and removal of fibrinogen and fetal bovine serum on hydroxyapatite (HAp) nanocrystal sensors was investigated by Fourier transform infrared (FTIR) spectroscopy and quartz crystal microbalance with dissipation (QCM-D) monitoring technique. The HAp nanocrystals were coated on a gold-coated quartz sensor by electrophoretic deposition. Proteins adsorbed on the HAp sensors were removed by (i) ammonia/hydrogen peroxide mixture (APM), (ii) ultraviolet light (UV), (iii) UV/APM, (iv) APM/UV and (v) sodium dodecyl sulfate (SDS) treatments. FTIR spectra of the reused surfaces revealed that the APM and SDS treatments left peptide fragments or the proteins adsorbed on the surfaces, whereas the other methods successfully removed the proteins. The QCM-D measurements indicated that in the removal treatments, fibrinogen was slowly adsorbed in the first cycle because of the change in surface wettability revealed by contact angle measurements. The SDS treatment was not effective in removing proteins. The APM or UV treatment decreased the frequency shifts for the reused HAp sensors. The UV/APM treatment did not induce the frequency shifts but decreased the dissipation shifts. Therefore, we conclude that the APM/UV treatment is the most useful method for reproducing protein adsorption behavior on HAp sensors.

Maximally asymmetric transbilayer distribution of anionic lipids alters the structure and interaction with lipids of an amyloidogenic protein dimer bound to the membrane surface

PubMed Central

Cheng, Sara Y.; Chou, George; Buie, Creighton; Vaughn, Mark W.; Compton, Campbell; Cheng, Kwan H.

2016-01-01

We used molecular dynamics simulations to explore the effects of asymmetric transbilayer distribution of anionic phosphatidylserine (PS) lipids on the structure of a protein on the membrane surface and subsequent protein–lipid interactions. Our simulation systems consisted of an amyloidogenic, beta-sheet rich dimeric protein (D42) absorbed to the phosphatidylcholine (PC) leaflet, or protein-contact PC leaflet, of two membrane systems: a single-component PC bilayer and double PC/PS bilayers. The latter comprised of a stable but asymmetric transbilayer distribution of PS in the presence of counterions, with a 1-component PC leaflet coupled to a 1-component PS leaflet in each bilayer. The maximally asymmetric PC/PS bilayer had a non-zero transmembrane potential (TMP) difference and higher lipid order packing, whereas the symmetric PC bilayer had a zero TMP difference and lower lipid order packing under physiologically relevant conditions. Analysis of the adsorbed protein structures revealed weaker protein binding, more folding in the N-terminal domain, more aggregation of the N- and C-terminal domains and larger tilt angle of D42 on the PC leaflet surface of the PC/PS bilayer versus the PC bilayer. Also, analysis of protein-induced membrane structural disruption revealed more localized bilayer thinning in the PC/PS versus PC bilayer. Although the electric field profile in the non-protein-contact PS leaflet of the PC/PS bilayer differed significantly from that in the non-protein-contact PC leaflet of the PC bilayer, no significant difference in the electric field profile in the protein-contact PC leaflet of either bilayer was evident. We speculate that lipid packing has a larger effect on the surface adsorbed protein structure than the electric field for a maximally asymmetric PC/PS bilayer. Our results support the mechanism that the higher lipid packing in a lipid leaflet promotes stronger protein–protein but weaker protein–lipid interactions for a dimeric protein on membrane surfaces. PMID:26827904

IRON-BINDING AND STORAGE PROTEINS IN SPUTUM

EPA Science Inventory

Induced sputum (IS) and bronchoalveolar lavage (BAL) sample different lung compartments, with IS obtaining secretions from the surfaces of the bronchial airways and BAL sampling secretions from the alveolar airspaces. Deposition of iron-containing particulate matter occurs prefer...

Paper-based microfluidic approach for surface-enhanced raman spectroscopy and highly reproducible detection of proteins beyond picomolar concentration.

PubMed

Saha, Arindam; Jana, Nikhil R

2015-01-14

Although microfluidic approach is widely used in various point of care diagnostics, its implementation in surface enhanced Raman spectroscopy (SERS)-based detection is challenging. This is because SERS signal depends on plasmonic nanoparticle aggregation induced generation of stable electromagnetic hot spots and in currently available microfluidic platform this condition is difficult to adapt. Here we show that SERS can be adapted using simple paper based microfluidic system where both the plasmonic nanomaterials and analyte are used in mobile phase. This approach allows analyte induced controlled particle aggregation and electromagnetic hot spot generation inside the microfluidic channel with the resultant SERS signal, which is highly reproducible and sensitive. This approach has been used for reproducible detection of protein in the pico to femtomolar concentration. Presented approach is simple, rapid, and cost-effective, and requires low sample volume. Method can be extended for SERS-based detection of other biomolecules.

Functional display of ice nucleation protein InaZ on the surface of bacterial ghosts.

PubMed

Kassmannhuber, Johannes; Rauscher, Mascha; Schöner, Lea; Witte, Angela; Lubitz, Werner

2017-09-03

In a concept study the ability to induce heterogeneous ice formation by Bacterial Ghosts (BGs) from Escherichia coli carrying ice nucleation protein InaZ from Pseudomonas syringae in their outer membrane was investigated by a droplet-freezing assay of ultra-pure water. As determined by the median freezing temperature and cumulative ice nucleation spectra it could be demonstrated that both the living recombinant E. coli and their corresponding BGs functionally display InaZ on their surface. Under the production conditions chosen both samples belong to type II ice-nucleation particles inducing ice formation at a temperature range of between -5.6 °C and -6.7 °C, respectively. One advantage for the application of such BGs over their living recombinant mother bacteria is that they are non-living native cell envelopes retaining the biophysical properties of ice nucleation and do no longer represent genetically modified organisms (GMOs).

Functional display of ice nucleation protein InaZ on the surface of bacterial ghosts

PubMed Central

Kassmannhuber, Johannes; Rauscher, Mascha; Schöner, Lea; Witte, Angela; Lubitz, Werner

2017-01-01

ABSTRACT In a concept study the ability to induce heterogeneous ice formation by Bacterial Ghosts (BGs) from Escherichia coli carrying ice nucleation protein InaZ from Pseudomonas syringae in their outer membrane was investigated by a droplet-freezing assay of ultra-pure water. As determined by the median freezing temperature and cumulative ice nucleation spectra it could be demonstrated that both the living recombinant E. coli and their corresponding BGs functionally display InaZ on their surface. Under the production conditions chosen both samples belong to type II ice-nucleation particles inducing ice formation at a temperature range of between −5.6 °C and −6.7 °C, respectively. One advantage for the application of such BGs over their living recombinant mother bacteria is that they are non-living native cell envelopes retaining the biophysical properties of ice nucleation and do no longer represent genetically modified organisms (GMOs). PMID:28121482

Ice-surface adsorption enhanced colligative effect of antifreeze proteins in ice growth inhibition

NASA Astrophysics Data System (ADS)

Mao, Yougang; Ba, Yong

2006-09-01

This Communication describes a mechanism to explain antifreeze protein's function to inhibit the growth of ice crystals. We propose that the adsorption of antifreeze protein (AFP) molecules on an ice surface induces a dense AFP-water layer, which can significantly decrease the mole fraction of the interfacial water and, thus, lower the temperature for a seed ice crystal to grow in a super-cooled AFP solution. This mechanism can also explain the nearly unchanged melting point for the ice crystal due to the AFP's ice-surface adsorption. A mathematical model combining the Langmuir theory of adsorption and the colligative effect of thermodynamics has been proposed to find the equilibrium constants of the ice-surface adsorptions, and the interfacial concentrations of AFPs through fitting the theoretical curves to the experimental thermal hysteresis data. This model has been demonstrated by using the experimental data of serial size-mutated beetle Tenebrio molitor (Tm) AFPs. It was found that the AFP's ice-surface adsorptions could increase the interfacial AFP's concentrations by 3 to 4 orders compared with those in the bulk AFP solutions.

Strong Antibody Responses Induced by Protein Antigens Conjugated onto the Surface of Lecithin-Based Nanoparticles

PubMed Central

Sloat, Brian R.; Sandoval, Michael A.; Hau, Andrew M.; He, Yongqun; Cui, Zhengrong

2009-01-01

An accumulation of research over the years has demonstrated the utility of nanoparticles as antigen carriers with adjuvant activity. Herein we defined the adjuvanticity of a novel lecithin-based nanoparticle engineered from emulsions. The nanoparticles were spheres of around 200 nm. Model protein antigens, bovine serum albumin (BSA) or Bacillus anthracis protective antigen (PA) protein, were covalently conjugated onto the nanoparticles. Mice immunized with the BSA-conjugated nanoparticles developed strong anti-BSA antibody responses comparable to that induced by BSA adjuvanted with incomplete Freund's adjuvant and 6.5-fold stronger than that induced by BSA adsorbed onto aluminum hydroxide. Immunization of mice with the PA-conjugated nanoparticles elicited a quick, strong, and durable anti-PA antibody response that afforded protection of the mice against a lethal dose of anthrax lethal toxin challenge. The potent adjuvanticity of the nanoparticles was likely due to their ability to move the antigens into local draining lymph nodes, to enhance the uptake of the antigens by antigen-presenting cells (APCs), and to activate APCs. This novel nanoparticle system has the potential to serve as a universal protein-based vaccine carrier capable of inducing strong immune responses. PMID:19729045

Uncovering the stoichiometry of Pyrococcus furiosus RNase P, a multi-subunit catalytic ribonucleoprotein complex, by surface-induced dissociation and ion mobility mass spectrometry.

PubMed

Ma, Xin; Lai, Lien B; Lai, Stella M; Tanimoto, Akiko; Foster, Mark P; Wysocki, Vicki H; Gopalan, Venkat

2014-10-20

We demonstrate that surface-induced dissociation (SID) coupled with ion mobility mass spectrometry (IM-MS) is a powerful tool for determining the stoichiometry of a multi-subunit ribonucleoprotein (RNP) complex assembled in a solution containing Mg(2+). We investigated Pyrococcus furiosus (Pfu) RNase P, an archaeal RNP that catalyzes tRNA 5' maturation. Previous step-wise, Mg(2+)-dependent reconstitutions of Pfu RNase P with its catalytic RNA subunit and two interacting protein cofactor pairs (RPP21⋅RPP29 and POP5⋅RPP30) revealed functional RNP intermediates en route to the RNase P enzyme, but provided no information on subunit stoichiometry. Our native MS studies with the proteins showed RPP21⋅RPP29 and (POP5⋅RPP30)2 complexes, but indicated a 1:1 composition for all subunits when either one or both protein complexes bind the cognate RNA. These results highlight the utility of SID and IM-MS in resolving conformational heterogeneity and yielding insights on RNP assembly. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Activation of endothelial-leukocyte adhesion molecule 1 (ELAM-1) gene transcription

DOE Office of Scientific and Technical Information (OSTI.GOV)

Montgomery, K.F.; Tarr, P.I.; Bomsztyk, K.

1991-08-01

Leukocyte adherence to endothelium is in part mediated by the transient expression of endothelial-leukocyte adhesion molecule 1 (ELAM-1) on endothelial surfaces stimulated by tumor necrosis factor {alpha} (TNF), interleukin (IL) 1, or bacterial lipopolysaccharide (LPS). The intracellular factors controlling induction of ELAM-1 mRNA and protein are unknown. In nuclear runoff experiments with cultured human umbilical vein endothelial cells (HUVEC), the authors demonstrate that transcriptional activation of the ELAM-1 gene occurs following stimulation with TNF. Sequence analysis of the 5{prime} flanking region of the ELAM-1 gene reveals consensus DNA-binding sequences for two known transcription factors, NF-{kappa}B and AP-1. Gel mobility shiftmore » assays demonstrate that TNF, IL-1, or LPS induces activation of NF-{kappa}B-like DNA binding activity in HUVEC. Phorbol 12-myristate 13-acetate, a known activator of protein kinase C (PKC), weakly induces NF-{kappa}B-like activity, ELAM-1 mRNA, and ELAM-1 surface expression in HUVEC. However, TNF, IL-1, and LPS do not activate PKC in HUVEC at doses that strongly induce NF-{kappa}B-like protein activation and ELAM-1 gene expression. PKC blockade with H7 does not inhibit activation of these NF-kB-like proteins but does inhibit ELAM-1 gene transcription. They conclude that PKC-independent activation of NF-{kappa}B in HUVEC with TNF, IL-1, or LPS is associated with, but not sufficient for, activation of ELAM-1 gene transcription.« less

Surface Induced Dissociation Yields Quaternary Substructure of Refractory Noncovalent Phosphorylase B and Glutamate Dehydrogenase Complexes

NASA Astrophysics Data System (ADS)

Ma, Xin; Zhou, Mowei; Wysocki, Vicki H.

2014-03-01

Ion mobility (IM) and tandem mass spectrometry (MS/MS) coupled with native MS are useful for studying noncovalent protein complexes. Collision induced dissociation (CID) is the most common MS/MS dissociation method. However, some protein complexes, including glycogen phosphorylase B kinase (PHB) and L-glutamate dehydrogenase (GDH) examined in this study, are resistant to dissociation by CID at the maximum collision energy available in the instrument. Surface induced dissociation (SID) was applied to dissociate the two refractory protein complexes. Different charge state precursor ions of the two complexes were examined by CID and SID. The PHB dimer was successfully dissociated to monomers and the GDH hexamer formed trimeric subcomplexes that are informative of its quaternary structure. The unfolding of the precursor and the percentages of the distinct products suggest that the dissociation pathways vary for different charge states. The precursors at lower charge states (+21 for PHB dimer and +27 for GDH hexamer) produce a higher percentage of folded fragments and dissociate more symmetrically than the precusors at higher charge states (+29 for PHB dimer and +39 for GDH hexamer). The precursors at lower charge state may be more native-like than the higher charge state because a higher percentage of folded fragments and a lower percentage of highly charged unfolded fragments are detected. The combination of SID and charge reduction is shown to be a powerful tool for quaternary structure analysis of refractory noncovalent protein complexes, as illustrated by the data for PHB dimer and GDH hexamer.

Mutant CCL2 Protein Coating Mitigates Wear Particle-Induced Bone Loss in a Murine Continuous Polyethylene Infusion Model

PubMed Central

Nabeshima, Akira; Pajarinen, Jukka; Lin, Tzu-hua; Jiang, Xinyi; Gibon, Emmanuel; Córdova, Luis A.; Loi, Florence; Lu, Laura; Jämsen, Eemeli; Egashira, Kensuke; Yang, Fan; Yao, Zhenyu; Goodman, Stuart B

2016-01-01

Wear particle-induced osteolysis limits the long-term survivorship of total joint replacement (TJR). Monocyte/macrophages are the key cells of this adverse reaction. Monocyte Chemoattractant Protein-1 (MCP-1/CCL2) is the most important chemokine regulating trafficking of monocyte/macrophages in particle-induced inflammation. 7ND recombinant protein is a mutant of CCL2 that inhibits CCL2 signaling. We have recently developed a layer-by-layer (LBL) coating platform on implant surfaces that can release biologically active 7ND. In this study, we investigated the effect of 7ND on wear particle-induced bone loss using the murine continuous polyethylene (PE) particle infusion model with 7ND coating of a titanium rod as a local drug delivery device. PE particles were infused into hollow titanium rods with or without 7ND coating implanted in the distal femur for 4 weeks. Specific groups were also injected with RAW 264.7 as the reporter macrophages. Wear particle-induced bone loss and the effects of 7ND were evaluated by microCT, immunohistochemical staining, and bioluminescence imaging. Local delivery of 7ND using the LBL coating decreased systemic macrophage recruitment, the number of osteoclasts and wear particle-induced bone loss. The development of a novel orthopaedic implant coating with anti-CCL2 protein may be a promising strategy to mitigate peri-prosthetic osteolysis. PMID:27918885

Antibody-Induced Internalization of the Human Respiratory Syncytial Virus Fusion Protein.

PubMed

Leemans, A; De Schryver, M; Van der Gucht, W; Heykers, A; Pintelon, I; Hotard, A L; Moore, M L; Melero, J A; McLellan, J S; Graham, B S; Broadbent, L; Power, U F; Caljon, G; Cos, P; Maes, L; Delputte, P

2017-07-15

Respiratory syncytial virus (RSV) infections remain a major cause of respiratory disease and hospitalizations among infants. Infection recurs frequently and establishes a weak and short-lived immunity. To date, RSV immunoprophylaxis and vaccine research is mainly focused on the RSV fusion (F) protein, but a vaccine remains elusive. The RSV F protein is a highly conserved surface glycoprotein and is the main target of neutralizing antibodies induced by natural infection. Here, we analyzed an internalization process of antigen-antibody complexes after binding of RSV-specific antibodies to RSV antigens expressed on the surface of infected cells. The RSV F protein and attachment (G) protein were found to be internalized in both infected and transfected cells after the addition of either RSV-specific polyclonal antibodies (PAbs) or RSV glycoprotein-specific monoclonal antibodies (MAbs), as determined by indirect immunofluorescence staining and flow-cytometric analysis. Internalization experiments with different cell lines, well-differentiated primary bronchial epithelial cells (WD-PBECs), and RSV isolates suggest that antibody internalization can be considered a general feature of RSV. More specifically for RSV F, the mechanism of internalization was shown to be clathrin dependent. All RSV F-targeted MAbs tested, regardless of their epitopes, induced internalization of RSV F. No differences could be observed between the different MAbs, indicating that RSV F internalization was epitope independent. Since this process can be either antiviral, by affecting virus assembly and production, or beneficial for the virus, by limiting the efficacy of antibodies and effector mechanism, further research is required to determine the extent to which this occurs in vivo and how this might impact RSV replication. IMPORTANCE Current research into the development of new immunoprophylaxis and vaccines is mainly focused on the RSV F protein since, among others, RSV F-specific antibodies are able to protect infants from severe disease, if administered prophylactically. However, antibody responses established after natural RSV infections are poorly protective against reinfection, and high levels of antibodies do not always correlate with protection. Therefore, RSV might be capable of interfering, at least partially, with antibody-induced neutralization. In this study, a process through which surface-expressed RSV F proteins are internalized after interaction with RSV-specific antibodies is described. One the one hand, this antigen-antibody complex internalization could result in an antiviral effect, since it may interfere with virus particle formation and virus production. On the other hand, this mechanism may also reduce the efficacy of antibody-mediated effector mechanisms toward infected cells. Copyright © 2017 American Society for Microbiology.

Antibody-Induced Internalization of the Human Respiratory Syncytial Virus Fusion Protein

PubMed Central

Leemans, A.; De Schryver, M.; Van der Gucht, W.; Heykers, A.; Pintelon, I.; Hotard, A. L.; Moore, M. L.; Melero, J. A.; McLellan, J. S.; Graham, B. S.; Broadbent, L.; Power, U. F.; Caljon, G.; Cos, P.; Maes, L.

2017-01-01

ABSTRACT Respiratory syncytial virus (RSV) infections remain a major cause of respiratory disease and hospitalizations among infants. Infection recurs frequently and establishes a weak and short-lived immunity. To date, RSV immunoprophylaxis and vaccine research is mainly focused on the RSV fusion (F) protein, but a vaccine remains elusive. The RSV F protein is a highly conserved surface glycoprotein and is the main target of neutralizing antibodies induced by natural infection. Here, we analyzed an internalization process of antigen-antibody complexes after binding of RSV-specific antibodies to RSV antigens expressed on the surface of infected cells. The RSV F protein and attachment (G) protein were found to be internalized in both infected and transfected cells after the addition of either RSV-specific polyclonal antibodies (PAbs) or RSV glycoprotein-specific monoclonal antibodies (MAbs), as determined by indirect immunofluorescence staining and flow-cytometric analysis. Internalization experiments with different cell lines, well-differentiated primary bronchial epithelial cells (WD-PBECs), and RSV isolates suggest that antibody internalization can be considered a general feature of RSV. More specifically for RSV F, the mechanism of internalization was shown to be clathrin dependent. All RSV F-targeted MAbs tested, regardless of their epitopes, induced internalization of RSV F. No differences could be observed between the different MAbs, indicating that RSV F internalization was epitope independent. Since this process can be either antiviral, by affecting virus assembly and production, or beneficial for the virus, by limiting the efficacy of antibodies and effector mechanism, further research is required to determine the extent to which this occurs in vivo and how this might impact RSV replication. IMPORTANCE Current research into the development of new immunoprophylaxis and vaccines is mainly focused on the RSV F protein since, among others, RSV F-specific antibodies are able to protect infants from severe disease, if administered prophylactically. However, antibody responses established after natural RSV infections are poorly protective against reinfection, and high levels of antibodies do not always correlate with protection. Therefore, RSV might be capable of interfering, at least partially, with antibody-induced neutralization. In this study, a process through which surface-expressed RSV F proteins are internalized after interaction with RSV-specific antibodies is described. One the one hand, this antigen-antibody complex internalization could result in an antiviral effect, since it may interfere with virus particle formation and virus production. On the other hand, this mechanism may also reduce the efficacy of antibody-mediated effector mechanisms toward infected cells. PMID:28468888

Protein adsorption and excipient effects on kinetic stability of silicone oil emulsions.

PubMed

Ludwig, D Brett; Carpenter, John F; Hamel, Jean-Bernard; Randolph, Theodore W

2010-04-01

The effect of silicone oil on the stability of therapeutic protein formulations is of concern in the biopharmaceutical industry as more proteins are stored and delivered in prefilled syringes. Prefilled syringes provide convenience for medical professionals and patients, but prolonged exposure of proteins to silicone oil within prefilled syringes may be problematic. In this study, we characterize systems of silicone oil-in-aqueous buffer emulsions and model proteins in formulations containing surfactant, sodium chloride, or sucrose. For each of the formulations studied, silicone oil-induced loss of soluble protein, likely through protein adsorption onto the silicone oil droplet surface. Excipient addition affected both protein adsorption and emulsion stability. Addition of surfactant stabilized emulsions but decreased protein adsorption to silicone oil microdroplets. In contrast, addition of sodium chloride increased protein adsorption and decreased emulsion stability. Silicone oil droplets with adsorbed lysozyme rapidly agglomerated and creamed out of suspension. This decrease in the kinetic stability of the emulsion is ascribed to surface charge neutralization and a bridging flocculation phenomenon and illustrates the need to investigate not only the effects of silicone oil on protein stability, but also the effects of protein formulation variables on emulsion stability. 2009 Wiley-Liss, Inc. and the American Pharmacists Association

Identification of immunoreactive extracellular proteins of Streptococcus agalactiae in bovine mastitis.

PubMed

Trigo, Gabriela; Ferreira, Paula; Ribeiro, Niza; Dinis, Márcia; Andrade, Elva Bonifácio; Melo-Cristino, José; Ramirez, Mário; Tavares, Delfina

2008-11-01

Streptococcus agalactiae is a common pathogen that causes bovine mastitis. The aims of this study were to evaluate the antibody response against S. agalactiae extracellular proteins in the whey and serum of naturally infected bovines and to identify possible immunodominant extracellular antigens. IgG1 antibodies against S. agalactiae extracellular proteins were elevated in the whey and serum of naturally infected bovines. In the whey, the levels of IgG1 specific for S. agalactiae extracellular proteins were similar in infected and noninfected milk quarters from the same cow, and the production of antibodies specific for S. agalactiae extracellular proteins was induced only by infection with this bacterium. The immunoreactivity of extracellular proteins with bovine whey was clearly different in infected versus control animals. Group B protective surface protein and 5'-nucleotidase family protein were 2 major immunoreactive proteins that were detected only in the whey of infected cows, suggesting that these proteins may be important in the pathogenesis of S. agalactiae-induced mastitis. This information could be used to diagnose S. agalactiae infection. In addition, these antigens may be useful as carrier proteins for serotype-specific polysaccharides in conjugate vaccines.

Temperature-Induced Protein Secretion by Leishmania mexicana Modulates Macrophage Signalling and Function

PubMed Central

Hassani, Kasra; Antoniak, Elisabeth; Jardim, Armando; Olivier, Martin

2011-01-01

Protozoan parasites of genus Leishmania are the causative agents of leishmaniasis. These digenetic microorganisms undergo a marked environmental temperature shift (TS) during transmission from the sandfly vector (ambient temperature, 25–26°C) to the mammalian host (37°C). We have observed that this TS induces a rapid and dramatic increase in protein release from Leishmania mexicana (cutaneous leishmaniasis) within 4 h. Proteomic identification of the TS-induced secreted proteins revealed 72 proteins, the majority of which lack a signal peptide and are thus thought to be secreted via nonconventional mechanisms. Interestingly, this protein release is accompanied by alterations in parasite morphology including an augmentation in the budding of exovesicles from its surface. Here we show that the exoproteome of L. mexicana upon TS induces cleavage and activation of the host protein tyrosine phosphatases, specifically SHP-1 and PTP1-B, in a murine bone-marrow-derived macrophage cell line. Furthermore, translocation of prominent inflammatory transcription factors, namely NF-κB and AP-1 is altered. The exoproteome also caused inhibition of nitric oxide production, a crucial leishmanicidal function of the macrophage. Overall, our results provide strong evidence that within early moments of interaction with the mammalian host, L. mexicana rapidly releases proteins and exovesicles that modulate signalling and function of the macrophage. These modulations can result in attenuation of the inflammatory response and deactivation of the macrophage aiding the parasite in the establishment of infection. PMID:21559274

TRANSCRIPTIONAL UPREGULATION OF α2δ-1 ELEVATES ARTERIAL SMOOTH MUSCLE CELL CAV1.2 CHANNEL SURFACE EXPRESSION AND CEREBROVASCULAR CONSTRICTION IN GENETIC HYPERTENSION

PubMed Central

Bannister, John P.; Bulley, Simon; Narayanan, Damodaran; Thomas-Gatewood, Candice; Luzny, Patrik; Pachuau, Judith; Jaggar, Jonathan H.

2012-01-01

A hallmark of hypertension is an increase in arterial myocyte voltage-dependent Ca2+ (CaV1.2) currents that induces pathological vasoconstriction. CaV1.2 channels are heteromeric complexes comprising a pore forming CaV1.2α1 with auxiliary α2δ and β subunits. Molecular mechanisms that elevate CaV1.2 currents during hypertension and the potential contribution of CaV1.2 auxiliary subunits are unclear. Here, we investigated the pathological significance of α2δ subunits in vasoconstriction associated with hypertension. Age-dependent development of hypertension in spontaneously hypertensive rats (SHR) was associated with an unequal elevation in α2δ-1 and CaV1.2α1 mRNA and protein in cerebral artery myocytes, with α2δ-1 increasing more than CaV1.2α1. Other α2δ isoforms did not emerge in hypertension. Myocytes and arteries of hypertensive SHR displayed higher surface-localized α2δ-1 and CaV1.2α1 proteins, surface α2δ-1 to CaV1.2α1 ratio (α2δ-1:CaV1.2α1), CaV1.2 current-density and non-inactivating current, and pressure- and - depolarization-induced vasoconstriction than those of Wistar-Kyoto controls. Pregabalin, an α2δ-1 ligand, did not alter α2δ-1 or CaV1.2α1 total protein, but normalized α2δ-1 and CaV1.2α1 surface expression, surface α2δ-1:CaV1.2α1, CaV1.2 current-density and inactivation, and vasoconstriction in myocytes and arteries of hypertensive rats to control levels. Genetic hypertension is associated with an elevation in α2δ-1 expression that promotes surface trafficking of CaV1.2 channels in cerebral artery myocytes. This leads to an increase in CaV1.2 current-density and a reduction in current inactivation that induces vasoconstriction. Data also suggest that α2δ-1 targeting is a novel strategy that may be used to reverse pathological CaV1.2 channel trafficking to induce cerebrovascular dilation in hypertension. PMID:22949532

Transcriptional upregulation of α2δ-1 elevates arterial smooth muscle cell voltage-dependent Ca2+ channel surface expression and cerebrovascular constriction in genetic hypertension.

PubMed

Bannister, John P; Bulley, Simon; Narayanan, Damodaran; Thomas-Gatewood, Candice; Luzny, Patrik; Pachuau, Judith; Jaggar, Jonathan H

2012-10-01

A hallmark of hypertension is an increase in arterial myocyte voltage-dependent Ca2+ (CaV1.2) currents that induces pathological vasoconstriction. CaV1.2 channels are heteromeric complexes composed of a pore-forming CaV1.2α1 with auxiliary α2δ and β subunits. Molecular mechanisms that elevate CaV1.2 currents during hypertension and the potential contribution of CaV1.2 auxiliary subunits are unclear. Here, we investigated the pathological significance of α2δ subunits in vasoconstriction associated with hypertension. Age-dependent development of hypertension in spontaneously hypertensive rats was associated with an unequal elevation in α2δ-1 and CaV1.2α1 mRNA and protein in cerebral artery myocytes, with α2δ-1 increasing more than CaV1.2α1. Other α2δ isoforms did not emerge in hypertension. Myocytes and arteries of hypertensive spontaneously hypertensive rats displayed higher surface-localized α2δ-1 and CaV1.2α1 proteins, surface α2δ-1:CaV1.2α1 ratio, CaV1.2 current density and noninactivating current, and pressure- and depolarization-induced vasoconstriction than those of Wistar-Kyoto controls. Pregabalin, an α2δ-1 ligand, did not alter α2δ-1 or CaV1.2α1 total protein but normalized α2δ-1 and CaV1.2α1 surface expression, surface α2δ-1:CaV1.2α1, CaV1.2 current density and inactivation, and vasoconstriction in myocytes and arteries of hypertensive rats to control levels. Genetic hypertension is associated with an elevation in α2δ-1 expression that promotes surface trafficking of CaV1.2 channels in cerebral artery myocytes. This leads to an increase in CaV1.2 current-density and a reduction in current inactivation that induces vasoconstriction. Data also suggest that α2δ-1 targeting is a novel strategy that may be used to reverse pathological CaV1.2 channel trafficking to induce cerebrovascular dilation in hypertension.

Dynamic application of microprojection arrays to skin induces circulating protein extravasation for enhanced biomarker capture and detection.

PubMed

Coffey, Jacob W; Meliga, Stefano C; Corrie, Simon R; Kendall, Mark A F

2016-04-01

Surface modified microprojection arrays are a needle-free alternative to capture circulating biomarkers from the skin in vivo for diagnosis. The concentration and turnover of biomarkers in the interstitial fluid, however, may limit the amount of biomarker that can be accessed by microprojection arrays and ultimately their capture efficiency. Here we report that microprojection array insertion induces protein extravasation from blood vessels and increases the concentration of biomarkers in skin, which can synergistically improve biomarker capture. Regions of blood vessels in skin were identified in the upper dermis and subcutaneous tissue by multi-photon microscopy. Insertion of microprojection array designs with varying projection length (40-190 μm), density (5000-20,408 proj.cm(-2)) and array size (4-36 mm(2)) did not affect the degree of extravasation. Furthermore, the location of extravasated protein did not correlate with projection penetration to these highly vascularised regions, suggesting extravasation was not caused by direct puncture of blood vessels. Biomarker extravasation was also induced by dynamic application of flat control surfaces, and varied with the impact velocity, further supporting this conclusion. The extravasated protein distribution correlated well with regions of high mechanical stress generated during insertion, quantified by finite element models. Using this approach to induce extravasation prior to microprojection array-based biomarker capture, anti-influenza IgG was captured within a 2 min application time, demonstrating that extravasation can lead to rapid biomarker sampling and significantly improved microprojection array capture efficiency. These results have broad implications for the development of transdermal devices that deliver to and sample from the skin. Crown Copyright © 2016. Published by Elsevier Ltd. All rights reserved.

Mutations in Protein-Binding Hot-Spots on the Hub Protein Smad3 Differentially Affect Its Protein Interactions and Smad3-Regulated Gene Expression

PubMed Central

Schiro, Michelle M.; Stauber, Sara E.; Peterson, Tami L.; Krueger, Chateen; Darnell, Steven J.; Satyshur, Kenneth A.; Drinkwater, Norman R.; Newton, Michael A.; Hoffmann, F. Michael

2011-01-01

Background Hub proteins are connected through binding interactions to many other proteins. Smad3, a mediator of signal transduction induced by transforming growth factor beta (TGF-β), serves as a hub protein for over 50 protein-protein interactions. Different cellular responses mediated by Smad3 are the product of cell-type and context dependent Smad3-nucleated protein complexes acting in concert. Our hypothesis is that perturbation of this spectrum of protein complexes by mutation of single protein-binding hot-spots on Smad3 will have distinct consequences on Smad3-mediated responses. Methodology/Principal Findings We mutated 28 amino acids on the surface of the Smad3 MH2 domain and identified 22 Smad3 variants with reduced binding to subsets of 17 Smad3-binding proteins including Smad4, SARA, Ski, Smurf2 and SIP1. Mutations defective in binding to Smad4, e.g., D408H, or defective in nucleocytoplasmic shuttling, e.g., W406A, were compromised in modulating the expression levels of a Smad3-dependent reporter gene or six endogenous Smad3-responsive genes: Mmp9, IL11, Tnfaip6, Fermt1, Olfm2 and Wnt11. However, the Smad3 mutants Y226A, Y297A, W326A, K341A, and E267A had distinct differences on TGF-β signaling. For example, K341A and Y226A both reduced the Smad3-mediated activation of the reporter gene by ∼50% but K341A only reduced the TGF-β inducibilty of Olfm2 in contrast to Y226A which reduced the TGF-β inducibility of all six endogenous genes as severely as the W406A mutation. E267A had increased protein binding but reduced TGF-β inducibility because it caused higher basal levels of expression. Y297A had increased TGF-β inducibility because it caused lower Smad3-induced basal levels of gene expression. Conclusions/Significance Mutations in protein binding hot-spots on Smad3 reduced the binding to different subsets of interacting proteins and caused a range of quantitative changes in the expression of genes induced by Smad3. This approach should be useful for unraveling which Smad3 protein complexes are critical for specific biological responses. PMID:21949838

Mutations in protein-binding hot-spots on the hub protein Smad3 differentially affect its protein interactions and Smad3-regulated gene expression.

PubMed

Schiro, Michelle M; Stauber, Sara E; Peterson, Tami L; Krueger, Chateen; Darnell, Steven J; Satyshur, Kenneth A; Drinkwater, Norman R; Newton, Michael A; Hoffmann, F Michael

2011-01-01

Hub proteins are connected through binding interactions to many other proteins. Smad3, a mediator of signal transduction induced by transforming growth factor beta (TGF-β), serves as a hub protein for over 50 protein-protein interactions. Different cellular responses mediated by Smad3 are the product of cell-type and context dependent Smad3-nucleated protein complexes acting in concert. Our hypothesis is that perturbation of this spectrum of protein complexes by mutation of single protein-binding hot-spots on Smad3 will have distinct consequences on Smad3-mediated responses. We mutated 28 amino acids on the surface of the Smad3 MH2 domain and identified 22 Smad3 variants with reduced binding to subsets of 17 Smad3-binding proteins including Smad4, SARA, Ski, Smurf2 and SIP1. Mutations defective in binding to Smad4, e.g., D408H, or defective in nucleocytoplasmic shuttling, e.g., W406A, were compromised in modulating the expression levels of a Smad3-dependent reporter gene or six endogenous Smad3-responsive genes: Mmp9, IL11, Tnfaip6, Fermt1, Olfm2 and Wnt11. However, the Smad3 mutants Y226A, Y297A, W326A, K341A, and E267A had distinct differences on TGF-β signaling. For example, K341A and Y226A both reduced the Smad3-mediated activation of the reporter gene by ∼50% but K341A only reduced the TGF-β inducibilty of Olfm2 in contrast to Y226A which reduced the TGF-β inducibility of all six endogenous genes as severely as the W406A mutation. E267A had increased protein binding but reduced TGF-β inducibility because it caused higher basal levels of expression. Y297A had increased TGF-β inducibility because it caused lower Smad3-induced basal levels of gene expression. Mutations in protein binding hot-spots on Smad3 reduced the binding to different subsets of interacting proteins and caused a range of quantitative changes in the expression of genes induced by Smad3. This approach should be useful for unraveling which Smad3 protein complexes are critical for specific biological responses.

Protective effect of surface layer proteins isolated from four Lactobacillus strains on hydrogen-peroxide-induced HT-29 cells oxidative stress.

PubMed

Zhao, Bin-Bin; Meng, Jun; Zhang, Qiu-Xiang; Kang, Ting-Ting; Lu, Rong-Rong

2017-09-01

The objective of this study was to explore the antioxidant effect of the surface layer proteins (SLPs) and their mechanism. We investigated four SLPs which were extracted from L. casei zhang, L. rhamnosus, L. gasseri and L. acidophilus NCFM respectively using LiCl. The protective effect of SLPs on H 2 O 2 -induced HT-29 cells oxidative injury was investigated. As results, SLPs (100μg/mL) could significantly mitigate HT-29 cells cytotoxicity, improve the activities of total antioxidant capacity (T-AOC), catalase (CAT) and superoxide dismutase (SOD), decrease the contents of malondialdehyde (MDA) and lactate dehydrogenase (LDH), compared with H 2 O 2 -induced group (P<0.05). Furthermore, SLPs were also shown to attenuate the apoptosis rate (10.94-24.03%, P<0.01), suppress the elevation of intracellular reactive oxygen species (ROS) and calcium levels, restore mitochondrial membrane potential (MMP) and block the activation of apoptosis-related proteins of caspase-3 and caspase-9 (P<0.05). Considering all the parameters analyzed, we concluded that Lactobacillus SLPs play an essential role in the antioxidant capacity of HT-29 cells induced by H 2 O 2 , and the mechanism could be attributed to SLPs' ability to enhance the activity of the intracellular antioxidant enzyme system, reduce ROS accumulation and to inhibit apoptosis by regulating mitochondrial pathway. Copyright © 2017 Elsevier B.V. All rights reserved.

Reorganization of lipid domain distribution in giant unilamellar vesicles upon immobilization with different membrane tethers.

PubMed

Sarmento, M J; Prieto, M; Fernandes, Fábio

2012-11-01

Characterization of phase coexistence in biologically relevant lipid mixtures is often carried out through confocal microscopy of giant unilamellar lipid vesicles (GUVs), loaded with fluorescent membrane probes. This last analysis is generally limited to the vesicle hemisphere further away from the coverslip, in order to avoid artifacts induced by the interaction with the solid surface, and immobilization of vesicles is in many cases required in order to carry out intensity, lifetime or single-molecule based microscopy. This is generally achieved through the use of membrane tethers adhering to a coverslip surface. Here, we aimed to determine whether GUV immobilization through membrane tethers induces changes in lipid domain distribution within liposomes displaying coexistence of lipid lamellar phases. Confocal imaging and a Förster resonance energy transfer (FRET) methodology showed that biotinylated phospholipids present significantly different membrane phase partition behavior upon protein binding, depending on the presence or absence of a linker between the lipid headgroup and the biotinyl moiety. Membrane phases enriched in a membrane tether displayed in some cases a dramatically increased affinity for the immobilization surface, effectively driving sorting of lipid domains to the adherent membrane area, and in some cases complete sequestering of a lipid phase to the interaction surface was observed. On the light of these results, we conclude that tethering of lipid membranes to protein surfaces has the potential to drastically reorganize the distribution of lipid domains, and this reorganization is solely dictated by the partition properties of the protein-tether complex. Copyright © 2012 Elsevier B.V. All rights reserved.

Prion protein induced signaling cascades in monocytes

DOE Office of Scientific and Technical Information (OSTI.GOV)

Krebs, Bjarne; Dorner-Ciossek, Cornelia; Schmalzbauer, Ruediger

2006-02-03

Prion proteins play a central role in transmission and pathogenesis of transmissible spongiform encephalopathies. The cellular prion protein (PrP{sup C}), whose physiological function remains elusive, is anchored to the surface of a variety of cell types including neurons and cells of the lymphoreticular system. In this study, we investigated the response of a mouse monocyte/macrophage cell line to exposure with PrP{sup C} fusion proteins synthesized with a human Fc-tag. PrP{sup C} fusion proteins showed an attachment to the surface of monocyte/macrophages in nanomolar concentrations. This was accompanied by an increase of cellular tyrosine phosphorylation as a result of activated signalingmore » pathways. Detailed investigations exhibited activation of downstream pathways through a stimulation with PrP fusion proteins, which include phosphorylation of ERK{sub 1,2} and Akt kinase. Macrophages opsonize and present antigenic structures, contact lymphocytes, and deliver cytokines. The findings reported here may become the basis of understanding the molecular function of PrP{sup C} in monocytes and macrophages.« less

Photo-induced formation of nitrous acid (HONO) on protein surfaces

NASA Astrophysics Data System (ADS)

Meusel, Hannah; Elshorbany, Yasin; Bartels-Rausch, Thorsten; Selzle, Kathrin; Lelieveld, Jos; Ammann, Markus; Pöschl, Ulrich; Su, Hang; Cheng, Yafang

2014-05-01

The study of nitrous acid (HONO) is of great interest, as the photolysis of HONO leads to the OH radical, which is the most important oxidant in the troposphere. HONO is directly emitted by combustion of fossil fuel and from soil biogenic nitrite (Su et al., 2011), and can also be formed by gas phase reactions of NO and OH and heterogeneous reactions of NO2. Previous atmospheric measurements have shown unexpectedly high HONO concentrations during daytime. Measured mixing ratios were about one order of magnitude higher than model simulations (Kleffmann et al. 2005, Vogel et al. 2003). The additional daytime source of HONO might be attributed to the photolysis of adsorbed nitric acid or heterogeneous photochemistry of NO2 on organic substrates, such as humic acids or polyphenolic compounds (Stemmler et al., 2006), or indirectly through nitration of phenols and subsequent photolysis of nitrophenols (Sosedova et al., 2011, Bejan et al., 2006). An important reactive surface for the heterogeneous formation of HONO could involve proteins, which are ubiquitous in the environment. They are part of coarse biological aerosol particles like pollen grains, fine particles (fragments of pollen, microorganism, plant debris) and dissolved in rainwater, soil and road dust (Miguel et al. 1999). In this project a thin film of bovine serum albumin (BSA), a model protein with 67 kDa and 21 tyrosine residues per molecule, is irradiated and exposed to nitrogen dioxide in humidified nitrogen. The formation of HONO is measured with long path absorption photometry (LOPAP). The generated HONO is in the range of 100 to 1100 ppt depending on light intensity, NO2 concentration and film thickness. Light induced HONO formation on protein surfaces is stable over the 20-hours experiment of irradiation and exposure. On the other hand, light activated proteins reacting with NO2 form nitrated proteins, as detected by liquid chromatography (LC-DAD). Our experiments on tetranitromethane (TNM) nitrated ovalbumin (OVA) also show a clear light induced decomposition of nitrated proteins with HONO identified as one of the major products. This suggests a shortening of the lifetime of nitrated proteins during daytime. Our results indicate an important role of light to the fate of proteins, and through HONO, important OH precursors. Proteins and nitrated proteins on aerosol and ground surfaces may therefore influence the atmospheric chemistry and contribute to the oxidation capacity. References Bejan, I. et al., Physical Chemistry Chemical Physics 2006, 8 (17), 2028-2035. Kleffmann, J. et al., Geophysical Research Letters 2005, 32 (5). Miguel, A. G. et al., Environmental Science & Technology 1999, 33 (23), 4159-4168. Sosedova, Y., et al., Photochemical and Photobiological Sciences 2011, 10, 1680-1690. Stemmler, K. et al., Nature 2006, 440 (7081), 195-198. Su et al., Science 2010, 333, 1616-1618. Vogel, B. et al., Atmospheric Environment 2003, 37 (21), 2957-2966.

Function of Platelet-Induced Epithelial Attachment at Titanium Surfaces Inhibits Microbial Colonization.

PubMed

Maeno, M; Lee, C; Kim, D M; Da Silva, J; Nagai, S; Sugawara, S; Nara, Y; Kihara, H; Nagai, M

2017-06-01

The aim of this study was to evaluate the barrier function of platelet-induced epithelial sheets on titanium surfaces. The lack of functional peri-implant epithelial sealing with basal lamina (BL) attachment at the interface of the implant and the adjacent epithelium allows for bacterial invasion, which may lead to peri-implantitis. Although various approaches have been reported to combat bacterial infection by surface modifications to titanium, none of these have been successful in a clinical application. In our previous study, surface modification with protease-activated receptor 4-activating peptide (PAR4-AP), which induced platelet activation and aggregation, was successful in demonstrating epithelial attachment via BL and epithelial sheet formation on the titanium surface. We hypothesized that the platelet-induced epithelial sheet on PAR4-AP-modified titanium surfaces would reduce bacterial attachment, penetration, and invasion. Titanium surface was modified with PAR4-AP and incubated with platelet-rich plasma (PRP). The aggregated platelets released collagen IV, a critical BL component, onto the PAR4-AP-modified titanium surface. Then, human gingival epithelial cells were seeded on the modified titanium surface and formed epithelial sheets. Green fluorescent protein (GFP)-expressing Escherichia coli was cultured onto PAR4-AP-modified titanium with and without epithelial sheet formation. While Escherichia coli accumulated densely onto the PAR4-AP titanium lacking epithelial sheet, few Escherichia coli were observed on the epithelial sheet on the PAR4-AP surface. No bacterial invasion into the interface of the epithelial sheet and the titanium surface was observed. These in vitro results indicate the efficacy of a platelet-induced epithelial barrier that functions to prevent bacterial attachment, penetration, and invasion on PAR4-AP-modified titanium.

Antigenic characterization of bovine ephemeral fever rhabdovirus G and GNS glycoproteins expressed from recombinant baculoviruses.

PubMed

Johal, Jasjit; Gresty, Karryn; Kongsuwan, Kritaya; Walker, Peter J

2008-01-01

Recombinant baculoviruses expressing the BEFV envelope glycoprotein G and non-structural glycoprotein G(NS) were constructed. The G protein expressed in insect cells was located on the cell surface and induced spontaneous cell fusion at mildly acidic pH. The expressed G protein reacted with MAbs to continuous and conformational neutralization sites (G1, G2, G3b and G4), but not to conformational site G3a. The expressed G(NS) protein was also located on the cell surface but did not exhibit fusogenic activity. The G(NS) protein reacted with polyclonal antiserum produced from vaccinia-virus-expressed recombinant G(NS) but did not react with G protein antibodies. A His(6)-tagged, soluble form of the G protein was expressed and purified by Ni(2+)-NTA chromatography. The purified G protein reacted with BEFV-neutralizing MAbs to all continuous and conformational antigenic sites. The highly protective characteristics of the native BEFV G protein suggest that the secreted, baculovirus-expressed product may be a useful vaccine antigen.

Protein Corona Prevents TiO2 Phototoxicity.

PubMed

Garvas, Maja; Testen, Anze; Umek, Polona; Gloter, Alexandre; Koklic, Tilen; Strancar, Janez

2015-01-01

TiO2 nanoparticles have generally low toxicity in the in vitro systems although some toxicity is expected to originate in the TiO2-associated photo-generated radical production, which can however be modulated by the radical trapping ability of the serum proteins. To explore the role of serum proteins in the phototoxicity of the TiO2 nanoparticles we measure viability of the exposed cells depending on the nanoparticle and serum protein concentrations. Fluorescence and spin trapping EPR spectroscopy reveal that the ratio between the nanoparticle and protein concentrations determines the amount of the nanoparticles' surface which is not covered by the serum proteins and is proportional to the amount of photo-induced radicals. Phototoxicity thus becomes substantial only at the protein concentration being too low to completely coat the nanotubes' surface. These results imply that TiO2 nanoparticles should be applied with ligands such as proteins when phototoxic effects are not desired - for example in cosmetics industry. On the other hand, the nanoparticles should be used in serum free medium or any other ligand free medium, when phototoxic effects are desired - as for efficient photodynamic cancer therapy.

Type IV pili mechanochemically regulate virulence factors in Pseudomonas aeruginosa.

PubMed

Persat, Alexandre; Inclan, Yuki F; Engel, Joanne N; Stone, Howard A; Gitai, Zemer

2015-06-16

Bacteria have evolved a wide range of sensing systems to appropriately respond to environmental signals. Here we demonstrate that the opportunistic pathogen Pseudomonas aeruginosa detects contact with surfaces on short timescales using the mechanical activity of its type IV pili, a major surface adhesin. This signal transduction mechanism requires attachment of type IV pili to a solid surface, followed by pilus retraction and signal transduction through the Chp chemosensory system, a chemotaxis-like sensory system that regulates cAMP production and transcription of hundreds of genes, including key virulence factors. Like other chemotaxis pathways, pili-mediated surface sensing results in a transient response amplified by a positive feedback that increases type IV pili activity, thereby promoting long-term surface attachment that can stimulate additional virulence and biofilm-inducing pathways. The methyl-accepting chemotaxis protein-like chemosensor PilJ directly interacts with the major pilin subunit PilA. Our results thus support a mechanochemical model where a chemosensory system measures the mechanically induced conformational changes in stretched type IV pili. These findings demonstrate that P. aeruginosa not only uses type IV pili for surface-specific twitching motility, but also as a sensor regulating surface-induced gene expression and pathogenicity.

Cell surface control of the multiubiquitination and deubiquitination of high-affinity immunoglobulin E receptors.

PubMed Central

Paolini, R; Kinet, J P

1993-01-01

Multiubiquitination of proteins is a critical step leading to selective degradation for many polypeptides. Therefore, activation-induced multiubiquitination of cell surface receptors, such as the platelet-derived growth factor (PDGF) receptor and the T cell antigen (TCR) receptor, may correspond to a degradation pathway for ligand-receptor complexes. Here we show that the antigen-induced engagement of high-affinity immunoglobulin E receptors (Fc epsilon RI) results in the immediate multiubiquitination of Fc epsilon RI beta and gamma chains. This ubiquitination is independent of receptor phosphorylation and is restricted to activated receptors. Surprisingly, receptor multiubiquitination is immediately reversible when receptors are disengaged. Therefore, multiubiquitination and deubiquitination of Fc epsilon RI receptors is controlled at the cell surface by receptor engagement and disengagement. The rapidity, specificity and, most importantly, the reversibility of the activation-induced receptor multiubiquitination suggest that this process may turn on/off a cell surface receptor signaling function thus far unsuspected. Images PMID:8382611

Directed assembly of binary monolayers with a high protein affinity: infrared reflection absorption spectroscopy (IRRAS) and surface plasmon resonance (SPR).

PubMed

Du, Xuezhong; Wang, Yuchun

2007-03-08

Infrared reflection absorption spectroscopy (IRRAS) and surface plasmon resonance (SPR) techniques have been employed to investigate human serum albumin (HSA) binding to binary monolayers of zwitterionic dipalmitoylphosphatidylcholine (DPPC) and cationic dioctadecyldimethylammonium bromide (DOMA). At the air-water interface, the favorable electrostatic interaction between DPPC and DOMA leads to a dense chain packing. The tilt angle of the hydrocarbon chains decreases with increasing mole fraction of DOMA (X(DOMA)) in the monolayers at the surface pressure 30 mN/m: DPPC ( approximately 30 degrees ), X(DOMA) = 0.1 ( approximately 15 degrees ), and X(DOMA) = 0.3 ( approximately 0 degrees ). Negligible protein binding to the DPPC monolayer is observed in contrast to a significant binding to the binary monolayers. After HSA binding, the hydrocarbon chains at X(DOMA) = 0.1 undergo an increase in tilt angle from 15 degrees to 25 approximately 30 degrees , and the chains at X(DOMA) = 0.3 remain almost unchanged. The two components in the monolayers deliver through lateral reorganization, induced by the protein in the subphase, to form multiple interaction sites favorable for protein binding. The surfaces with a high protein affinity are created through the directed assembly of binary monolayers for use in biosensing.

Radio frequency glow discharge-induced acidification of fluoropolymers.

PubMed

Krawczyk, Benjamin M; Baltrusaitis, Jonas; Yoder, Colin M; Vargo, Terrence G; Bowden, Ned B; Kader, Khalid N

2011-12-01

Fluoropolymer surfaces are unique in view of the fact that they are quite inert, have low surface energies, and possess high thermal stabilities. Attempts to modify fluoropolymer surfaces have met with difficulties in that it is difficult to control the modification to maintain bulk characteristics of the polymer. In a previously described method, the replacement of a small fraction of surface fluorine by acid groups through radio frequency glow discharge created a surface with unexpected reactivity allowing for attachment of proteins in their active states. The present study demonstrates that 1-ethyl-3-[3-dimethylaminopropyl] carbodiimide hydrochloride (EDC) reacts with the acid groups on fluoropolymer surfaces in a novel reaction not previously described. This reaction yields an excellent leaving group in which a primary amine on proteins can substitute to form a covalent bond between a protein and these surfaces. In an earlier study, we demonstrated that collagen IV could be deposited on a modified PTFE surface using EDC as a linker. Once collagen IV is attached to the surface, it assembles to form a functional stratum resembling collagen IV in native basement membrane. In this study, we show data suggesting that the fluorine to carbon ratio determines the acidity of the fluoropolymer surfaces and how well collagen IV attaches to and assembles on four different fluoropolymer surfaces. Copyright © 2011 Wiley Periodicals, Inc.

Plasma immersion ion implantation of polyurethane shape memory polymer: Surface properties and protein immobilization

NASA Astrophysics Data System (ADS)

Cheng, Xinying; Kondyurin, Alexey; Bao, Shisan; Bilek, Marcela M. M.; Ye, Lin

2017-09-01

Polyurethane-type shape memory polymers (SMPU) are promising biomedical implant materials due to their ability to recover to a predetermined shape from a temporary shape induced by thermal activation close to human body temperature and their advantageous mechanical properties including large recovery strains and low recovery stresses. Plasma Immersion Ion Implantation (PIII) is a surface modification process using energetic ions that generates radicals in polymer surfaces leading to carbonisation and oxidation and the ability to covalently immobilise proteins without the need for wet chemistry. Here we show that PIII treatment of SMPU significantly enhances its bioactivity making SMPU suitable for applications in permanent implantable biomedical devices. Scanning Electron Microscopy (SEM), contact angle measurements, surface energy measurements, attenuated total reflection Fourier transform infrared (ATR-FTIR) spectroscopy and X-ray photoelectron spectroscopy (XPS) were used to characterise the PIII modified surface, including its after treatment aging kinetics and its capability to covalently immobilise protein directly from solution. The results show a substantial improvement in wettability and dramatic changes of surface chemical composition dependent on treatment duration, due to the generation of radicals and subsequent oxidation. The SMPU surface, PIII treated for 200s, achieved a saturated level of covalently immobilized protein indicating that a full monolayer coverage was achieved. We conclude that PIII is a promising and efficient surface modification method to enhance the biocompatibility of SMPU for use in medical applications that demand bioactivity for tissue integration and stability in vivo.

A Novel Staphylococcus aureus Vaccine: Iron Surface Determinant B Induces Rapid Antibody Responses in Rhesus Macaques and Specific Increased Survival in a Murine S. aureus Sepsis Model

PubMed Central

Kuklin, Nelly A.; Clark, Desmond J.; Secore, Susan; Cook, James; Cope, Leslie D.; McNeely, Tessie; Noble, Liliane; Brown, Martha J.; Zorman, Julie K.; Wang, Xin Min; Pancari, Gregory; Fan, Hongxia; Isett, Kevin; Burgess, Bruce; Bryan, Janine; Brownlow, Michelle; George, Hugh; Meinz, Maria; Liddell, Mary E.; Kelly, Rosemarie; Schultz, Loren; Montgomery, Donna; Onishi, Janet; Losada, Maria; Martin, Melissa; Ebert, Timothy; Tan, Charles Y.; Schofield, Timothy L.; Nagy, Eszter; Meineke, Andreas; Joyce, Joseph G.; Kurtz, Myra B.; Caulfield, Michael J.; Jansen, Kathrin U.; McClements, William; Anderson, Annaliesa S.

2006-01-01

Staphylococcus aureus is a major cause of nosocomial infections worldwide, and the rate of resistance to clinically relevant antibiotics, such as methicillin, is increasing; furthermore, there has been an increase in the number of methicillin-resistant S. aureus community-acquired infections. Effective treatment and prevention strategies are urgently needed. We investigated the potential of the S. aureus surface protein iron surface determinant B (IsdB) as a prophylactic vaccine against S. aureus infection. IsdB is an iron-sequestering protein that is conserved in diverse S. aureus clinical isolates, both methicillin resistant and methicillin sensitive, and it is expressed on the surface of all isolates tested. The vaccine was highly immunogenic in mice when it was formulated with amorphous aluminum hydroxyphosphate sulfate adjuvant, and the resulting antibody responses were associated with reproducible and significant protection in animal models of infection. The specificity of the protective immune responses in mice was demonstrated by using an S. aureus strain deficient for IsdB and HarA, a protein with a high level of identity to IsdB. We also demonstrated that IsdB is highly immunogenic in rhesus macaques, inducing a more-than-fivefold increase in antibody titers after a single immunization. Based on the data presented here, IsdB has excellent prospects for use as a vaccine against S. aureus disease in humans. PMID:16552052

A novel Staphylococcus aureus vaccine: iron surface determinant B induces rapid antibody responses in rhesus macaques and specific increased survival in a murine S. aureus sepsis model.

PubMed

Kuklin, Nelly A; Clark, Desmond J; Secore, Susan; Cook, James; Cope, Leslie D; McNeely, Tessie; Noble, Liliane; Brown, Martha J; Zorman, Julie K; Wang, Xin Min; Pancari, Gregory; Fan, Hongxia; Isett, Kevin; Burgess, Bruce; Bryan, Janine; Brownlow, Michelle; George, Hugh; Meinz, Maria; Liddell, Mary E; Kelly, Rosemarie; Schultz, Loren; Montgomery, Donna; Onishi, Janet; Losada, Maria; Martin, Melissa; Ebert, Timothy; Tan, Charles Y; Schofield, Timothy L; Nagy, Eszter; Meineke, Andreas; Joyce, Joseph G; Kurtz, Myra B; Caulfield, Michael J; Jansen, Kathrin U; McClements, William; Anderson, Annaliesa S

2006-04-01

Staphylococcus aureus is a major cause of nosocomial infections worldwide, and the rate of resistance to clinically relevant antibiotics, such as methicillin, is increasing; furthermore, there has been an increase in the number of methicillin-resistant S. aureus community-acquired infections. Effective treatment and prevention strategies are urgently needed. We investigated the potential of the S. aureus surface protein iron surface determinant B (IsdB) as a prophylactic vaccine against S. aureus infection. IsdB is an iron-sequestering protein that is conserved in diverse S. aureus clinical isolates, both methicillin resistant and methicillin sensitive, and it is expressed on the surface of all isolates tested. The vaccine was highly immunogenic in mice when it was formulated with amorphous aluminum hydroxyphosphate sulfate adjuvant, and the resulting antibody responses were associated with reproducible and significant protection in animal models of infection. The specificity of the protective immune responses in mice was demonstrated by using an S. aureus strain deficient for IsdB and HarA, a protein with a high level of identity to IsdB. We also demonstrated that IsdB is highly immunogenic in rhesus macaques, inducing a more-than-fivefold increase in antibody titers after a single immunization. Based on the data presented here, IsdB has excellent prospects for use as a vaccine against S. aureus disease in humans.

Surface properties of heat-induced soluble soy protein aggregates of different molecular masses.

PubMed

Guo, Fengxian; Xiong, Youling L; Qin, Fang; Jian, Huajun; Huang, Xiaolin; Chen, Jie

2015-02-01

Suspensions (2% and 5%, w/v) of soy protein isolate (SPI) were heated at 80, 90, or 100 °C for different time periods to produce soluble aggregates of different molecular sizes to investigate the relationship between particle size and surface properties (emulsions and foams). Soluble aggregates generated in these model systems were characterized by gel permeation chromatography and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Heat treatment increased surface hydrophobicity, induced SPI aggregation via hydrophobic interaction and disulfide bonds, and formed soluble aggregates of different sizes. Heating of 5% SPI always promoted large-size aggregate (LA; >1000 kDa) formation irrespective of temperature, whereas the aggregate size distribution in 2% SPI was temperature dependent: the LA fraction progressively rose with temperature (80→90→100 °C), corresponding to the attenuation of medium-size aggregates (MA; 670 to 1000 kDa) initially abundant at 80 °C. Heated SPI with abundant LA (>50%) promoted foam stability. LA also exhibited excellent emulsifying activity and stabilized emulsions by promoting the formation of small oil droplets covered with a thick interfacial protein layer. However, despite a similar influence on emulsion stability, MA enhanced foaming capacity but were less capable of stabilizing emulsions than LA. The functionality variation between heated SPI samples is clearly related to the distribution of aggregates that differ in molecular size and surface activity. The findings may encourage further research to develop functional SPI aggregates for various commercial applications. © 2015 Institute of Food Technologists®

Cell Surface Translocation of Annexin A2 Facilitates Glutamate-induced Extracellular Proteolysis*

PubMed Central

Valapala, Mallika; Maji, Sayantan; Borejdo, Julian; Vishwanatha, Jamboor K.

2014-01-01

Glutamate-induced elevation in intracellular Ca2+ has been implicated in excitotoxic cell death. Neurons respond to increased glutamate levels by activating an extracellular proteolytic cascade involving the components of the plasmin-plasminogen system. AnxA2 is a Ca2+-dependent phospholipid binding protein and serves as an extracellular proteolytic center by recruiting the tissue plasminogen activator and plasminogen and mediating the localized generation of plasmin. Ratiometric Ca2+ imaging and time-lapse confocal microscopy demonstrated glutamate-induced Ca2+ influx. We showed that glutamate translocated both endogenous and AnxA2-GFP to the cell surface in a process dependent on the activity of the NMDA receptor. Glutamate-induced translocation of AnxA2 is dependent on the phosphorylation of tyrosine 23 at the N terminus, and mutation of tyrosine 23 to a non-phosphomimetic variant inhibits the translocation process. The cell surface-translocated AnxA2 forms an active plasmin-generating complex, and this activity can be neutralized by a hexapeptide directed against the N terminus. These results suggest an involvement of AnxA2 in potentiating glutamate-induced cell death processes. PMID:24742684

Electrodiffusion of lipids on membrane surfaces.

PubMed

Zhou, Y C

2012-05-28

Lateral translocation of lipids and proteins is a universal process on membrane surfaces. Local aggregation or organization of lipids and proteins can be induced when the random lateral motion is mediated by the electrostatic interactions and membrane curvature. Although the lateral diffusion rates of lipids on membranes of various compositions are measured and the electrostatic free energies of predetermined protein-membrane-lipid systems can be computed, the process of the aggregation and the evolution to the electrostatically favorable states remain largely undetermined. Here we propose an electrodiffusion model, based on the variational principle of the free energy functional, for the self-consistent lateral drift-diffusion of multiple species of charged lipids on membrane surfaces. Finite sizes of lipids are modeled to enforce the geometrical constraint of the lipid concentration on membrane surfaces. A surface finite element method is developed to appropriate the Laplace-Beltrami operators in the partial differential equations of the model. Our model properly describes the saturation of lipids on membrane surfaces, and correctly predicts that the MARCKS peptide can consistently sequester three multivalent phosphatidylinositol 4,5-bisphosphate lipids through its basic amino acid residues, regardless of a wide range of the percentage of monovalent phosphatidylserine in the membrane.

Electrodiffusion of lipids on membrane surfaces

NASA Astrophysics Data System (ADS)

Zhou, Y. C.

2012-05-01

Lateral translocation of lipids and proteins is a universal process on membrane surfaces. Local aggregation or organization of lipids and proteins can be induced when the random lateral motion is mediated by the electrostatic interactions and membrane curvature. Although the lateral diffusion rates of lipids on membranes of various compositions are measured and the electrostatic free energies of predetermined protein-membrane-lipid systems can be computed, the process of the aggregation and the evolution to the electrostatically favorable states remain largely undetermined. Here we propose an electrodiffusion model, based on the variational principle of the free energy functional, for the self-consistent lateral drift-diffusion of multiple species of charged lipids on membrane surfaces. Finite sizes of lipids are modeled to enforce the geometrical constraint of the lipid concentration on membrane surfaces. A surface finite element method is developed to appropriate the Laplace-Beltrami operators in the partial differential equations of the model. Our model properly describes the saturation of lipids on membrane surfaces, and correctly predicts that the MARCKS peptide can consistently sequester three multivalent phosphatidylinositol 4,5-bisphosphate lipids through its basic amino acid residues, regardless of a wide range of the percentage of monovalent phosphatidylserine in the membrane.

Determination of the surface charge density and temperature dependence of purple membrane by electric force microscopy.

PubMed

Du, Huiwen; Li, Denghua; Wang, Yibing; Wang, Chenxuan; Zhang, Dongdong; Yang, Yan-lian; Wang, Chen

2013-08-29

We report here the measurement of the temperature-dependent surface charge density of purple membrane (PM) by using electrostatic force microscopy (EFM). The surface charge density was measured to be 3.4 × 10(5) e/cm(2) at room temperature and reaches the minimum at around 52 °C. The initial decrease of the surface charge density could be attributed to the reduced dipole alignment because of the thermally induced protein mobility in PM. The increase of charge density at higher temperature could be ascribed to the weakened interaction between proteins and the lipids, which leads to the exposure of the charged amino acids. This work could be a benefit to the direct assessment of the structural stability and electric properties of biological membranes at the nanoscale.

Neutralization of West Nile virus by cross-linking of its surface proteins with Fab fragments of the human monoclonal antibody CR4354

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kaufmann, Bärbel; Vogt, Matthew R.; Goudsmit, Jaap

2010-11-15

Many flaviviruses are significant human pathogens, with the humoral immune response playing an essential role in restricting infection and disease. CR4354, a human monoclonal antibody isolated from a patient, neutralizes West Nile virus (WNV) infection at a postattachment stage in the viral life-cycle. Here, we determined the structure of WNV complexed with Fab fragments of CR4354 using cryoelectron microscopy. The outer glycoprotein shell of a mature WNV particle is formed by 30 rafts of three homodimers of the viral surface protein E. CR4354 binds to a discontinuous epitope formed by protein segments from two neighboring E molecules, but does notmore » cause any detectable structural disturbance on the viral surface. The epitope occurs at two independent positions within an icosahedral asymmetric unit, resulting in 120 binding sites on the viral surface. The cross-linking of the six E monomers within one raft by four CR4354 Fab fragments suggests that the antibody neutralizes WNV by blocking the pH-induced rearrangement of the E protein required for virus fusion with the endosomal membrane.« less

C-terminal of human histamine H1 receptors regulates their agonist-induced clathrin-mediated internalization and G-protein signaling.

PubMed

Hishinuma, Shigeru; Nozawa, Hiroki; Akatsu, Chizuru; Shoji, Masaru

2016-11-01

It has been suggested that the agonist-induced internalization of G-protein-coupled receptors from the cell surface into intracellular compartments regulates cellular responsiveness. We previously reported that G q/11 -protein-coupled human histamine H 1 receptors internalized via clathrin-dependent mechanisms upon stimulation with histamine. However, the molecular determinants of H 1 receptors responsible for agonist-induced internalization remain unclear. In this study, we evaluated the roles of the intracellular C-terminal of human histamine H 1 receptors tagged with hemagglutinin (HA) at the N-terminal in histamine-induced internalization in Chinese hamster ovary cells. The histamine-induced internalization was evaluated by the receptor binding assay with [ 3 H]mepyramine and confocal immunofluorescence microscopy with an anti-HA antibody. We found that histamine-induced internalization was inhibited under hypertonic conditions or by pitstop, a clathrin terminal domain inhibitor, but not by filipin or nystatin, disruptors of the caveolar structure and function. The histamine-induced internalization was also inhibited by truncation of a single amino acid, Ser487, located at the end of the intracellular C-terminal of H 1 receptors, but not by its mutation to alanine. In contrast, the receptor-G-protein coupling, which was evaluated by histamine-induced accumulation of [ 3 H]inositol phosphates, was potentiated by truncation of Ser487, but was lost by its mutation to alanine. These results suggest that the intracellular C-terminal of human H 1 receptors, which only comprises 17 amino acids (Cys471-Ser487), plays crucial roles in both clathrin-dependent internalization of H 1 receptors and G-protein signaling, in which truncation of Ser487 and its mutation to alanine are revealed to result in biased signaling toward activation of G-proteins and clathrin-mediated internalization, respectively. © 2016 International Society for Neurochemistry.

Sulfatide-Hsp70 Interaction Promotes Hsp70 Clustering and Stabilizes Binding to Unfolded Protein

PubMed Central

Harada, Yoichiro; Sato, Chihiro; Kitajima, Ken

2015-01-01

The 70-kDa heat shock protein (Hsp70), one of the major stress-inducible molecular chaperones, is localized not only in the cytosol, but also in extracellular milieu in mammals. Hsp70 interacts with various cell surface glycolipids including sulfatide (3'-sulfogalactosphingolipid). However, the molecular mechanism, as well as the biological relevance, underlying the glycolipid-Hsp70 interaction is unknown. Here we report that sulfatide promotes Hsp70 oligomerization through the N-terminal ATPase domain, which stabilizes the binding of Hsp70 to unfolded protein in vitro. We find that the Hsp70 oligomer has apparent molecular masses ranging from 440 kDa to greater than 669 kDa. The C-terminal peptide-binding domain is dispensable for the sulfatide-induced oligomer formation. The oligomer formation is impaired in the presence of ATP, while the Hsp70 oligomer, once formed, is unable to bind to ATP. These results suggest that sulfatide locks Hsp70 in a high-affinity state to unfolded proteins by clustering the peptide-binding domain and blocking the binding to ATP that induces the dissociation of Hsp70 from protein substrates. PMID:25989600

Generation of neutralising antibodies against porcine endogenous retroviruses (PERVs)

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kaulitz, Danny; Fiebig, Uwe; Eschricht, Magdalena

2011-03-01

Antibodies neutralising porcine endogenous retroviruses (PERVs) were induced in different animal species by immunisation with the transmembrane envelope protein p15E. These antibodies recognised epitopes, designated E1, in the fusion peptide proximal region (FPPR) of p15E, and E2 in the membrane proximal external region (MPER). E2 is localised in a position similar to that of an epitope in the transmembrane envelope protein gp41 of the human immunodeficiency virus-1 (HIV-1), recognised by the monoclonal antibody 4E10 that is broadly neutralising. To detect neutralising antibodies specific for PERV, a novel assay was developed, which is based on quantification of provirus integration by real-timemore » PCR. In addition, for the first time, highly effective neutralising antibodies were obtained by immunisation with the surface envelope protein of PERV. These data indicate that neutralising antibodies can be induced by immunisation with both envelope proteins.« less

Protein-resistant cross-linked poly(vinyl alcohol) micropatterns via photolithography using removable polyoxometalate photocatalyst.

PubMed

Pavli, Pagona; Petrou, Panagiota S; Douvas, Antonios M; Dimotikali, Dimitra; Kakabakos, Sotirios E; Argitis, Panagiotis

2014-10-22

In the last years, there has been an increasing interest in controlling the protein adsorption properties of surfaces because this control is crucial for the design of biomaterials. On the other hand, controlled immobilization of proteins is also important for their application as solid surfaces in immunodiagnostics and biosensors. Herein we report a new protein patterning method where regions of the substrate are covered by a hydrophilic film that minimizes protein adsorption. Particularly, poly(vinyl alcohol) (PVA) cross-linked structures created by an especially developed photolithographic process are proved to prevent protein physisorption and they are used as a guide for selective protein adsorption on the uncovered areas of a protein adsorbing substrate such as polystyrene. The PVA cross-linking is induced by photo-oxidation using, as a catalyst, polyoxometalate (H3PW12O40 or α-(NH4)6P2W18O62), which is removed using a methyl alcohol/water mixed solvent as the developer. We demonstrate that the polystyrene and the cross-linked PVA exhibit dramatically different performances in terms of protein physisorption. In particular, the polystyrene areas presented up to 130 times higher protein binding capacity than the PVA ones, whereas the patterning resolution could easily reach dimensions of a few micrometers. The proposed approach can be applied on any substrate where PVA films can be coated for controlling protein adsorption onto surface areas custom defined by the user.

Differential expression of cruzipain- and gp63-like molecules in the phytoflagellate trypanosomatid Phytomonas serpens induced by exogenous proteins.

PubMed

Elias, Camila G R; Chagas, Michel G; Souza-Gonçalves, Ana Luiza; Pascarelli, Bernardo M O; d'Avila-Levy, Claudia M; Branquinha, Marta H; Santos, André L S

2012-01-01

Phytomonas serpens synthesizes metallo- and cysteine-proteases that are related to gp63 and cruzipain, respectively, two virulence factors produced by pathogenic trypanosomatids. Here, we described the cellular distribution of gp63- and cruzipain-like molecules in P. serpens through immunocytochemistry and confocal fluorescence microscopy. Both proteases were detected in distinct cellular compartments, presenting co-localization in membrane domains and intracellular regions. Subsequently, we showed that exogenous proteins modulated the production of both protease classes, but in different ways. Regarding the metalloprotease, only fetal bovine serum (FBS) influenced the gp63 expression, reducing its surface exposition (≈30%). Conversely, the cruzipain-like molecule was differentially modulated according to the proteins: human and bovine albumins reduced its expression around 50% and 35%, respectively; mucin and FBS did not alter its production, while IgG and hemoglobin drastically enhanced its surface exposition around 7- and 11-fold, respectively. Additionally, hemoglobin induced an augmentation in the cell-associated cruzipain-like activity in a dose-dependent manner. A twofold increase of the secreted cruzipain-like protein was detected after parasite incubation with 1% hemoglobin compared to the parasites incubated in PBS-glucose. The results showed the ability of P. serpens in modulating the expression and the activity of proteolytic enzymes after exposition to exogenous proteins, with emphasis in its cruzipain-like molecules. Copyright © 2011 Elsevier Inc. All rights reserved.

Regulated internalization of caveolae

PubMed Central

1994-01-01

Caveolae are specialized invaginations of the plasma membrane which have been proposed to play a role in diverse cellular processes such as endocytosis and signal transduction. We have developed an assay to determine the fraction of internal versus plasma membrane caveolae. The GPI-anchored protein, alkaline phosphatase, was clustered in caveolae after antibody-induced crosslinking at low temperature and then, after various treatments, the relative amount of alkaline phosphatase on the cell surface was determined. Using this assay we were able to show a time- and temperature-dependent decrease in cell-surface alkaline phosphatase activity which was dependent on antibody-induced clustering. The decrease in cell surface alkaline phosphatase activity was greatly accelerated by the phosphatase inhibitor, okadaic acid, but not by a protein kinase C activator. Internalization of clustered alkaline phosphatase in the presence or absence of okadaic acid was blocked by cytochalasin D and by the kinase inhibitor staurosporine. Electron microscopy confirmed that okadaic acid induced removal of caveolae from the cell surface. In the presence of hypertonic medium this was followed by the redistribution of groups of caveolae to the center of the cell close to the microtubule-organizing center. This process was reversible, blocked by cytochalasin D, and the centralization of the caveolar clusters was shown to be dependent on an intact microtubule network. Although the exact mechanism of internalization remains unknown, the results show that caveolae are dynamic structures which can be internalized into the cell. This process may be regulated by kinase activity and require an intact actin network. PMID:7962085

Hypoxia-Induced Mitogenic Factor Promotes Cardiac Hypertrophy via Calcium-Dependent and Hypoxia-Inducible Factor-1α Mechanisms.

PubMed

Kumar, Santosh; Wang, Gang; Liu, Wenjuan; Ding, Wenwen; Dong, Ming; Zheng, Na; Ye, Hongyu; Liu, Jie

2018-06-11

HIMF (hypoxia-induced mitogenic factor/found in inflammatory zone 1/resistin like α) is a secretory and cytokine-like protein and serves as a critical stimulator of hypoxia-induced pulmonary hypertension. With a role for HIMF in heart disease unknown, we explored the possible roles for HIMF in cardiac hypertrophy by overexpressing and knocking down HIMF in cardiomyocytes and characterizing HIMF gene ( himf ) knockout mice. We found that HIMF mRNA and protein levels were upregulated in phenylephrine-stimulated cardiomyocyte hypertrophy and our mouse model of transverse aortic constriction-induced cardiac hypertrophy, as well as in human hearts with dilated cardiomyopathy. Furthermore, HIMF overexpression could induce cardiomyocyte hypertrophy, as characterized by elevated protein expression of hypertrophic biomarkers (ANP [atrial natriuretic peptide] and β-MHC [myosin heavy chain-β]) and increased cell-surface area compared with controls. Conversely, HIMF knockdown prevented phenylephrine-induced cardiomyocyte hypertrophy and himf ablation in knockout mice significantly attenuated transverse aortic constriction-induced hypertrophic remodeling and cardiac dysfunction. HIMF overexpression increased the cytosolic Ca 2+ concentration and activated the CaN-NFAT (calcineurin-nuclear factor of activated T cell) and MAPK (mitogen-activated protein kinase) pathways; this effect could be prevented by reducing cytosolic Ca 2+ concentration with L-type Ca 2+ channel blocker nifedipine or inhibiting the CaSR (Ca 2+ sensing receptor) with Calhex 231. Furthermore, HIMF overexpression increased HIF-1α (hypoxia-inducible factor) expression in neonatal rat ventricular myocytes, and HIMF knockout inhibited HIF-1α upregulation in transverse aortic constriction mice. Knockdown of HIF-1α attenuated HIMF-induced cardiomyocyte hypertrophy. In conclusion, HIMF has a critical role in the development of cardiac hypertrophy, and targeting HIMF may represent a potential therapeutic strategy. © 2018 American Heart Association, Inc.

Angiotensin II induces kidney inflammatory injury and fibrosis through binding to myeloid differentiation protein-2 (MD2).

PubMed

Xu, Zheng; Li, Weixin; Han, Jibo; Zou, Chunpeng; Huang, Weijian; Yu, Weihui; Shan, Xiaoou; Lum, Hazel; Li, Xiaokun; Liang, Guang

2017-03-21

Growing evidence indicates that angiotensin II (Ang II), a potent biologically active product of RAS, is a key regulator of renal inflammation and fibrosis. In this study, we tested the hypothesis that Ang II induces renal inflammatory injury and fibrosis through interaction with myeloid differentiation protein-2 (MD2), the accessory protein of toll-like receptor 4 (TLR4) of the immune system. Results indicated that in MD2 -/- mice, the Ang II-induced renal fibrosis, inflammation and kidney dysfunction were significantly reduced compared to control Ang II-infused wild-type mice. Similarly, in the presence of small molecule MD2 specific inhibitor L6H21 or siRNA-MD2, the Ang II-induced increases of pro-fibrotic and pro-inflammatory molecules were prevented in tubular NRK-52E cells. MD2 blockade also inhibited activation of NF-κB and ERK. Moreover, MD2 blockade prevented the Ang II-stimulated formation of the MD2/TLR4/MyD88 signaling complex, as well as the increased surface binding of Ang II in NRK-52E cells. In addition, Ang II directly bound recombinant MD2 protein, rather than TLR4 protein. We conclude that MD2 is a significant contributor in the Ang II-induced kidney inflammatory injury in chronic renal diseases. Furthermore, MD2 inhibition could be a new and important therapeutic strategy for preventing progression of chronic renal diseases.

Clostridium perfringens Epsilon Toxin: A Malevolent Molecule for Animals and Man?

PubMed Central

Stiles, Bradley G.; Barth, Gillian; Barth, Holger; Popoff, Michel R.

2013-01-01

Clostridium perfringens is a prolific, toxin-producing anaerobe causing multiple diseases in humans and animals. One of these toxins is epsilon, a 33 kDa protein produced by Clostridium perfringens (types B and D) that induces fatal enteric disease of goats, sheep and cattle. Epsilon toxin (Etx) belongs to the aerolysin-like toxin family. It contains three distinct domains, is proteolytically-activated and forms oligomeric pores on cell surfaces via a lipid raft-associated protein(s). Vaccination controls Etx-induced disease in the field. However, therapeutic measures are currently lacking. This review initially introduces C. perfringens toxins, subsequently focusing upon the Etx and its biochemistry, disease characteristics in various animals that include laboratory models (in vitro and in vivo), and finally control mechanisms (vaccines and therapeutics). PMID:24284826

Clostridium perfringens epsilon toxin: a malevolent molecule for animals and man?

PubMed

Stiles, Bradley G; Barth, Gillian; Barth, Holger; Popoff, Michel R

2013-11-12

Clostridium perfringens is a prolific, toxin-producing anaerobe causing multiple diseases in humans and animals. One of these toxins is epsilon, a 33 kDa protein produced by Clostridium perfringens (types B and D) that induces fatal enteric disease of goats, sheep and cattle. Epsilon toxin (Etx) belongs to the aerolysin-like toxin family. It contains three distinct domains, is proteolytically-activated and forms oligomeric pores on cell surfaces via a lipid raft-associated protein(s). Vaccination controls Etx-induced disease in the field. However, therapeutic measures are currently lacking. This review initially introduces C. perfringens toxins, subsequently focusing upon the Etx and its biochemistry, disease characteristics in various animals that include laboratory models (in vitro and in vivo), and finally control mechanisms (vaccines and therapeutics).

Maximally asymmetric transbilayer distribution of anionic lipids alters the structure and interaction with lipids of an amyloidogenic protein dimer bound to the membrane surface.

PubMed

Cheng, Sara Y; Chou, George; Buie, Creighton; Vaughn, Mark W; Compton, Campbell; Cheng, Kwan H

2016-03-01

We used molecular dynamics simulations to explore the effects of asymmetric transbilayer distribution of anionic phosphatidylserine (PS) lipids on the structure of a protein on the membrane surface and subsequent protein-lipid interactions. Our simulation systems consisted of an amyloidogenic, beta-sheet rich dimeric protein (D42) absorbed to the phosphatidylcholine (PC) leaflet, or protein-contact PC leaflet, of two membrane systems: a single-component PC bilayer and double PC/PS bilayers. The latter comprised of a stable but asymmetric transbilayer distribution of PS in the presence of counterions, with a 1-component PC leaflet coupled to a 1-component PS leaflet in each bilayer. The maximally asymmetric PC/PS bilayer had a non-zero transmembrane potential (TMP) difference and higher lipid order packing, whereas the symmetric PC bilayer had a zero TMP difference and lower lipid order packing under physiologically relevant conditions. Analysis of the adsorbed protein structures revealed weaker protein binding, more folding in the N-terminal domain, more aggregation of the N- and C-terminal domains and larger tilt angle of D42 on the PC leaflet surface of the PC/PS bilayer versus the PC bilayer. Also, analysis of protein-induced membrane structural disruption revealed more localized bilayer thinning in the PC/PS versus PC bilayer. Although the electric field profile in the non-protein-contact PS leaflet of the PC/PS bilayer differed significantly from that in the non-protein-contact PC leaflet of the PC bilayer, no significant difference in the electric field profile in the protein-contact PC leaflet of either bilayer was evident. We speculate that lipid packing has a larger effect on the surface adsorbed protein structure than the electric field for a maximally asymmetric PC/PS bilayer. Our results support the mechanism that the higher lipid packing in a lipid leaflet promotes stronger protein-protein but weaker protein-lipid interactions for a dimeric protein on membrane surfaces. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

Autodisplay: Development of an Efficacious System for Surface Display of Antigenic Determinants in Salmonella Vaccine Strains

PubMed Central

Kramer, Uwe; Rizos, Konstantin; Apfel, Heiko; Autenrieth, Ingo B.; Lattemann, Claus T.

2003-01-01

To optimize antigen delivery by Salmonella vaccine strains, a system for surface display of antigenic determinants was established by using the autotransporter secretion pathway of gram-negative bacteria. A modular system for surface display allowed effective targeting of heterologous antigens or fragments thereof to the bacterial surface by the autotransporter domain of AIDA-I, the Escherichia coli adhesin involved in diffuse adherence. A major histocompatibility complex class II-restricted epitope, comprising amino acids 74 to 86 of the Yersinia enterocolitica heat shock protein Hsp60 (Hsp6074-86), was fused to the AIDA-I autotransporter domain, and the resulting fusion protein was expressed at high levels on the cell surface of E. coli and Salmonella enterica serovar Typhimurium. Colonization studies in mice vaccinated with Salmonella strains expressing AIDA-I fusion proteins demonstrated high genetic stability of the generated vaccine strain in vivo. Furthermore, a pronounced T-cell response against Yersinia Hsp6074-86 was induced in mice vaccinated with a Salmonella vaccine strain expressing the Hsp6074-86-AIDA-I fusion protein. This was shown by monitoring Yersinia Hsp60-stimulated IFN-γ secretion and proliferation of splenic T cells isolated from vaccinated mice. These results demonstrate that the surface display of antigenic determinants by the autotransporter pathway deserves special attention regarding the application in live attenuated Salmonella vaccine strains. PMID:12654812

Autodisplay: development of an efficacious system for surface display of antigenic determinants in Salmonella vaccine strains.

PubMed

Kramer, Uwe; Rizos, Konstantin; Apfel, Heiko; Autenrieth, Ingo B; Lattemann, Claus T

2003-04-01

To optimize antigen delivery by Salmonella vaccine strains, a system for surface display of antigenic determinants was established by using the autotransporter secretion pathway of gram-negative bacteria. A modular system for surface display allowed effective targeting of heterologous antigens or fragments thereof to the bacterial surface by the autotransporter domain of AIDA-I, the Escherichia coli adhesin involved in diffuse adherence. A major histocompatibility complex class II-restricted epitope, comprising amino acids 74 to 86 of the Yersinia enterocolitica heat shock protein Hsp60 (Hsp60(74-86)), was fused to the AIDA-I autotransporter domain, and the resulting fusion protein was expressed at high levels on the cell surface of E. coli and Salmonella enterica serovar Typhimurium. Colonization studies in mice vaccinated with Salmonella strains expressing AIDA-I fusion proteins demonstrated high genetic stability of the generated vaccine strain in vivo. Furthermore, a pronounced T-cell response against Yersinia Hsp60(74-86) was induced in mice vaccinated with a Salmonella vaccine strain expressing the Hsp60(74-86)-AIDA-I fusion protein. This was shown by monitoring Yersinia Hsp60-stimulated IFN-gamma secretion and proliferation of splenic T cells isolated from vaccinated mice. These results demonstrate that the surface display of antigenic determinants by the autotransporter pathway deserves special attention regarding the application in live attenuated Salmonella vaccine strains.

Kinetics of Surface-Driven Self-Assembly and Fatigue-Induced Disassembly of a Virus-Based Nanocoating.

PubMed

Valbuena, Alejandro; Mateu, Mauricio G

2017-02-28

Self-assembling protein layers provide a "bottom-up" approach for precisely organizing functional elements at the nanoscale over a large solid surface area. The design of protein sheets with architecture and physical properties suitable for nanotechnological applications may be greatly facilitated by a thorough understanding of the principles that underlie their self-assembly and disassembly. In a previous study, the hexagonal lattice formed by the capsid protein (CA) of human immunodeficiency virus (HIV) was self-assembled as a monomolecular layer directly onto a solid substrate, and its mechanical properties and dynamics at equilibrium were analyzed by atomic force microscopy. Here, we use atomic force microscopy to analyze the kinetics of self-assembly of the planar CA lattice on a substrate and of its disassembly, either spontaneous or induced by materials fatigue. Both self-assembly and disassembly of the CA layer are cooperative reactions that proceed until a phase equilibrium is reached. Self-assembly requires a critical protein concentration and is initiated by formation of nucleation points on the substrate, followed by lattice growth and eventual merging of CA patches into a continuous monolayer. Disassembly of the CA layer showed hysteresis and appears to proceed only after large enough defects (nucleation points) are formed in the lattice, whose number is largely increased by inducing materials fatigue that depends on mechanical load and its frequency. Implications of the kinetic results obtained for a better understanding of self-assembly and disassembly of the HIV capsid and protein-based two-dimensional nanomaterials and the design of anti-HIV drugs targeting (dis)assembly and biocompatible nanocoatings are discussed. Copyright © 2017 Biophysical Society. Published by Elsevier Inc. All rights reserved.

An attack of the plant parasite Cuscuta reflexa induces the expression of attAGP, an attachment protein of the host tomato.

PubMed

Albert, Markus; Belastegui-Macadam, Xana; Kaldenhoff, Ralf

2006-11-01

Dodder or Cuscutaceae are holoparasitic plants subsisting on other dicotyledonous plants. The infection process is initiated by adherence of Cuscuta prehaustoria to the host surface, followed by penetration attempts by hyphae. In the case of a successful infection, these organs connect the parasite's vascular tissue to that of the host. Here we show that contact of Cuscuta reflexa prehaustoria to tomato induces the expression of a new arabinogalactan protein (AGP), attAGP, in the tomato precisely at the site of dodder attack. We show that attAGP is a plasma membrane-bound cell wall-localized protein. Using the RNAi technique and attAGP-targeted virus-induced gene silencing, we observed a correlation between attAGP expression level and force of attachment of the parasite to host tomatoes. If the expression level of attAGP was reduced, the C. reflexa attachment capability was significantly reduced, too. We conclude that C. reflexa infection induced a signal in the host leading to expression of tomato attAGP, which promotes the parasite's adherence.

Protein diffusiophoresis and salt osmotic diffusion in aqueous solutions.

PubMed

Annunziata, Onofrio; Buzatu, Daniela; Albright, John G

2012-10-25

Diffusion of a solute can be induced by the concentration gradient of another solute in solution. This transport mechanism is known as cross-diffusion. We have investigated cross-diffusion in a ternary protein-salt-water system. Specifically, we measured the two cross-diffusion coefficients for the lysozyme-NaCl-water system at 25 °C and pH 4.5 as a function of protein and salt concentrations by Rayleigh interferometry. One cross-diffusion coefficient characterizes salt osmotic diffusion induced by a protein concentration gradient, and is related to protein-salt thermodynamic interactions as described by the theories of Donnan membrane equilibrium and protein preferential hydration. The other cross-diffusion coefficient characterizes protein diffusiophoresis induced by a salt concentration gradient, and is described as the difference between a preferential-interaction coefficient and a transport parameter. We first relate our experimental results to the protein net charge and the thermodynamic excess of water near the protein surface. We then extract the Stefan-Maxwell diffusion coefficient describing protein-salt interactions in water. We find that the value of this coefficient is negative, contrary to the friction interpretation of Stefan-Maxwell equations. This result is explained by considering protein hydration. Finally, protein diffusiophoresis is quantitatively examined by considering electrophoretic and hydration effects on protein migration and utilized to accurately estimate lysozyme electrophoretic mobility. To our knowledge, this is the first time that protein diffusiophoresis has been experimentally characterized and a protein-salt Stefan-Maxwell diffusion coefficient reported. This work represents a significant contribution for understanding and modeling the effect of concentration gradients in protein-salt aqueous systems relevant to diffusion-based mass-transfer technologies and transport in living systems.

Protein Transfer Into Human Cells by VSV-G-induced Nanovesicles

PubMed Central

Mangeot, Philippe-Emmanuel; Dollet, Sandra; Girard, Mathilde; Ciancia, Claire; Joly, Stéphane; Peschanski, Marc; Lotteau, Vincent

2011-01-01

Identification of new techniques to express proteins into mammal cells is of particular interest for both research and medical purposes. The present study describes the use of engineered vesicles to deliver exogenous proteins into human cells. We show that overexpression of the spike glycoprotein of the vesicular stomatitis virus (VSV-G) in human cells induces the release of fusogenic vesicles named gesicles. Biochemical and functional studies revealed that gesicles incorporated proteins from producer cells and could deliver them to recipient cells. This protein-transduction method allows the direct transport of cytoplasmic, nuclear or surface proteins in target cells. This was demonstrated by showing that the TetR transactivator and the receptor for the murine leukemia virus (MLV) envelope [murine cationic amino acid transporter-1 (mCAT-1)] were efficiently delivered by gesicles in various cell types. We further shows that gesicle-mediated transfer of mCAT-1 confers to human fibroblasts a robust permissiveness to ecotropic vectors, allowing the generation of human-induced pluripotent stem cells in level 2 biosafety facilities. This highlights the great potential of mCAT-1 gesicles to increase the safety of experiments using retro/lentivectors. Besides this, gesicles is a versatile tool highly valuable for the nongenetic delivery of functions such as transcription factors or genome engineering agents. PMID:21750535

A novel anti-CD22 scFv-apoptin fusion protein induces apoptosis in malignant B-cells.

PubMed

Agha Amiri, Solmaz; Shahhosseini, Soraya; Zarei, Najmeh; Khorasanizadeh, Dorsa; Aminollahi, Elahe; Rezaie, Faegheh; Zargari, Mehryar; Azizi, Mohammad; Khalaj, Vahid

2017-12-01

CD22 marker is a highly internalizing antigen which is located on the surface of B-cells and is being used as a promising target for treatment of B cell malignancies. Monoclonal antibodies targeting CD22 have been introduced and some are currently under investigation in clinical trials. Building on the success of antibody drug conjugates, we developed a fusion protein consisting of a novel anti-CD22 scFv and apoptin and tested binding and therapeutic effects in lymphoma cells. The recombinant protein was expressed in E. coli and successfully purified and refolded. In vitro binding analysis by immunofluorescence and flow cytometry demonstrated that the recombinant protein specifically binds to CD22 positive Raji cells but not to CD22 negative Jurkat cells. The cytotoxic properties of scFv-apoptin were assessed by an MTT assay and Annexin V/PI flow cytometry analysis and showed that the recombinant protein induced apoptosis preferentially in Raji cells with no detectable effects in Jurkat cells. Our findings indicated that the recombinant anti-CD22 scFv-apoptin fusion protein could successfully cross the cell membrane and induce apoptosis with high specificity, make it as a promising molecule for immunotherapy of B-cell malignancies.

Deinococcus Mn2+ -Peptide Complex: A Novel Approach to Alphavirus Vaccine Development

DTIC Science & Technology

2016-08-05

immunogenicity loss due to oxidative damage to the surface proteins at the high doses of radiation required for complete virus inactivation. Thus, we...bacteria Deinococcus radiodurans) in the present study which selectively protects proteins but not the nucleic acid from the radiation - induced...presence of MDP have significant epitope preservation even at supra-lethal doses of radiation . Irradiated viruses were found to be completely

Differences in the Pathways of Proteins Unfolding Induced by Urea and Guanidine Hydrochloride: Molten Globule State and Aggregates

PubMed Central

Povarova, Olga I.; Kuznetsova, Irina M.; Turoverov, Konstantin K.

2010-01-01

It was shown that at low concentrations guanidine hydrochloride (GdnHCl) can cause aggregation of proteins in partially folded state and that fluorescent dye 1-anilinonaphthalene-8-sulfonic acid (ANS) binds with these aggregates rather than with hydrophobic clusters on the surface of protein in molten globule state. That is why the increase in ANS fluorescence intensity is often recorded in the pathway of protein denaturation by GdnHCl, but not by urea. So what was previously believed to be the molten globule state in the pathway of protein denaturation by GdnHCl, in reality, for some proteins represents the aggregates of partially folded molecules. PMID:21152408

Detection and size analysis of proteins with switchable DNA layers.

PubMed

Rant, Ulrich; Pringsheim, Erika; Kaiser, Wolfgang; Arinaga, Kenji; Knezevic, Jelena; Tornow, Marc; Fujita, Shozo; Yokoyama, Naoki; Abstreiter, Gerhard

2009-04-01

We introduce a chip-compatible scheme for the label-free detection of proteins in real-time that is based on the electrically driven conformation switching of DNA oligonucleotides on metal surfaces. The switching behavior is a sensitive indicator for the specific recognition of IgG antibodies and antibody fragments, which can be detected in quantities of less than 10(-18) mol on the sensor surface. Moreover, we show how the dynamics of the induced molecular motion can be monitored by measuring the high-frequency switching response. When proteins bind to the layer, the increase in hydrodynamic drag slows the switching dynamics, which allows us to determine the size of the captured proteins. We demonstrate the identification of different antibody fragments by means of their kinetic fingerprint. The switchDNA method represents a generic approach to simultaneously detect and size target molecules using a single analytical platform.

Activation of amphibian oocytes by sperm extracts.

PubMed

Bonilla, F; Ajmat, M T; Sánchez Toranzo, G; Zelarayán, L; Oterino, J; Bühler, M I

2008-11-01

In the fertilization of most animals, egg activation is accompanied by an increase in cytoplasmatic Ca2+; however, the mechanism through which the fertilizing sperm induce this phenomenon is still controversial. An increase in intracellular free Ca2+ is required to trigger egg activation events, a process that includes cortical granule exocytosis, resumption and completion of meiosis and DNA replication, and culminates in the first mitotic cleavage. In this work, we investigated the effect of microinjection and incubation of different fractions of homologous sperm extract on the activation of Bufo arenarum oocytes matured in vitro. Two heat treatment-sensitive fractions obtained by chromatography were able to induce oocyte activation. The sperm fraction, which contained a 24 kDa protein, induced 90% activation when it was microinjected into the oocytes. Whilst the sperm fraction, which contained a 36 kDa protein, was able to induce about 70% activation only when it was applied on the oocyte surface.

Immunodominance and Functional Activities of Antibody Responses to Inactivated West Nile Virus and Recombinant Subunit Vaccines in Mice▿

PubMed Central

Zlatkovic, Juergen; Stiasny, Karin; Heinz, Franz X.

2011-01-01

Factors controlling the dominance of antibody responses to specific sites in viruses and/or protein antigens are ill defined but can be of great importance for the induction of potent immune responses to vaccines. West Nile virus and other related important human-pathogenic flaviviruses display the major target of neutralizing antibodies, the E protein, in an icosahedral shell at the virion surface. Potent neutralizing antibodies were shown to react with the upper surface of domain III (DIII) of this protein. Using the West Nile virus system, we conducted a study on the immunodominance and functional quality of E-specific antibody responses after immunization of mice with soluble protein E (sE) and isolated DIII in comparison to those after immunization with inactivated whole virions. With both virion and sE, the neutralizing response was dominated by DIII-specific antibodies, but the functionality of these antibodies was almost four times higher after virion immunization. Antibodies induced by the isolated DIII had an at least 15-fold lower specific neutralizing activity than those induced by the virion, and only 50% of these antibodies were able to bind to virus particles. Our results suggest that immunization with the tightly packed E in virions focuses the DIII antibody response to the externally exposed sites of this domain which are the primary targets for virus neutralization, different from sE and isolated DIII, which also display protein surfaces that are cryptic in the virion. Despite its low potency for priming, DIII was an excellent boosting antigen, suggesting novel vaccination strategies that strengthen and focus the antibody response to critical neutralizing sites in DIII. PMID:21147919

Preparation and testing of a Haemophilus influenzae Type b/Hepatitis B surface antigen conjugate vaccine.

PubMed

An, So Jung; Woo, Joo Sung; Chae, Myung Hwa; Kothari, Sudeep; Carbis, Rodney

2015-03-24

The majority of conjugate vaccines focus on inducing an antibody response to the polysaccharide antigen and the carrier protein is present primarily to induce a T-cell dependent response. In this study conjugates consisting of poly(ribosylribitolphosphate) (PRP) purified from Haemophilus influenzae Type b bound to Hepatitis B virus surface antigen (HBsAg) virus like particles were prepared with the aim of inducing an antibody response to not only the PRP but also the HBsAg. A conjugate consisting of PRP bound to HBsAg via an adipic acid dihydrazide (ADH) spacer induced strong IgG antibodies to both the PRP and HBsAg. When conjugation was performed without the ADH spacer the induction of an anti-PRP response was equivalent to that seen by conjugate with the ADH spacer, however, a negligible anti-HBsAg response was induced. For comparison, PRP was conjugated to diphtheria toxoid (DT) and Vi polysaccharide purified from Salmonella Typhi conjugated to HBsAg both using an ADH spacer. The PRPAH-DT conjugate induced strong anti-PRP and anti-DT responses, the Vi-AHHBsAg conjugate induced a good anti-HBsAg response but not as strong as that induced by the PRPAH-HBsAg conjugate. This study demonstrated that in mice it was possible to induce robust antibody responses to both polysaccharide and carrier protein provided the conjugate has certain physico-chemical properties. A PRPAH-HBsAg conjugate with the capacity to induce anti-PRP and anti-HBsAg responses could be incorporated into a multivalent pediatric vaccine and simplify formulation of such a vaccine. Copyright © 2015 Elsevier Ltd. All rights reserved.

Counterion effects in protein nanoparticle electrostatic binding: a theoretical study.

PubMed

Ghosh, Goutam

2015-04-01

Effects of counterions on the folding conformation of proteins, bound electrostatically on the surface of charge-ligand functionalized nanoparticles, have been investigated based on the protein folding energy calculation. The folding energy of a protein has been taken as a sum of the short range interaction energies, like, the van der Waals attraction and the hydrogen bond energies, and the long range coulomb interaction energy. On electrostatic binding, counterions associated with surface ligands of nanoparticles diffuse into bound proteins through the medium of dispersion. As a result, bound proteins partially unfold, as observed in circular dichroism experiments, which has been realized using the "charge-dipole" and the "charge-induced dipole" interactions of counterions with polar and non-polar residues, respectively. The effect of counterions solvation in the dispersing medium, e.g., water, which causes water molecules to polarize around the counterions, has also been considered. The folding energy of bound proteins has been seen to decrease proportionally with the increasing number of diffusion of counterions and their polarizability. Copyright © 2015 Elsevier B.V. All rights reserved.

Effect of DOPE and cholesterol on the protein adsorption onto lipid nanoparticles

NASA Astrophysics Data System (ADS)

Caracciolo, Giulio; Pozzi, Daniela; Capriotti, Anna Laura; Cavaliere, Chiara; Laganà, Aldo

2013-03-01

Upon administration, nanoparticles (NPs) are exposed to biological fluids from which they adsorb proteins and other biomolecules to form a "protein corona". NP-protein interactions are still poorly understood and quantitative studies to characterize them remain scarce. Here, we have investigated the effect of neutral dioleoylphosphatidylethanolamine (DOPE) and cholesterol on the adsorption of human plasma proteins onto the surface of 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP)-based cationic liposomes of 100 nm in diameter. Quantitative analysis of the protein corona revealed that replacing cationic DOTAP lipids with neutral lipids, being indifferently DOPE or cholesterol, reduces the affinity of fibrinogen, prothrombin, vitamin K, and vitronectin for the lipid surface. On the other side, DOPE specifically promotes the adsorption of apolipoproteins and serum albumin, while cholesterol induces the preferential binding of immunoglobulins and complement proteins. The results of this study will help to explain why NPs of different lipid compositions have a dramatic difference in their in vivo transfection efficiency and will be useful for design of lipid NPs with optimal circulation profiles.

High hydrophobic amino acid exposure is responsible of the neurotoxic effects induced by E200K or D202N disease-related mutations of the human prion protein.

PubMed

Corsaro, Alessandro; Thellung, Stefano; Bucciarelli, Tonino; Scotti, Luca; Chiovitti, Katia; Villa, Valentina; D'Arrigo, Cristina; Aceto, Antonio; Florio, Tullio

2011-03-01

Mutations in prion protein are thought to be causative of inherited prion diseases favoring the spontaneous conversion of the normal prion protein into the scrapie-like pathological prion protein. We previously reported that, by controlled thermal denaturation, human prion protein fragment 90-231 acquires neurotoxic properties when transformed in a β-rich conformation, resembling the scrapie-like conformation. In this study we generated prion protein fragment 90-231 bearing mutations identified in familial prion diseases (D202N and E200K), to analyze their role in the induction of a neurotoxic conformation. Prion protein fragment 90-231(wild type) and the D202N mutant were not toxic in native conformation but induced cell death only after thermal denaturation. Conversely, prion protein fragment 90-231(E200K) was highly toxic in its native structure, suggesting that E200K mutation per se favors the acquisition of a peptide neurotoxic conformation. To identify the structural determinants of prion protein fragment 90-231 toxicity, we show that while the wild type peptide is structured in α-helix, hPrP90-231 E200K is spontaneously refolded in a β-structured conformer characterized by increased proteinase K resistance and propensity to generate fibrils. However, the most significant difference induced by E200K mutation in prion protein fragment 90-231 structure in native conformation we observed, was an increase in the exposure of hydrophobic amino-acids on protein surface that was detected in wild type and D202N proteins only after thermal denaturation. In conclusion, we propose that increased hydrophobicity is one of the main determinants of toxicity induced by different mutations in prion protein-derived peptides. Copyright © 2010 Elsevier Ltd. All rights reserved.

CXCL4 is a novel nickel-binding protein and augments nickel allergy.

PubMed

Kuroishi, T; Bando, K; Tanaka, Y; Shishido, K; Kinbara, M; Ogawa, T; Muramoto, K; Endo, Y; Sugawara, S

2017-08-01

Nickel (Ni) is the most frequent metal allergen and induces a TH 1 -dependent type-IV allergy. Although Ni 2+ is considered to bind to endogenous proteins, it currently remains unclear whether these Ni-binding proteins are involved in Ni allergy in vivo. We previously reported the adjuvant effects of lipopolysaccharide (LPS) in a Ni allergy mouse model. As LPS induces a number of inflammatory mediators, we hypothesized that Ni-binding protein(s) are also induced by LPS. The objective of this study was to purify and identify Ni-binding protein(s) from serum taken from LPS-injected mice (referred as LPS serum) and examined the augmenting effects of these Ni-binding protein(s) on Ni allergy in an in vivo model. BALB/cA mice were sensitized with an i.p. injection of NiCl 2 and LPS. Ten days after sensitization, mice were challenged with NiCl 2 by an i.d. injection into ear pinnae. Ni-binding protein(s) were purified by Ni-affinity column chromatography and gel filtration. Lipopolysaccharide serum, but not serum taken from saline-injected mice, augmented ear swelling induced by Ni-allergic inflammation. Ni-binding, but not non-binding fraction, purified from LPS serum augmented Ni-allergic inflammation. Mass spectrometry and Western blotting detected CXCL4 in the active fraction. A batch analysis with Ni-sepharose and a surface plasmon resonance analysis revealed direct binding between CXCL4 and Ni 2+ . Recombinant CXCL4 augmented Ni-allergic inflammation and exerted adjuvant effects at the sensitization phase. These results indicate that CXCL4 is a novel Ni-binding protein that augments Ni allergy at the elicitation and sensitization phases. This is the first study to demonstrate that the Ni-binding protein augments Ni allergy in vivo. © 2017 John Wiley & Sons Ltd.

The Plasma Membrane is Compartmentalized by a Self-Similar Cortical Actin Fractal

NASA Astrophysics Data System (ADS)

Sadegh, Sanaz; Higgin, Jenny; Mannion, Patrick; Tamkun, Michael; Krapf, Diego

A broad range of membrane proteins display anomalous diffusion on the cell surface. Different methods provide evidence for obstructed subdiffusion and diffusion on a fractal space, but the underlying structure inducing anomalous diffusion has never been visualized due to experimental challenges. We addressed this problem by imaging the cortical actin at high resolution while simultaneously tracking individual membrane proteins in live mammalian cells. Our data show that actin introduces barriers leading to compartmentalization of the plasma membrane and that membrane proteins are transiently confined within actin fences. Furthermore, superresolution imaging shows that the cortical actin is organized into a self-similar fractal. These results present a hierarchical nanoscale picture of the plasma membrane and demonstrate direct interactions between the actin cortex and the cell surface.

Bioactivity evolution of the surface functionalized bioactive glasses.

PubMed

Magyari, Klára; Baia, Lucian; Vulpoi, Adriana; Simon, Simion; Popescu, Octavian; Simon, Viorica

2015-02-01

The formation of a calcium phosphate layer on the surface of the SiO2 -CaO-P2 O5 glasses after immersion in simulated body fluid (SBF) generally demonstrates the bioactivity of these materials. Grafting of the surface by chemical bonding can minimize the structural changes in protein adsorbed on the surface. Therefore, in this study our interest was to evaluate the bioactivity and blood biocompatibility of the SiO2 -CaO-P2 O5 glasses after their surface modification by functionalization with aminopropyl-triethoxysilane and/or by fibrinogen. It is shown that the fibrinogen adsorbed on the glass surfaces induces a growing of the apatite-like layer. It is also evidenced that the protein content from SBF influences the growth of the apatite-like layer. Furthermore, the good blood compatibility of the materials after fibrinogen and bovine serum albumin adsorption is proved from the assessment of the β-sheet-β-turn ratio. © 2014 Wiley Periodicals, Inc.

Comparative analysis of 2,4,6-trinitrotoluene (TNT)-induced cellular responses and proteomes in Pseudomonas sp. HK-6 in two types of media.

PubMed

Cho, Yun-Seok; Lee, Bheong-Uk; Kahng, Hyung-Yeel; Oh, Kye-Heon

2009-04-01

TNT-induced cellular responses and proteomes in Pseudomonas sp. HK-6 were comparatively analyzed in two different media: basal salts (BS) and Luria broth (LB). HK-6 cells could not degrade more than 0.5 mM TNT with BS medium, while in LB medium, they exhibited the enhanced capability to degrade as much as 3.0 mM TNT. Analysis of total cellular fatty acids in HK-6 cells suggested that the relative abundance of several saturated or unsaturated fatty acids is altered under TNT-mediated stress conditions. Scanning electron microscopy showed the presence of perforations, irregular rod formations, and wrinkled extracellular surfaces in cells under TNT stress. Proteomic analysis of soluble protein fractions from HK-6 cultures grown with TNT as a substrate revealed 11 protein spots induced by TNT. Among these, seven proteins (including Alg8, AlgB, NirB, and the AhpC/Tsa family) were detected only in LB medium containing TNT. The proteins AspS, Tsf, and assimilatory nitrate reductase were increasingly expressed only in BS medium containing TNT. The protein dGTPase was found to be induced and expressed when cells were grown in either type of TNT-containing media. These results provide a better understanding of the cytotoxicity and survival mechanism used by Pseudomonas sp. HK-6 when placed under TNT stress conditions.

High intensity ultrasound treatment of faba bean (Vicia faba L.) protein: Effect on surface properties, foaming ability and structural changes.

PubMed

Martínez-Velasco, Alejandro; Lobato-Calleros, Consuelo; Hernández-Rodríguez, Blanca E; Román-Guerrero, Angélica; Alvarez-Ramirez, Jose; Vernon-Carter, E Jaime

2018-06-01

Response surface methodology was used for establishing the amplitude (72.67%) and time (17.29 min) high-intensity ultrasound (HIUS) conditions leading to an optimized faba bean protein isolate (OFPI) with lower interfacial tension, zeta potential and viscosity, and higher solubility than native faba bean protein isolate (NFPI). OFPI showed significantly higher adsorption dynamics at the air-water interface, and produced foam with significant smaller bubble diameter, higher overrun, stability and yield stress, and lower liquid drainage than NFPI. Fourier Transform Spectroscopy (FT-IR) revealed that the secondary structure of OFPI deferred from NFPI in terms of increases in β conformations (6.61% β-sheet, 19.6% β-turn, 0.8% anti-parallel β-sheet) and decreases in inter-molecular aggregates (43.54%). Multienzyme study pinpointed that the structural changes could have induced a decrease on the relative protein digestibility of OFPI respect that of NFPI. The results of this work demonstrate that HIUS technology improves the surface and foaming properties of faba bean protein isolate, which may favour the revalorisation of this crop. Copyright © 2018 Elsevier B.V. All rights reserved.

Small-Molecule Sigma1 Modulator Induces Autophagic Degradation of PD-L1.

PubMed

Maher, Christina M; Thomas, Jeffrey D; Haas, Derick A; Longen, Charles G; Oyer, Halley M; Tong, Jane Y; Kim, Felix J

2018-02-01

Emerging evidence suggests that Sigma1 ( SIGMAR1 , also known as sigma-1 receptor) is a unique ligand-regulated integral membrane scaffolding protein that contributes to cellular protein and lipid homeostasis. Previously, we demonstrated that some small-molecule modulators of Sigma1 alter endoplasmic reticulum (ER)-associated protein homeostasis pathways in cancer cells, including the unfolded protein response and autophagy. Programmed death-ligand 1 (PD-L1) is a type I integral membrane glycoprotein that is cotranslationally inserted into the ER and is processed and transported through the secretory pathway. Once at the surface of cancer cells, PD-L1 acts as a T-cell inhibitory checkpoint molecule and suppresses antitumor immunity. Here, we demonstrate that in Sigma1-expressing triple-negative breast and androgen-independent prostate cancer cells, PD-L1 protein levels were suppressed by RNAi knockdown of Sigma1 and by small-molecule inhibition of Sigma1. Sigma1-mediated action was confirmed by pharmacologic competition between Sigma1-selective inhibitor and activator ligands. When administered alone, the Sigma1 inhibitor decreased cell surface PD-L1 expression and suppressed functional interaction of PD-1 and PD-L1 in a coculture of T cells and cancer cells. Conversely, the Sigma1 activator increased PD-L1 cell surface expression, demonstrating the ability to positively and negatively modulate Sigma1 associated PD-L1 processing. We discovered that the Sigma1 inhibitor induced degradation of PD-L1 via autophagy, by a mechanism distinct from bulk macroautophagy or general ER stress-associated autophagy. Finally, the Sigma1 inhibitor suppressed IFNγ-induced PD-L1. Our data demonstrate that small-molecule Sigma1 modulators can be used to regulate PD-L1 in cancer cells and trigger its degradation by selective autophagy. Implications: Sigma1 modulators sequester and eliminate PD-L1 by autophagy, thus preventing functional PD-L1 expression at the cell surface. This posits Sigma1 modulators as novel therapeutic agents in PD-L1/PD-1 blockade strategies that regulate the tumor immune microenvironment. Visual Overview: http://mcr.aacrjournals.org/content/molcanres/16/2/243/F1.large.jpg Mol Cancer Res; 16(2); 243-55. ©2017 AACR . ©2017 American Association for Cancer Research.

Water-mediated influence of a crowded environment on internal vibrations of a protein molecule.

PubMed

Kuffel, Anna; Zielkiewicz, Jan

2016-02-14

The influence of crowding on the protein inner dynamics is examined by putting a single protein molecule close to one or two neighboring protein molecules. The presence of additional molecules influences the amplitudes of protein fluctuations. Also, a weak dynamical coupling of collective velocities of surface atoms of proteins separated by a layer of water is detected. The possible mechanisms of these phenomena are described. The cross-correlation function of the collective velocities of surface atoms of two proteins was decomposed into the Fourier series. The amplitude spectrum displays a peak at low frequencies. Also, the results of principal component analysis suggest that the close presence of an additional protein molecule influences the high-amplitude, low-frequency modes in the most prominent way. This part of the spectrum covers biologically important protein motions. The neighbor-induced changes in the inner dynamics of the protein may be connected with the changes in the velocity power spectrum of interfacial water. The additional protein molecule changes the properties of solvation water and in this way it can influence the dynamics of the second protein. It is suggested that this phenomenon may be described, at first approximation, by a damped oscillator driven by an external random force. This model was successfully applied to conformationally rigid Choristoneura fumiferana antifreeze protein molecules.

Neutrophil cell surface receptors and their intracellular signal transduction pathways☆

PubMed Central

Futosi, Krisztina; Fodor, Szabina; Mócsai, Attila

2013-01-01

Neutrophils play a critical role in the host defense against bacterial and fungal infections, but their inappropriate activation also contributes to tissue damage during autoimmune and inflammatory diseases. Neutrophils express a large number of cell surface receptors for the recognition of pathogen invasion and the inflammatory environment. Those include G-protein-coupled chemokine and chemoattractant receptors, Fc-receptors, adhesion receptors such as selectins/selectin ligands and integrins, various cytokine receptors, as well as innate immune receptors such as Toll-like receptors and C-type lectins. The various cell surface receptors trigger very diverse signal transduction pathways including activation of heterotrimeric and monomeric G-proteins, receptor-induced and store-operated Ca2 + signals, protein and lipid kinases, adapter proteins and cytoskeletal rearrangement. Here we provide an overview of the receptors involved in neutrophil activation and the intracellular signal transduction processes they trigger. This knowledge is crucial for understanding how neutrophils participate in antimicrobial host defense and inflammatory tissue damage and may also point to possible future targets of the pharmacological therapy of neutrophil-mediated autoimmune or inflammatory diseases. PMID:23994464

Kinetic stabilization of Bacillus licheniformis alpha-amylase through introduction of hydrophobic residues at the surface.

PubMed

Machius, Mischa; Declerck, Nathalie; Huber, Robert; Wiegand, Georg

2003-03-28

It is generally assumed that in proteins hydrophobic residues are not favorable at solvent-exposed sites, and that amino acid substitutions on the surface have little effect on protein thermostability. Contrary to these assumptions, we have identified hyperthermostable variants of Bacillus licheniformis alpha-amylase (BLA) that result from the incorporation of hydrophobic residues at the surface. Under highly destabilizing conditions, a variant combining five stabilizing mutations unfolds 32 times more slowly and at a temperature 13 degrees C higher than the wild-type. Crystal structure analysis at 1.7 A resolution suggests that stabilization is achieved through (a) extension of the concept of increased hydrophobic packing, usually applied to cavities, to surface indentations, (b) introduction of favorable aromatic-aromatic interactions on the surface, (c) specific stabilization of intrinsic metal binding sites, and (d) stabilization of a beta-sheet by introducing a residue with high beta-sheet forming propensity. All mutated residues are involved in forming complex, cooperative interaction networks that extend from the interior of the protein to its surface and which may therefore constitute "weak points" where BLA unfolding is initiated. This might explain the unexpectedly large effect induced by some of the substitutions on the kinetic stability of BLA. Our study shows that substantial protein stabilization can be achieved by stabilizing surface positions that participate in underlying cooperatively formed substructures. At such positions, even the apparently thermodynamically unfavorable introduction of hydrophobic residues should be explored.

Design and construction of glutamine binding proteins with a self-adhering capability to unmodified hydrophobic surfaces as reagentless fluorescence sensing devices.

PubMed

Wada, Akira; Mie, Masayasu; Aizawa, Masuo; Lahoud, Pedro; Cass, Anthony E G; Kobatake, Eiry

2003-12-31

The chemically and genetically remodeling of proteins with ligand binding specificities can be utilized to synthesize various protein-based microsensors for detecting single biomolecules. Here, we describe the construction and characterization of fluorophore-labeled glutamine binding proteins (QBP) and derivatives coupled to the independently designed hydrophobic polypeptide (E12) that can adhere onto solid surfaces via hydrophobic interactions. The single cysteine mutant (N160C QBP) modified with the three environmentally sensitive fluorescent dyes (IAANS, acrylodan, and IANBD ester) showed increased changes in fluorescence intensity induced by glutamine binding. The use of these conjugates as reagentless fluorescence sensors enables us to determine the glutamine concentrations (0.1-50 microM) in homogeneous solution. The fusion of N160C QBP with E12, (Gly4-Ser)n spacers (GSn), and IANBD resulted in the novel fluorescence sensing elements having an adhering capability to hydrophobic surfaces of unmodified microplates. In ELISA and fluorescence experiments for the microplates treated with a series of the conjugates, IANBD-labeled N160C QBP-GS1-E12 displayed the best reproducibility in adhesion onto the hydrophobic surfaces and the precise correlation between fluorescence changes and glutamine concentrations. The performance of the biosensor-attached microplate for glutamine titrations demonstrated that the hydrophobic interaction of E12 with solid surfaces is useful for effective immobilization of proteins that need specific conformational movements in recognizing particular biomolecules. Therefore, the technique using E12 as a surface-linking domain for protein adhesion onto unmodified substrates could be applied effectively to prepare microplates/arrays for a wide variety of high-throughput assays on chemical and biological samples.

Visible light-induced insulin aggregation on surfaces via photoexcitation of bound thioflavin T.

PubMed

Chouchane, Karim; Pignot-Paintrand, Isabelle; Bruckert, Franz; Weidenhaupt, Marianne

2018-04-01

Insulin is known to form amyloid aggregates when agitated in a hydrophobic container. Amyloid aggregation is routinely measured by the fluorescence of the conformational dye thioflavin T, which, when incorporated into amyloid fibers, fluoresces at 480 nm. The kinetics of amyloid aggregation in general is characterized by an initial lag-phase, during which aggregative nuclei form on the hydrophobic surface. These nuclei then lead to the formation of fibrils presenting a rapid growth during the elongation phase. Here we describe a novel mechanism of insulin amyloid aggregation which is surprisingly devoid of a lag-time for nucleation. The excitation of thioflavin T by visible light at 440 nm induces the aggregation of thioflavin T-positive insulin fibrils on hydrophobic surfaces in the presence of strong agitation and at physiological pH. This process is material surface-induced and depends on the fact that surface-adsorbed insulin can bind thioflavin T. Light-induced insulin aggregation kinetics is thioflavin T-mediated and is based on an energy transfer from visible light to the protein via thioflavin T. It relies on a constant supply of thioflavin T and insulin from the solution to the aggregate. The growth rate increases with the irradiance and with the concentration of thioflavin T. The supply of insulin seems to be the limiting factor of aggregate growth. This light-induced aggregation process allows the formation of local surface-bound aggregation patterns. Copyright © 2018 Elsevier B.V. All rights reserved.

Exogenous cathepsin V protein protects human cardiomyocytes HCM from angiotensin Ⅱ-Induced hypertrophy.

PubMed

Huang, Kun; Gao, Lu; Yang, Ming; Wang, Jiliang; Wang, Zheng; Wang, Lin; Wang, Guobin; Li, Huili

2017-08-01

Angiotensin (Ang) Ⅱ-induced cardiac hypertrophy can deteriorate to heart failure, a leading cause of mortality. Endogenous Cathepsin V (CTSV) has been reported to be cardioprotective against hypertrophy. However, little is known about the effect of exogenous CTSV on cardiac hypertrophy. We used the human cardiomyocytes HCM as a cell model to investigate the effects of exogenous CTSV on Ang Ⅱ-induced cardiac cell hypertrophy. Cell surface area and expression of classical markers of hypertrophy were analyzed. We further explored the mechanism of CTSV cardioprotective by assessing the levels and activities of PI3K/Akt/mTOR and MAPK signaling pathway proteins. We found that pre-treating cardiomyocytes with CTSV could significantly inhibit Ang Ⅱ-induced hypertrophy. The mRNA expression of hypertrophy markers ANP, BNP and β-MHC was obviously elevated in Ang Ⅱ-treated cardiac cells. Whereas, exogenous CTSV effectively halted this elevation. Further study revealed that the protective effects of exogenous CTSV might be mediated by repressing the phosphorylation of proteins in the PI3K/Akt/mTOR and MAPK pathways. Based on our results, we concluded that exogenous CTSV inhibited Ang Ⅱ-induced hypertrophy in HCM cells by inhibiting PI3K/Akt/mTOR. This study provides experimental evidence for the application of CTSV protein for the treatment of cardiac hypertrophy. Copyright © 2017 Elsevier Ltd. All rights reserved.

Shigella outer membrane protein PSSP-1 is broadly protective against Shigella infection.

PubMed

Kim, Jae-Ouk; Rho, Semi; Kim, Su Hee; Kim, Heejoo; Song, Hyo Jin; Kim, Eun Jin; Kim, Ryang Yeo; Kim, Eun Hye; Sinha, Anuradha; Dey, Ayan; Yang, Jae Seung; Song, Man Ki; Nandy, Ranjan Kumar; Czerkinsky, Cecil; Kim, Dong Wook

2015-04-01

In developing countries, Shigella is a primary cause of diarrhea in infants and young children. Although antibiotic therapy is an effective treatment for shigellosis, therapeutic options are narrowing due to the emergence of antibiotic resistance. Thus, preventive vaccination could become the most efficacious approach for controlling shigellosis. We have identified several conserved protein antigens that are shared by multiple Shigella serotypes and species. Among these, one antigen induced cross-protection against experimental shigellosis, and we have named it pan-Shigella surface protein 1 (PSSP-1). PSSP-1-induced protection requires a mucosal administration route and coadministration of an adjuvant. When PSSP-1 was administered intranasally, it induced cross-protection against Shigella flexneri serotypes 2a, 5a, and 6, Shigella boydii, Shigella sonnei, and Shigella dysenteriae serotype 1. Intradermally administered PSSP-1 induced strong serum antibody responses but failed to induce protection in the mouse lung pneumonia model. In contrast, intranasal administration elicited efficient local and systemic antibody responses and production of interleukin 17A and gamma interferon. Interestingly, blood samples from patients with recent-onset shigellosis showed variable but significant mucosal antibody responses to other conserved Shigella protein antigens but not to PSSP-1. We suggest that PSSP-1 is a promising antigen for a broadly protective vaccine against Shigella. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Ursolic acid, a natural pentacyclic triterpenoid, inhibits intracellular trafficking of proteins and induces accumulation of intercellular adhesion molecule-1 linked to high-mannose-type glycans in the endoplasmic reticulum

PubMed Central

Mitsuda, Satoshi; Yokomichi, Tomonobu; Yokoigawa, Junpei; Kataoka, Takao

2014-01-01

Ursolic acid (3β-hydroxy-urs-12-en-28-oic acid) is a natural pentacyclic triterpenoid that is present in many plants, including medicinal herbs, and foods. Ursolic acid was initially identified as an inhibitor of the expression of intercellular adhesion molecule-1 (ICAM-1) in response to interleukin-1α (IL-1α). We report here a novel biological activity: ursolic acid inhibits intracellular trafficking of proteins. Ursolic acid markedly inhibited the IL-1α-induced cell-surface ICAM-1 expression in human cancer cell lines and human umbilical vein endothelial cells. By contrast, ursolic acid exerted weak inhibitory effects on the IL-1α-induced ICAM-1 expression at the protein level. Surprisingly, we found that ursolic acid decreased the apparent molecular weight of ICAM-1 and altered the structures of N-linked oligosaccharides bound to ICAM-1. Ursolic acid induced the accumulation of ICAM-1 in the endoplasmic reticulum, which was linked mainly to high-mannose-type glycans. Moreover, in ursolic-acid-treated cells, the Golgi apparatus was fragmented into pieces and distributed over the cells. Thus, our results reveal that ursolic acid inhibits intracellular trafficking of proteins and induces the accumulation of ICAM-1 linked to high-mannose-type glycans in the endoplasmic reticulum. PMID:24649404

Tetraspanin Tspan9 regulates platelet collagen receptor GPVI lateral diffusion and activation

PubMed Central

Haining, Elizabeth J.; Matthews, Alexandra L.; Noy, Peter J.; Romanska, Hanna M.; Harris, Helen J.; Pike, Jeremy; Morowski, Martina; Gavin, Rebecca L.; Yang, Jing; Milhiet, Pierre-Emmanuel; Berditchevski, Fedor; Nieswandt, Bernhard; Poulter, Natalie S.; Watson, Steve P.; Tomlinson, Michael G.

2017-01-01

Abstract The tetraspanins are a superfamily of four-transmembrane proteins, which regulate the trafficking, lateral diffusion and clustering of the transmembrane proteins with which they interact. We have previously shown that tetraspanin Tspan9 is expressed on platelets. Here we have characterised gene-trap mice lacking Tspan9. The mice were viable with normal platelet numbers and size. Tspan9-deficient platelets were specifically defective in aggregation and secretion induced by the platelet collagen receptor GPVI, despite normal surface GPVI expression levels. A GPVI activation defect was suggested by partially impaired GPVI-induced protein tyrosine phosphorylation. In mechanistic experiments, Tspan9 and GPVI co-immunoprecipitated and co-localised, but super-resolution imaging revealed no defects in collagen-induced GPVI clustering on Tspan9-deficient platelets. However, single particle tracking using total internal reflection fluorescence microscopy showed that GPVI lateral diffusion was reduced by approximately 50% in the absence of Tspan9. Therefore, Tspan9 plays a fine-tuning role in platelet activation by regulating GPVI membrane dynamics. PMID:28032533

Effect of SDS on human hair: Study on the molecular structure and morphology.

PubMed

Singh, Bhawana; Umapathy, Siva

2011-05-01

This paper presents a model study to understand the effect of surfactants on the physicochemical properties of human hair. FT-IR ATR spectroscopy has been employed to understand the chemical changes induced by sodium dodecyl sulfate (SDS) on human scalp hair. In particular, the SDS induced changes in the secondary structure of protein present in the outer protective layer of hair, i.e. cuticle, have been investigated. Conformational changes in the secondary structure of protein were studied by curve fitting of the amide I band after every phase of SDS treatment. It has been found that SDS brings rearrangements in the protein backbone conformations by transforming β -sheet structure to random coil and β -turn. Additionally, AFM and SEM studies were carried out to understand the morphological changes induced on the hair surface. SEM and AFM images demonstrated the rupture and partial erosion of cuticle sublayers. Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Internalization and intracellular retention of CD4 are two separate functions of the human immunodeficiency virus type 1 Nef protein.

PubMed

Giolo, Giorgia; Neri, Francesca; Casartelli, Nicoletta; Potestà, Marina; Belleudi, Francesca; Torrisi, Maria Rosaria; Doria, Margherita

2007-11-01

The pathogenic Nef protein of the human immunodeficiency virus type 1 (HIV-1) downregulates CD4 by inducing its endocytosis and by inhibiting the transport of the receptor to the cell membrane. By means of in vivo-selected mutations, we show that L37, P78 and E177 residues of Nef are required for its effect on CD4 internalization and recycling but dispensable for Nef-induced retention and degradation of intracellular CD4. Of note, the function of Nef on the anterograde transport of newly synthesized CD4 molecules is irrelevant in cells with a slow constitutive CD4 turnover such as T cell lines. Moreover, we show that a mutated CD4 that is unresponsive to Nef-mediated endocytosis, CD4LL(144)AA, is retained intracellularly and degraded by Nef like wild-type CD4. Thus, Nef's abilities to enhance endocytosis and induce intracellular retention of CD4 are mediated by separate protein surfaces and occur through distinct mechanisms.

Flavonoids inhibit cytokine-induced endothelial cell adhesion protein gene expression.

PubMed Central

Gerritsen, M. E.; Carley, W. W.; Ranges, G. E.; Shen, C. P.; Phan, S. A.; Ligon, G. F.; Perry, C. A.

1995-01-01

Treatment of human endothelial cells with cytokines such as interleukin-1, tumor necrosis factor-alpha (TNF-alpha) or interferon-gamma induces the expression of specific leukocyte adhesion molecules on the endothelial cell surface. Interfering with either leukocyte adhesion or adhesion protein upregulation is an important therapeutic target as evidenced by the potent anti-inflammatory actions of neutralizing antibodies to these ligands in various animal models and in patients. In the present study we report that cotreatment of human endothelial cells with certain hydroxyflavones and flavanols blocks cytokine-induced ICAM-1, VCAM-1, and E-selectin expression on human endothelial cells. One of the most potent flavones, apigenin, exhibited a dose- and time-dependent, reversible effect on adhesion protein expression as well as inhibiting adhesion protein upregulation at the transcriptional level. Apigenin also inhibited IL-1 alpha-induced prostaglandin synthesis and TNF-alpha-induced IL-6 and IL-8 production, suggesting that the hydroxyflavones may act as general inhibitors of cytokine-induced gene expression. Although apigenin did not inhibit TNF-alpha-induced nuclear translocation of NF-kappa B(p50(NFKB1)/p65(RelA)) we found this flavonoid did inhibit TNF-alpha induced beta-galactosidase activity in SW480 cells stably transfected with a beta-galactosidase reporter construct driven by four NF-kappa B elements, suggesting an action on NF-kappa B transcriptional activation. Adhesion of leukocytes to cytokine-treated endothelial cells was blocked in endothelial cells cotreated with apigenin. Finally, apigenin demonstrated potent anti-inflammatory activity in carrageenan induced rat paw edema and delayed type hypersensitivity in the mouse. We conclude that flavonoids offer important therapeutic potential for the treatment of a variety of inflammatory diseases involving an increase in leukocyte adhesion and trafficking. Images Figure 7 Figure 8 Figure 11 PMID:7543732

Fluorescence probe techniques to monitor protein adsorption-induced conformation changes on biodegradable polymers.

PubMed

Benesch, Johan; Hungerford, Graham; Suhling, Klaus; Tregidgo, Carolyn; Mano, João F; Reis, Rui L

2007-08-15

The study of protein adsorption and any associated conformational changes on interaction with biomaterials is of great importance in the area of implants and tissue constructs. This study aimed to evaluate some fluorescent techniques to probe protein conformation on a selection of biodegradable polymers currently under investigation for biomedical applications. Because of the fluorescence emanating from the polymers, the use of monitoring intrinsic protein fluorescence was precluded. A highly solvatochromic fluorescent dye, Nile red, and a well-known protein label, fluorescein isothiocyanate, were employed to study the adsorption of serum albumin to polycaprolactone and to some extent also to two starch-containing polymer blends (SPCL and SEVA-C). A variety of fluorescence techniques, steady state, time resolved, and imaging were employed. Nile red was found to leach from the protein, while fluorescein isothiocyanate proved useful in elucidating a conformational change in the protein and the observation of protein aggregates adsorbed to the polymer surface. These effects were seen by making use of the phenomenon of energy migration between the fluorescent tags to monitor interprobe distance and the use of fluorescence lifetime imaging to ascertain the surface packing of the protein on polymer.

AMP-activated protein kinase-mediated glucose transport as a novel target of tributyltin in human embryonic carcinoma cells.

PubMed

Yamada, Shigeru; Kotake, Yaichiro; Sekino, Yuko; Kanda, Yasunari

2013-05-01

Organotin compounds such as tributyltin (TBT) are known to cause various forms of cytotoxicity, including developmental toxicity and neurotoxicity. However, the molecular target of the toxicity induced by nanomolar levels of TBT has not been identified. In the present study, we found that exposure to 100 nM TBT induced growth arrest in human pluripotent embryonic carcinoma cell line NT2/D1. Since glucose provides metabolic energy, we focused on the glycolytic system. We found that exposure to TBT reduced the levels of both glucose-6-phosphate and fructose-6-phosphate. To investigate the effect of TBT exposure on glycolysis, we examined glucose transporter (GLUT) activity. TBT exposure inhibited glucose uptake via a decrease in the level of cell surface-bound GLUT1. Furthermore, we examined the effect of AMP-activated protein kinase (AMPK), which is known to regulate glucose transport by facilitating GLUT translocation. Treatment with the potent AMPK activator, AICAR, restored the TBT-induced reduction in cell surface-bound GLUT1 and glucose uptake. In conclusion, these results suggest that exposure to nanomolar levels of TBT causes growth arrest by targeting glycolytic systems in human embryonic carcinoma cells. Thus, understanding the energy metabolism may provide new insights into the mechanisms of metal-induced cytotoxicity.

Interaction of lysozyme protein with different sized silica nanoparticles and their resultant structures

NASA Astrophysics Data System (ADS)

Yadav, Indresh; Aswal, V. K.; Kohlbrecher, J.

2016-05-01

The interaction of model protein-lysozyme with three different sized anionic silica nanoparticles has been studied by UV-vis spectroscopy, dynamic light scattering (DLS) and small-angle neutron scattering (SANS). The surface area and curvature of the nanoparticles change with size, which significantly influence their interaction with protein. The lysozyme adsorbs on the surface of the nanoparticles due to electrostatic attraction and leads to the phase transformation from one phase (clear) to two-phase (turbid) of the nanoparticle-protein system. The dominance of lysozyme induced short-range attraction over long-range electrostatic repulsion between nanoparticles is responsible for phase transformation and modeled by the two-Yukawa potential. The magnitude of the attractive interaction increases with the size of the nanoparticles as a result the phase transformation commences relatively at lower concentration of lysozyme. The structure of the nanoparticle-protein system in two-phase is characterized by the diffusion limited aggregate type of mass fractal morphology.

Heterogeneous Nucleation of Protein Crystals on Fluorinated Layered Silicate

PubMed Central

Ino, Keita; Udagawa, Itsumi; Iwabata, Kazuki; Takakusagi, Yoichi; Kubota, Munehiro; Kurosaka, Keiichi; Arai, Kazuhito; Seki, Yasutaka; Nogawa, Masaya; Tsunoda, Tatsuo; Mizukami, Fujio; Taguchi, Hayao; Sakaguchi, Kengo

2011-01-01

Here, we describe an improved system for protein crystallization based on heterogeneous nucleation using fluorinated layered silicate. In addition, we also investigated the mechanism of nucleation on the silicate surface. Crystallization of lysozyme using silicates with different chemical compositions indicated that fluorosilicates promoted nucleation whereas the silicates without fluorine did not. The use of synthesized saponites for lysozyme crystallization confirmed that the substitution of hydroxyl groups contained in the lamellae structure for fluorine atoms is responsible for the nucleation-inducing property of the nucleant. Crystallization of twelve proteins with a wide range of pI values revealed that the nucleation promoting effect of the saponites tended to increase with increased substitution rate. Furthermore, the saponite with the highest fluorine content promoted nucleation in all the test proteins regardless of their overall net charge. Adsorption experiments of proteins on the saponites confirmed that the density of adsorbed molecules increased according to the substitution rate, thereby explaining the heterogeneous nucleation on the silicate surface. PMID:21818343

Interaction of lysozyme protein with different sized silica nanoparticles and their resultant structures

DOE Office of Scientific and Technical Information (OSTI.GOV)

Yadav, Indresh, E-mail: iykumarindresh288@gmail.com; Aswal, V. K.; Kohlbrecher, J.

The interaction of model protein-lysozyme with three different sized anionic silica nanoparticles has been studied by UV-vis spectroscopy, dynamic light scattering (DLS) and small-angle neutron scattering (SANS). The surface area and curvature of the nanoparticles change with size, which significantly influence their interaction with protein. The lysozyme adsorbs on the surface of the nanoparticles due to electrostatic attraction and leads to the phase transformation from one phase (clear) to two-phase (turbid) of the nanoparticle-protein system. The dominance of lysozyme induced short-range attraction over long-range electrostatic repulsion between nanoparticles is responsible for phase transformation and modeled by the two-Yukawa potential. Themore » magnitude of the attractive interaction increases with the size of the nanoparticles as a result the phase transformation commences relatively at lower concentration of lysozyme. The structure of the nanoparticle-protein system in two-phase is characterized by the diffusion limited aggregate type of mass fractal morphology.« less

BcXYG1, a Secreted Xyloglucanase from Botrytis cinerea, Triggers Both Cell Death and Plant Immune Responses.

PubMed

Zhu, Wenjun; Ronen, Mordechi; Gur, Yonatan; Minz-Dub, Anna; Masrati, Gal; Ben-Tal, Nir; Savidor, Alon; Sharon, Itai; Eizner, Elad; Valerius, Oliver; Braus, Gerhard H; Bowler, Kyle; Bar-Peled, Maor; Sharon, Amir

2017-09-01

In search of Botrytis cinerea cell death-inducing proteins, we found a xyloglucanase (BcXYG1) that induced strong necrosis and a resistance response in dicot plants. Expression of the BcXYG1 gene was strongly induced during the first 12 h post inoculation, and analysis of disease dynamics using PathTrack showed that a B. cinerea strain overexpressing BcXYG1 produced early local necrosis, supporting a role of BcXYG1 as an early cell death-inducing factor. The xyloglucanase activity of BcXYG1 was not necessary for the induction of necrosis and plant resistance, as a mutant of BcXYG1 lacking the xyloglucanase enzymatic activity retained both functions. Residues in two exposed loops on the surface of BcXYG1 were found to be necessary for the induction of cell death but not to induce plant resistance. Further analyses showed that BcXYG1 is apoplastic and possibly interacts with the proteins of the plant cell membrane and also that the BcXYG1 cell death-promoting signal is mediated by the leucine-rich repeat receptor-like kinases BAK1 and SOBIR1. Our findings support the role of cell death-inducing proteins in establishing the infection of necrotrophic pathogens and highlight the recognition of fungal apoplastic proteins by the plant immune system as an important mechanism of resistance against this class of pathogens. © 2017 American Society of Plant Biologists. All Rights Reserved.

Involvement of protein kinase C in the modulation of morphine-induced analgesia and the inhibitory effects of exposure to 60-hz magnetic fields in the land snail, Cepaea nemoralis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kavaliers, M.; Ossenkopp, K.P.

1990-02-26

One of the more consistent and dramatic effects of exposure to magnetic fields is the attenuation of morphine-induced analgesia. Results of previous studies have implicated alterations in calcium channel functioning and Ca{sup ++} flux in the mediation of these effects. It is generally accepted that Ca{sup ++}-activated-phospholipid-dependent protein kinase (Protein kinase C; PKC) plays an important role in relaying trans-membrane signaling in diverse Ca{sup ++} dependent cellular processes. In experiment 1 we observed that morphine-induced analgesia in the land snail, Cepaea nemoralis, as measured by the latency of an avoidance behavior to a warmed surface, was reduced by the PKCmore » activator, SC-9, and was enhanced by the PKC inhibitors, H-7 and H-9. In contrast, HA-10004, a potent inhibitor of other protein kinases, but only a very weak inhibitor of PKC, had no effect on morphine-induced analgesia. In experiment 2 exposure of snails for 30 minutes to a 1.0 gauss (rms) 60-Hz magnetic field reduced morphine-induced analgesia. This inhibitory effect of the magnetic field was reduced by the PKC inhibitors, H-7 and H-9, and was augmented by the PKC activator SC-9. These results suggest that: (i) PKC is involved in the modulation of morphine-induced analgesia and, (ii) the inhibitory effects of magnetic fields involve PKC.« less

Protein covalent immobilization via its scarce thiol versus abundant amine groups: Effect on orientation, cell binding domain exposure and conformational lability.

PubMed

Ba, O M; Hindie, M; Marmey, P; Gallet, O; Anselme, K; Ponche, A; Duncan, A C

2015-10-01

Quantity, orientation, conformation and covalent linkage of naturally cell adhesive proteins adsorbed or covalently linked to a surface, are known to influence the preservation of their subsequent long term cell adhesion properties and bioactivity. In the present work, we explore two different strategies for the covalent linking of plasma fibronectin (pFN) - used as a cell adhesive model protein, onto a polystyrene (PS) surface. One is aimed at tethering the protein to the surface in a semi-oriented fashion (via one of the 4 free thiol reactive groups on the protein) with a heterofunctional coupling agent (SSMPB method). The other aims to immobilize the protein in a more random fashion by reaction between the abundant pendant primary amine bearing amino acids of the pFN and activated carboxylic surface functions obtained after glutaric anhydride surface treatment (GA method). The overall goal will be to verify the hypothesis of a correlation between covalent immobilization of a model cell adhesive protein to a PS surface in a semi-oriented configuration (versus randomly oriented) with promotion of enhanced exposure of the protein's cell binding domain. This in turn would lead to enhanced cell adhesion. Ideally the goal is to elaborate substrates exhibiting a long term stable protein monolayer with preserved cell adhesive properties and bioactivity for biomaterial and/or cell adhesion commercial plate applications. However, the initial restrictive objective of this paper is to first quantitatively and qualitatively investigate the reversibly (merely adsorbed) versus covalently irreversibly bound protein to the surface after the immobilization procedure. Although immobilized surface amounts were similar (close to the monolayer range) for all immobilization approaches, covalent grafting showed improved retention and stronger "tethering" of the pFN protein to the surface (roughly 40%) after SDS rinsing compared to that for mere adsorption (0%) suggesting an added value to the covalent grafting immobilization methods. However no differences in exposure of the cell binding domains were observed (ELISA results) before SDS rinsing, suggesting that pFN protein grafting to the surface is initially kinetically driven be a stochastic random adsorption phenomenon. Covalent grafting acts in the final stage as a process that simply tethers and stabilizes (or freezes) the initial conformation/orientation of the adsorbed protein on the surface. In addition covalent linkage via the SSMPB approach is likely favored by surface-induce exposure of one of the normally hidden free thiol group pair, thus optimizing covalent linkage to the surface. However after SDS rinsing, this "tethering"/"freezing" effect was significantly more prominent for the GA grafting approach (due to greater number of potential covalent links between the protein and the surface) compared to that for the SSMPB approach. This hypothesis was buttressed by the improved resistance to denaturation (smaller conformational lability) for the GA compared to the SMPB approach and improved exposure of the cell binding domain for the former (>50%) even after SDS rinsing. These results are promising in that they suggest covalent tethering of fibronectin to PS substrate in a monolayer range, with significantly improved irreversible protein surface bonding via both approaches (compared to that for mere adsorption). The latter are likely applicable to a wide range of proteins. Copyright © 2015 Elsevier B.V. All rights reserved.

Templating Biomineralization: Surface Directed Protein Self-assembly and External Magnetic Field Stimulation of Osteoblasts

NASA Astrophysics Data System (ADS)

Ba, Xiaolan

Biomineralization is a wide-spread phenomenon in the biological systems, which is the process of mineral formation by organisms through interaction between its organic contents and the inorganic minerals. The process is essential in a broad spectrum of biological phenomena ranging from bone and tooth formation to pathological mineralization under hypoxic conditions or cancerous formations. In this thesis I studied biomineralization at the earliest stages in order to obtain a better understanding of the fundamental principals involved. This knowledge is essential if we want to engineer devices which will increase bone regeneration or prevent unwanted mineral deposits. Extracellular matrix (ECM) proteins play an essential role during biomineralization in bone and engineered tissues. In this dissertation, I present an approach to mimic the ECM in vitro to probe the interactions of these proteins with calcium phosphate mineral and with each other. Early stage of mineralization is investigated by mechanical properties of the protein fibers using Scanning Probe Microscopy (SPM) and Shear Modulation Force Microscopy (SMFM). The development of mineral crystals on the protein matrices is also characterized by Scanning Electron Microscopy (SEM), Transmission Electron Microscopy (TEM) and Grazing Incidence X-ray Diffraction (GIXRD). The results demonstrate complementary actions of the two ECM proteins to collect cations and template calcium phosphate mineral, respectively. Magnets have been clinically used as an "induction source" in various bone or orthodontic treatments. However, the mechanism and effects of magnetic fields remain unclear. In this dissertation, I also undertake the present investigation to study the effects of 150 mT static magnetic fields (SMF) on ECM development and cell biomineralization using MC3T3-E1 osteobalst-like cells. Early stage of biomineralization is characterized by SPM, SMFM and confocal laser scanning microscopy (CSLM). Late stage of biomineralization is investigated by SEM, GIXRD and energy dispersive X-ray spectroscopy (EDXS). Gene expression during the exposure of SMF is also studies by RT-PCR. The results indicated that exposure to SMF induces osteoblasts to produce larger quantities of HA, with higher degree of crystalline order. The controlling and understanding of protein on the surface is of great interest in biomedical application such as implant medicine, biosensor design, food processing, and chromatographic separations. The adsorbed protein onto the surface significantly determines the performance of biomaterials in a biological environment. Recent studies have suggested that the preservation of the native secondary structure of protein adsorbed is essential for biological application. In order to manipulate protein adsorption and design biocompatible materials, the mechanisms underlying protein-surface interactions, especially how surface properties of materials induce conformational changes of adsorbed proteins, needs to be well understood. Here we demonstrated that even though SPS is a necessary condition, it is not sufficient. We show that low substrate conductivity as well as proper salt concentration are also critical in sustained protein adsorption continuously. These factors allow one to pattern regions of different conducting properties and for the first time patterns physiologically relevant protein structures. Here we show that we can achieve patterned biomineralized regimes, both with plasma proteins in a simple and robust manner without additional functionalization or application of electrochemical gradients. Since the data indicate that the patterns just need to differ in electrical conductivity, rather than surface chemistry, we propose that the creation of transient image charges, due to incomplete charge screening, may be responsible for sustain the driving force for continual protein absorption.

TLR-2 Recognizes Propionibacterium acnes CAMP Factor 1 from Highly Inflammatory Strains

PubMed Central

Ollagnier, Guillaume; Désiré, Nathalie; Sayon, Sophie; Raingeaud, Jöel; Marcelin, Anne-Geneviève; Calvez, Vincent; Khammari, Amir; Batteux, Frédéric; Dréno, Brigitte; Dupin, Nicolas

2016-01-01

Background Propionibacterium acnes (P. acnes) is an anaerobic, Gram-positive bacteria encountered in inflammatory acne lesions, particularly in the pilosebaceous follicle. P. acnes triggers a strong immune response involving keratinocytes, sebocytes and monocytes, the target cells during acne development. Lipoteicoic acid and peptidoglycan induce the inflammatory reaction, but no P. acnes surface protein interacting with Toll-like receptors has been identified. P. acnes surface proteins have been extracted by lithium stripping and shown to induce CXCL8 production by keratinocytes. Methodology and principal findings Far-western blotting identified two surface proteins, of 24.5- and 27.5-kDa in size, specifically recognized by TLR2. These proteins were characterized, by LC-MS/MS, as CAMP factor 1 devoid of its signal peptide sequence, as shown by N-terminal sequencing. Purified CAMP factor 1 induces CXCL8 production by activating the CXCL8 gene promoter, triggering the synthesis of CXCL8 mRNA. Antibodies against TLR2 significantly decreased the CXCL8 response. For the 27 P. acnes strains used in this study, CAMP1-TLR2 binding intensity was modulated and appeared to be strong in type IB and II strains, which produced large amounts of CXCL8, whereas most of the type IA1 and IA2 strains presented little or no CAMP1-TLR2 binding and low levels of CXCL8 production. The nucleotide sequence of CAMP factor displays a major polymorphism, defining two distinct genetic groups corresponding to CAMP factor 1 with 14 amino-acid changes from strains phylotyped II with moderate and high levels of CAMP1-TLR2 binding activity, and CAMP factor 1 containing 0, 1 or 2 amino-acid changes from strains phylotyped IA1, IA2, or IB presenting no, weak or moderate CAMP1-TLR2 binding. Conclusions Our findings indicate that CAMP factor 1 may contribute to P. acnes virulence, by amplifying the inflammation reaction through direct interaction with TLR2. PMID:27902761

TLR-2 Recognizes Propionibacterium acnes CAMP Factor 1 from Highly Inflammatory Strains.

PubMed

Lheure, Coralie; Grange, Philippe Alain; Ollagnier, Guillaume; Morand, Philippe; Désiré, Nathalie; Sayon, Sophie; Corvec, Stéphane; Raingeaud, Jöel; Marcelin, Anne-Geneviève; Calvez, Vincent; Khammari, Amir; Batteux, Frédéric; Dréno, Brigitte; Dupin, Nicolas

2016-01-01

Propionibacterium acnes (P. acnes) is an anaerobic, Gram-positive bacteria encountered in inflammatory acne lesions, particularly in the pilosebaceous follicle. P. acnes triggers a strong immune response involving keratinocytes, sebocytes and monocytes, the target cells during acne development. Lipoteicoic acid and peptidoglycan induce the inflammatory reaction, but no P. acnes surface protein interacting with Toll-like receptors has been identified. P. acnes surface proteins have been extracted by lithium stripping and shown to induce CXCL8 production by keratinocytes. Far-western blotting identified two surface proteins, of 24.5- and 27.5-kDa in size, specifically recognized by TLR2. These proteins were characterized, by LC-MS/MS, as CAMP factor 1 devoid of its signal peptide sequence, as shown by N-terminal sequencing. Purified CAMP factor 1 induces CXCL8 production by activating the CXCL8 gene promoter, triggering the synthesis of CXCL8 mRNA. Antibodies against TLR2 significantly decreased the CXCL8 response. For the 27 P. acnes strains used in this study, CAMP1-TLR2 binding intensity was modulated and appeared to be strong in type IB and II strains, which produced large amounts of CXCL8, whereas most of the type IA1 and IA2 strains presented little or no CAMP1-TLR2 binding and low levels of CXCL8 production. The nucleotide sequence of CAMP factor displays a major polymorphism, defining two distinct genetic groups corresponding to CAMP factor 1 with 14 amino-acid changes from strains phylotyped II with moderate and high levels of CAMP1-TLR2 binding activity, and CAMP factor 1 containing 0, 1 or 2 amino-acid changes from strains phylotyped IA1, IA2, or IB presenting no, weak or moderate CAMP1-TLR2 binding. Our findings indicate that CAMP factor 1 may contribute to P. acnes virulence, by amplifying the inflammation reaction through direct interaction with TLR2.

On the Hofmeister effect: fluctuations at the protein-water interface and the surface tension.

PubMed

Bogár, Ferenc; Bartha, Ferenc; Násztor, Zoltán; Fábián, László; Leitgeb, Balázs; Dér, András

2014-07-24

We performed molecular dynamics simulations on the tryptophane-cage miniprotein using a nonpolarizable force field, in order to model the effect of concentrated water solutions of neutral salts on protein conformation, which is a manifestation of Hofmeister effects. From the equilibrium values and the fluctuations of the solvent accessible surface area of the miniprotein, the salt-induced changes of the mean value of protein-water interfacial tension were determined. At 300 K, the chaotropic ClO4(-) and NO3(-) decreased the interfacial tension according to their position in the Hofmeister series (by approximately 5 and 2.7 mN/m, respectively), while the kosmotropic F(-) increased it (by 1 mN/m). These values were compared to those obtained from the Gibbs equation using the excess surface adsorption calculated from the probability distribution of the water molecules and ions around the miniprotein, and the two sets were found to be very close to each other. Our results present a direct evidence for the central role of interfacial tension and fluctuations at the protein-water interface in Hofmeister phenomena, and provide a computational method for the determination of the protein-water interfacial tension, establishing a link between the phenomenological and microscopic description of protein-water interfaces.

Mesoscale computational studies of membrane bilayer remodeling by curvature-inducing proteins

PubMed Central

Ramakrishnan, N.; Sunil Kumar, P. B.; Radhakrishnan, Ravi

2014-01-01

Biological membranes constitute boundaries of cells and cell organelles. These membranes are soft fluid interfaces whose thermodynamic states are dictated by bending moduli, induced curvature fields, and thermal fluctuations. Recently, there has been a flood of experimental evidence highlighting active roles for these structures in many cellular processes ranging from trafficking of cargo to cell motility. It is believed that the local membrane curvature, which is continuously altered due to its interactions with myriad proteins and other macromolecules attached to its surface, holds the key to the emergent functionality in these cellular processes. Mechanisms at the atomic scale are dictated by protein-lipid interaction strength, lipid composition, lipid distribution in the vicinity of the protein, shape and amino acid composition of the protein, and its amino acid contents. The specificity of molecular interactions together with the cooperativity of multiple proteins induce and stabilize complex membrane shapes at the mesoscale. These shapes span a wide spectrum ranging from the spherical plasma membrane to the complex cisternae of the Golgi apparatus. Mapping the relation between the protein-induced deformations at the molecular scale and the resulting mesoscale morphologies is key to bridging cellular experiments across the various length scales. In this review, we focus on the theoretical and computational methods used to understand the phenomenology underlying protein-driven membrane remodeling. Interactions at the molecular scale can be computationally probed by all atom and coarse grained molecular dynamics (MD, CGMD), as well as dissipative particle dynamics (DPD) simulations, which we only describe in passing. We choose to focus on several continuum approaches extending the Canham - Helfrich elastic energy model for membranes to include the effect of curvature-inducing proteins and explore the conformational phase space of such systems. In this description, the protein is expressed in the form of a spontaneous curvature field. The approaches include field theoretical methods limited to the small deformation regime, triangulated surfaces and particle-based computational models to investigate the large-deformation regimes observed in the natural state of many biological membranes. Applications of these methods to understand the properties of biological membranes in homogeneous and inhomogeneous environments of proteins, whose underlying curvature fields are either isotropic or anisotropic, are discussed. The diversity in the curvature fields elicits a rich variety of morphological states, including tubes, discs, branched tubes, and caveola. Mapping the thermodynamic stability of these states as a function of tuning parameters such as concentration and strength of curvature induction of the proteins is discussed. The relative stabilities of these self-organized shapes are examined through free-energy calculations. The suite of methods discussed here can be tailored to applications in specific cellular settings such as endocytosis during cargo trafficking and tubulation of filopodial structures in migrating cells, which makes these methods a powerful complement to experimental studies. PMID:25484487

Mesoscale computational studies of membrane bilayer remodeling by curvature-inducing proteins.

PubMed

Ramakrishnan, N; Sunil Kumar, P B; Radhakrishnan, Ravi

2014-10-01

Biological membranes constitute boundaries of cells and cell organelles. These membranes are soft fluid interfaces whose thermodynamic states are dictated by bending moduli, induced curvature fields, and thermal fluctuations. Recently, there has been a flood of experimental evidence highlighting active roles for these structures in many cellular processes ranging from trafficking of cargo to cell motility. It is believed that the local membrane curvature, which is continuously altered due to its interactions with myriad proteins and other macromolecules attached to its surface, holds the key to the emergent functionality in these cellular processes. Mechanisms at the atomic scale are dictated by protein-lipid interaction strength, lipid composition, lipid distribution in the vicinity of the protein, shape and amino acid composition of the protein, and its amino acid contents. The specificity of molecular interactions together with the cooperativity of multiple proteins induce and stabilize complex membrane shapes at the mesoscale. These shapes span a wide spectrum ranging from the spherical plasma membrane to the complex cisternae of the Golgi apparatus. Mapping the relation between the protein-induced deformations at the molecular scale and the resulting mesoscale morphologies is key to bridging cellular experiments across the various length scales. In this review, we focus on the theoretical and computational methods used to understand the phenomenology underlying protein-driven membrane remodeling. Interactions at the molecular scale can be computationally probed by all atom and coarse grained molecular dynamics (MD, CGMD), as well as dissipative particle dynamics (DPD) simulations, which we only describe in passing. We choose to focus on several continuum approaches extending the Canham - Helfrich elastic energy model for membranes to include the effect of curvature-inducing proteins and explore the conformational phase space of such systems. In this description, the protein is expressed in the form of a spontaneous curvature field. The approaches include field theoretical methods limited to the small deformation regime, triangulated surfaces and particle-based computational models to investigate the large-deformation regimes observed in the natural state of many biological membranes. Applications of these methods to understand the properties of biological membranes in homogeneous and inhomogeneous environments of proteins, whose underlying curvature fields are either isotropic or anisotropic, are discussed. The diversity in the curvature fields elicits a rich variety of morphological states, including tubes, discs, branched tubes, and caveola. Mapping the thermodynamic stability of these states as a function of tuning parameters such as concentration and strength of curvature induction of the proteins is discussed. The relative stabilities of these self-organized shapes are examined through free-energy calculations. The suite of methods discussed here can be tailored to applications in specific cellular settings such as endocytosis during cargo trafficking and tubulation of filopodial structures in migrating cells, which makes these methods a powerful complement to experimental studies.

Mesoscale computational studies of membrane bilayer remodeling by curvature-inducing proteins

NASA Astrophysics Data System (ADS)

Ramakrishnan, N.; Sunil Kumar, P. B.; Radhakrishnan, Ravi

2014-10-01

Biological membranes constitute boundaries of cells and cell organelles. These membranes are soft fluid interfaces whose thermodynamic states are dictated by bending moduli, induced curvature fields, and thermal fluctuations. Recently, there has been a flood of experimental evidence highlighting active roles for these structures in many cellular processes ranging from trafficking of cargo to cell motility. It is believed that the local membrane curvature, which is continuously altered due to its interactions with myriad proteins and other macromolecules attached to its surface, holds the key to the emergent functionality in these cellular processes. Mechanisms at the atomic scale are dictated by protein-lipid interaction strength, lipid composition, lipid distribution in the vicinity of the protein, shape and amino acid composition of the protein, and its amino acid contents. The specificity of molecular interactions together with the cooperativity of multiple proteins induce and stabilize complex membrane shapes at the mesoscale. These shapes span a wide spectrum ranging from the spherical plasma membrane to the complex cisternae of the Golgi apparatus. Mapping the relation between the protein-induced deformations at the molecular scale and the resulting mesoscale morphologies is key to bridging cellular experiments across various length scales. In this review, we focus on the theoretical and computational methods used to understand the phenomenology underlying protein-driven membrane remodeling. Interactions at the molecular scale can be computationally probed by all atom and coarse grained molecular dynamics (MD, CGMD), as well as dissipative particle dynamics (DPD) simulations, which we only describe in passing. We choose to focus on several continuum approaches extending the Canham-Helfrich elastic energy model for membranes to include the effect of curvature-inducing proteins and explore the conformational phase space of such systems. In this description, the protein is expressed in the form of a spontaneous curvature field. The approaches include field theoretical methods limited to the small deformation regime, triangulated surfaces and particle-based computational models to investigate the large-deformation regimes observed in the natural state of many biological membranes. Applications of these methods to understand the properties of biological membranes in homogeneous and inhomogeneous environments of proteins, whose underlying curvature fields are either isotropic or anisotropic, are discussed. The diversity in the curvature fields elicits a rich variety of morphological states, including tubes, discs, branched tubes, and caveola. Mapping the thermodynamic stability of these states as a function of tuning parameters such as concentration and strength of curvature induction of the proteins is discussed. The relative stabilities of these self-organized shapes are examined through free-energy calculations. The suite of methods discussed here can be tailored to applications in specific cellular settings such as endocytosis during cargo trafficking and tubulation of filopodial structures in migrating cells, which makes these methods a powerful complement to experimental studies.

Membrane protein GARP is a receptor for latent TGF-beta on the surface of activated human Treg.

PubMed

Stockis, Julie; Colau, Didier; Coulie, Pierre G; Lucas, Sophie

2009-12-01

Human Treg and Th clones secrete the latent form of TGF-beta, in which the mature TGF-beta protein is bound to the latency-associated peptide (LAP), and is thereby prevented from binding to the TGF-beta receptor. We previously showed that upon TCR stimulation, human Treg clones but not Th clones produce active TGF-beta and bear LAP on their surface. Here, we show that latent TGF-beta, i.e. both LAP and mature TGF-beta, binds to glycoprotein A repetitions predominant (GARP), a transmembrane protein containing leucine rich repeats, which is present on the surface of stimulated Treg clones but not on Th clones. Membrane localization of latent TGF-beta mediated by binding to GARP may be necessary for the ability of Treg to activate TGF-beta upon TCR stimulation. However, it is not sufficient as lentiviral-mediated expression of GARP in human Th cells induces binding of latent TGF-beta to the cell surface, but does not result in the production of active TGF-beta upon stimulation of these Th cells.

A surface transporter family conveys the trypanosome differentiation signal.

PubMed

Dean, Samuel; Marchetti, Rosa; Kirk, Kiaran; Matthews, Keith R

2009-05-14

Microbial pathogens use environmental cues to trigger the developmental events needed to infect mammalian hosts or transmit to disease vectors. The parasites causing African sleeping sickness respond to citrate or cis-aconitate (CCA) to initiate life-cycle development when transmitted to their tsetse fly vector. This requires hypersensitization of the parasites to CCA by exposure to low temperature, conditions encountered after tsetse fly feeding at dusk or dawn. Here we identify a carboxylate-transporter family, PAD (proteins associated with differentiation), required for perception of this differentiation signal. Consistent with predictions for the response of trypanosomes to CCA, PAD proteins are expressed on the surface of the transmission-competent 'stumpy-form' parasites in the bloodstream, and at least one member is thermoregulated, showing elevated expression and surface access at low temperature. Moreover, RNA-interference-mediated ablation of PAD expression diminishes CCA-induced differentiation and eliminates CCA hypersensitivity under cold-shock conditions. As well as being molecular transducers of the differentiation signal in these parasites, PAD proteins provide the first example of a surface marker able to discriminate the transmission stage of trypanosomes in their mammalian host.

The crystal structure of DR6 in complex with the amyloid precursor protein provides insight into death receptor activation

DOE Office of Scientific and Technical Information (OSTI.GOV)

Xu, Kai; Olsen, Olav; Tzvetkova-Robev, Dorothea

The amyloid precursor protein (APP) has garnered considerable attention due to its genetic links to Alzheimer's disease. Death receptor 6 (DR6) was recently shown to bind APP via the protein extracellular regions, stimulate axonal pruning, and inhibit synapse formation. Here, we report the crystal structure of the DR6 ectodomain in complex with the E2 domain of APP and show that it supports a model for APP-induced dimerization and activation of cell surface DR6.

The crystal structure of DR6 in complex with the amyloid precursor protein provides insight into death receptor activation

DOE PAGES

Xu, Kai; Olsen, Olav; Tzvetkova-Robev, Dorothea; ...

2015-04-02

The amyloid precursor protein (APP) has garnered considerable attention due to its genetic links to Alzheimer's disease. Death receptor 6 (DR6) was recently shown to bind APP via the protein extracellular regions, stimulate axonal pruning, and inhibit synapse formation. Here, we report the crystal structure of the DR6 ectodomain in complex with the E2 domain of APP and show that it supports a model for APP-induced dimerization and activation of cell surface DR6.

Wsc1 and Mid2 Are Cell Surface Sensors for Cell Wall Integrity Signaling That Act through Rom2, a Guanine Nucleotide Exchange Factor for Rho1

PubMed Central

Philip, Bevin; Levin, David E.

2001-01-01

Wsc1 and Mid2 are highly O-glycosylated cell surface proteins that reside in the plasma membrane of Saccharomyces cerevisiae. They have been proposed to function as mechanosensors of cell wall stress induced by wall remodeling during vegetative growth and pheromone-induced morphogenesis. These proteins are required for activation of the cell wall integrity signaling pathway that consists of the small G-protein Rho1, protein kinase C (Pkc1), and a mitogen-activated protein kinase cascade. We show here by two-hybrid experiments that the C-terminal cytoplasmic domains of Wsc1 and Mid2 interact with Rom2, a guanine nucleotide exchange factor (GEF) for Rho1. At least with regard to Wsc1, this interaction is mediated by the Rom2 N-terminal domain. This domain is distinct from the Rho1-interacting domain, suggesting that the GEF can interact simultaneously with a sensor and with Rho1. We also demonstrate that extracts from wsc1 and mid2 mutants are deficient in the ability to catalyze GTP loading of Rho1 in vitro, providing evidence that the function of the sensor-Rom2 interaction is to stimulate nucleotide exchange toward this G-protein. In a related line of investigation, we identified the PMT2 gene in a genetic screen for mutations that confer an additive cell lysis defect with a wsc1 null allele. Pmt2 is a member of a six-protein family in yeast that catalyzes the first step in O mannosylation of target proteins. We demonstrate that Mid2 is not mannosylated in a pmt2 mutant and that this modification is important for signaling by Mid2. PMID:11113201

Dexamethasone Suppresses Oxysterol-Induced Differentiation of Monocytic Cells

PubMed Central

Son, Yonghae; Kim, Bo-Young; Eo, Seong-Kug; Park, Young Chul; Kim, Koanhoi

2016-01-01

Oxysterol like 27-hydroxycholesterol (27OHChol) has been reported to induce differentiation of monocytic cells into a mature dendritic cell phenotype. We examined whether dexamethasone (Dx) affects 27OHChol-induced differentiation using THP-1 cells. Treatment of monocytic cells with Dx resulted in almost complete inhibition of transcription and surface expression of CD80, CD83, and CD88 induced by 27OHChol. Elevated surface levels of MHC class I and II molecules induced by 27OHChol were reduced to basal levels by treatment with Dx. A decreased endocytosis ability caused by 27OHChol was recovered by Dx. We also examined effects of Dx on expression of CD molecules involved in atherosclerosis. Increased levels of surface protein and transcription of CD105, CD137, and CD166 by treatment with 27OHChol were significantly inhibited by cotreatment with Dx. These results indicate that Dx inhibits 27OHChol-induced differentiation of monocytic cells into a mature dendritic cell phenotype and expression of CD molecules whose levels are associated with atherosclerosis. In addition, we examined phosphorylation of AKT induced by 27OHChol and effect of Dx, where cotreatment with Dx inhibited the phosphorylation of AKT. The current study reports that Dx regulates oxysterol-mediated dendritic cell differentiation of monocytic cells. PMID:27340507

Dexamethasone Suppresses Oxysterol-Induced Differentiation of Monocytic Cells.

PubMed

Son, Yonghae; Kim, Bo-Young; Eo, Seong-Kug; Park, Young Chul; Kim, Koanhoi

2016-01-01

Oxysterol like 27-hydroxycholesterol (27OHChol) has been reported to induce differentiation of monocytic cells into a mature dendritic cell phenotype. We examined whether dexamethasone (Dx) affects 27OHChol-induced differentiation using THP-1 cells. Treatment of monocytic cells with Dx resulted in almost complete inhibition of transcription and surface expression of CD80, CD83, and CD88 induced by 27OHChol. Elevated surface levels of MHC class I and II molecules induced by 27OHChol were reduced to basal levels by treatment with Dx. A decreased endocytosis ability caused by 27OHChol was recovered by Dx. We also examined effects of Dx on expression of CD molecules involved in atherosclerosis. Increased levels of surface protein and transcription of CD105, CD137, and CD166 by treatment with 27OHChol were significantly inhibited by cotreatment with Dx. These results indicate that Dx inhibits 27OHChol-induced differentiation of monocytic cells into a mature dendritic cell phenotype and expression of CD molecules whose levels are associated with atherosclerosis. In addition, we examined phosphorylation of AKT induced by 27OHChol and effect of Dx, where cotreatment with Dx inhibited the phosphorylation of AKT. The current study reports that Dx regulates oxysterol-mediated dendritic cell differentiation of monocytic cells.

Enhancement of Cell Membrane Invaginations, Vesiculation and Uptake of Macromolecules by Protonation of the Cell Surface

PubMed Central

Ben-Dov, Nadav; Korenstein, Rafi

2012-01-01

The different pathways of endocytosis share an initial step involving local inward curvature of the cell’s lipid bilayer. It has been shown that to generate membrane curvature, proteins or lipids enforce transversal asymmetry of the plasma membrane. Thus it emerges as a general phenomenon that transversal membrane asymmetry is the common required element for the formation of membrane curvature. The present study demonstrates that elevating proton concentration at the cell surface stimulates the formation of membrane invaginations and vesiculation accompanied by efficient uptake of macromolecules (Dextran-FITC, 70 kD), relative to the constitutive one. The insensitivity of proton induced uptake to inhibiting treatments and agents of the known endocytic pathways suggests the entry of macromolecules to proceeds via a yet undefined route. This is in line with the fact that neither ATP depletion, nor the lowering of temperature, abolishes the uptake process. In addition, fusion mechanism such as associated with low pH uptake of toxins and viral proteins can be disregarded by employing the polysaccharide dextran as the uptake molecule. The proton induced uptake increases linearly in the extracellular pH range of 6.5 to 4.5, and possesses a steep increase at the range of 4> pH>3, reaching a plateau at pH≤3. The kinetics of the uptake implies that the induced vesicles release their content to the cytosol and undergo rapid recycling to the plasma membrane. We suggest that protonation of the cell’s surface induces local charge asymmetries across the cell membrane bilayer, inducing inward curvature of the cell membrane and consequent vesiculation and uptake. PMID:22558127

Mitochondrial targeted HSP90 inhibitor Gamitrinib-TPP (G-TPP) induces PINK1/Parkin-dependent mitophagy.

PubMed

Fiesel, Fabienne C; James, Elle D; Hudec, Roman; Springer, Wolfdieter

2017-12-05

Loss-of-function mutations in PINK1 or PARKIN are associated with early-onset Parkinson's disease. Upon mitochondrial stress, PINK1 and Parkin together mediate a response that protects cells from the accumulation of harmful, damaged mitochondria. PINK1, the upstream kinase accumulates on the mitochondrial surface and recruits the E3 ubiquitin ligase Parkin on site to ubiquitylate substrate proteins. The joint activity of both to generate phosphorylated poly-ubiquitin chains on the mitochondrial surface induces the recruitment of autophagy receptors and eventually whole organelles are cleared by autophagy. While this pathway is generally accepted to occur upon chemical uncoupling of mitochondria, the (patho-) physiologic relevance has been questioned. However, few studies have indicated that PINK1 and Parkin are also activated upon accumulation of misfolded proteins in the mitochondrial lumen upon overexpression of ΔOTC (Ornithine transcarbamylase). Here, we used the mitochondrial targeted HSP90 inhibitor Gamitrinib-triphenylphosphonium (G-TPP), an anti-cancer agent, to chemically interfere with mitochondrial protein folding. G-TPP treatment induced PINK1 accumulation, ubiquitin phosphorylation at Ser65, Parkin activation and its recruitment to mitochondria was specific for mitochondrial HSP90 inhibition and largely independent of mitochondrial membrane depolarization. Mitophagy induction was observed by monitoring autophagy receptor recruitment and the mitoKeima reporter. Importantly, mitophagy was not only induced in cancer cells but also in primary human fibroblasts and thereof converted neurons. G-TPP treatment might represent a novel strategy to study PINK1 and Parkin-mediated mitochondrial quality control using a more physiologically relevant stress.

Ultraviolet-induced surface grafting of octafluoropentyl methacrylate on polyether ether ketone for inducing antibiofilm properties.

PubMed

Amdjadi, Parisa; Nojehdehian, Hanieh; Najafi, Farhood; Ghasemi, Amir; Seifi, Massoud; Dashtimoghadam, Erfan; Fahimipour, Farahnaz; Tayebi, Lobat

2017-07-01

Since octafluoropentyl methacrylate is an antifouling polymer, surface modification of polyether ether ketone with octafluoropentyl methacrylate is a practical approach to obtaining anti-biofilm biocompatible devices. In the current study, the surface treatment of polyether ether ketone by the use of ultraviolet irradiation, so as to graft (octafluoropentyl methacrylate) polymer chains, was initially implemented and then investigated. The Fourier-transform infrared and nuclear magnetic resonance spectra corroborated the appearance of new signals associated with the fluoroacrylate group. Thermogravimetric curves indicated enhanced asymmetry in the polymer structure due to the introduction of the said new groups. Measuring the peak area in differential scanning calorimetry experiments also showed additional bond formation. Static water contact angle measurements indicated a change in wettability to the more hydrophobic surface. The polyether ether ketone-octafluoropentyl methacrylate surface greatly reduced the protein adsorption. This efficient method can modulate and tune the surface properties of polyether ether ketone according to specific applications.

[Effect of Shexiang Baoxin Pills on isoprenaline-induced myocardial cell hypertrophy and Cx43 expression].

PubMed

Tang, Fen; Jiang, Zhentao; Tan, Wenting; Long, Junrong; Liu, Shengquan; Chu, Chun

2017-08-28

To observe the effects of Shexiang Baoxin Pill (SBP) on isoprenaline (Iso)-induced changes in myocardial cell volume, shape, and connexin 43 (Cx43) expression.
 Methods: H9C2 myocardial cells were randomly divided into a control group, a Iso group and a Iso+SBP group. After 72 h of culture, the average surface area of H9C2 cells was measured under phase contrast microscope. Bicinchoninic acid (BCA) protein assay was carried out to determine the concentration of proteins. The survival rate of myocardial cells was measured by methylthiazolyldiphenyl-tetrazolium bromide (MTT) assay, and the Cx43 expression was detected by Western blot.
 Results: The mean surface area and Cx43 concentration in Iso-treated myocardial cells were increased under the phase contrast microscope (P<0.05). Compared with the Iso group, the mean surface area was decreased, and the Cx43 concentration was reduced in the Iso+SBP group (both P<0.05). Compared with the control group, the Cx43 expression was obviously down-regulated in the H9C2 cells of the Iso group (P<0.05); while compared with the Iso group, the Cx43 expression was obviously up-regulated in the Iso+SBP group (P<0.05).
 Conclusion: Shexiang Baoxin Pills can prevent Iso-induced myocardial hypertrophy and down-regulate Cx43 expression.

Mucin- and carbohydrate-stimulated adhesion and subproteome changes of the probiotic bacterium Lactobacillus acidophilus NCFM.

PubMed

Celebioglu, Hasan Ufuk; Olesen, Sita Vaag; Prehn, Kennie; Lahtinen, Sampo J; Brix, Susanne; Abou Hachem, Maher; Svensson, Birte

2017-06-23

Adhesion to intestinal mucosa is a crucial property for probiotic bacteria. Adhesion is thought to increase host-bacterial interactions, thus potentially enabling health benefits to the host. Molecular events connected with adhesion and surface proteome changes were investigated for the probiotic Lactobacillus acidophilus NCFM cultured with established or emerging prebiotic carbohydrates as carbon source and in the presence of mucin, the glycoprotein of the epithelial mucus layer. Variation in adhesion to HT29-cells and mucin was associated with carbon source and mucin-induced subproteome abundancy differences. Specifically, while growth on fructooligosaccharides (FOS) only stimulated adhesion to intestinal HT-29 cells, cellobiose and polydextrose in addition increased adhesion to mucin. Adhesion to HT-29 cells increased by about 2-fold for bacteria grown on mucin-supplemented glucose. Comparative 2DE-MS surface proteome analysis showed different proteins in energy metabolism appearing on the surface, suggesting they exert moonlighting functions. Mucin-supplemented bacteria had relative abundance of pyruvate kinase and fructose-bisphosphate aldolase increased by about 2-fold while six spots with 3.2-2.1 fold reduced relative abundance comprised elongation factor G, phosphoglycerate kinase, BipAEFTU family GTP-binding protein, ribonucleoside triphosphate reductase, adenylosuccinate synthetase, 30S ribosomal protein S1, and manganese-dependent inorganic pyrophosphatase. Surface proteome of cellobiose- compared to glucose-grown L. acidophilus NCFM had phosphate starvation inducible protein stress-related, thermostable pullulanase, and elongation factor G increasing 4.4-2.4 fold, while GAPDH, elongation factor Ts, and pyruvate kinase were reduced by 2.0-1.5 fold in relative abundance. Addition of recombinant L. acidophilus NCFM elongation factor G and pyruvate kinase to a coated mucin layer significantly suppressed subsequent adhesion of the bacterium. Human diet is important for intestinal health and food components, especially non-digestible carbohydrates can beneficially modify the microbiota. In the present study, effects of emerging and established prebiotic carbohydrates on the probiotic potential of Lactobacillus acidophilus NCFM were investigated by testing adhesion to a mucin layer and intestinal cells, and comparing this with changes in abundancy of surface proteins thought to be important for host interactions. Increased adhesion was observed following culturing of the bacterium with fructooligosaccharides, cellobiose or polydextrose, as well as mucin-supplemented glucose as carbon source. Enhanced adhesion ability can prolong bacterial residence in GIT yielding positive health effects. Higher relative abundance of certain surface proteins under various conditions (i.e. grown on cellobiose or mucin-supplemented glucose) suggested involvement of these proteins in adhesion, as confirmed by competition in case of two recombinantly produced moonlighting proteins. Combination of Lactobacillus acidophilus NCFM with different carbohydrates revealed potential bacterial determinants of synbiotic interactions, including stimulation of adhesion. Copyright © 2017 Elsevier B.V. All rights reserved.

C-Type Lectin Receptor MCL Facilitates Mincle Expression and Signaling through Complex Formation.

PubMed

Miyake, Yasunobu; Masatsugu, Oh-hora; Yamasaki, Sho

2015-06-01

C-type lectin receptors expressed in APCs are recently defined pattern recognition receptors that play a crucial role in immune responses against pathogen-associated molecular patterns. Among pathogen-associated molecular patterns, cord factor (trehalose-6,6'-dimycolate [TDM]) is the most potent immunostimulatory component of the mycobacterial cell wall. Two C-type lectin receptors, macrophage-inducible C-type lectin (Mincle) and macrophage C-type lectin (MCL), are required for immune responses against TDM. Previous studies indicate that MCL is required for TDM-induced Mincle expression. However, the mechanism by which MCL induces Mincle expression has not been fully understood. In this study, we demonstrate that MCL interacts with Mincle to promote its surface expression. After LPS or zymosan stimulation, MCL-deficient bone marrow-derived dendritic cells (BMDCs) had a lower level of Mincle protein expression, although mRNA expression was comparable with wild-type BMDCs. Meanwhile, BMDCs from MCL transgenic mice showed an enhanced level of Mincle expression on the cell surface. MCL was associated with Mincle through the stalk region and this region was necessary and sufficient for the enhancement of Mincle expression. This interaction appeared to be mediated by the hydrophobic repeat of MCL, as substitution of four hydrophobic residues within the stalk region with serine (MCL(4S)) abolished the function to enhance the surface expression of Mincle. MCL(4S) mutant failed to restore the defective TDM responses in MCL-deficient BMDCs. These results suggest that MCL positively regulates Mincle expression through protein-protein interaction via its stalk region, thereby magnifying Mincle-mediated signaling. Copyright © 2015 by The American Association of Immunologists, Inc.

Identification of a New Class of Lipid Droplet-Associated Proteins in Plants1[C][W][OPEN

PubMed Central

Horn, Patrick J.; James, Christopher N.; Gidda, Satinder K.; Kilaru, Aruna; Dyer, John M.; Mullen, Robert T.; Ohlrogge, John B.; Chapman, Kent D.

2013-01-01

Lipid droplets in plants (also known as oil bodies, lipid bodies, or oleosomes) are well characterized in seeds, and oleosins, the major proteins associated with their surface, were shown to be important for stabilizing lipid droplets during seed desiccation and rehydration. However, lipid droplets occur in essentially all plant cell types, many of which may not require oleosin-mediated stabilization. The proteins associated with the surface of nonseed lipid droplets, which are likely to influence the formation, stability, and turnover of this compartment, remain to be elucidated. Here, we have combined lipidomic, proteomic, and transcriptomic studies of avocado (Persea americana) mesocarp to identify two new lipid droplet-associated proteins, which we named LDAP1 and LDAP2. These proteins are highly similar to each other and also to the small rubber particle proteins that accumulate in rubber-producing plants. An Arabidopsis (Arabidopsis thaliana) homolog to LDAP1 and LDAP2, At3g05500, was localized to the surface of lipid droplets after transient expression in tobacco (Nicotiana tabacum) cells that were induced to accumulate triacylglycerols. We propose that small rubber particle protein-like proteins are involved in the general process of binding and perhaps the stabilization of lipid-rich particles in the cytosol of plant cells and that the avocado and Arabidopsis protein members reveal a new aspect of the cellular machinery that is involved in the packaging of triacylglycerols in plant tissues. PMID:23821652

Surface-water Interface Induces Conformational Changes Critical for Protein Adsorption: Implications for Monolayer Formation of EAS Hydrophobin

PubMed Central

Ley, Kamron; Christofferson, Andrew; Penna, Matthew; Winkler, Dave; Maclaughlin, Shane; Yarovsky, Irene

2015-01-01

The class I hydrophobin EAS is part of a family of small, amphiphilic fungal proteins best known for their ability to self-assemble into stable monolayers that modify the hydrophobicity of a surface to facilitate further microbial growth. These proteins have attracted increasing attention for industrial and biomedical applications, with the aim of designing surfaces that have the potential to maintain their clean state by resisting non-specific protein binding. To gain a better understanding of this process, we have employed all-atom molecular dynamics to study initial stages of the spontaneous adsorption of monomeric EAS hydrophobin on fully hydroxylated silica, a commonly used industrial and biomedical substrate. Particular interest has been paid to the Cys3-Cys4 loop, which has been shown to exhibit disruptive behavior in solution, and the Cys7-Cys8 loop, which is believed to be involved in the aggregation of EAS hydrophobin at interfaces. Specific and water mediated interactions with the surface were also analyzed. We have identified two possible binding motifs, one which allows unfolding of the Cys7-Cys8 loop due to the surfactant-like behavior of the Cys3-Cys4 loop, and another which has limited unfolding due to the Cys3-Cys4 loop remaining disordered in solution. We have also identified intermittent interactions with water which mediate the protein adsorption to the surface, as well as longer lasting interactions which control the diffusion of water around the adsorption site. These results have shown that EAS behaves in a similar way at the air-water and surface-water interfaces, and have also highlighted the need for hydrophilic ligand functionalization of the silica surface in order to prevent the adsorption of EAS hydrophobin. PMID:26636091

Surface-water Interface Induces Conformational Changes Critical for Protein Adsorption: Implications for Monolayer Formation of EAS Hydrophobin.

PubMed

Ley, Kamron; Christofferson, Andrew; Penna, Matthew; Winkler, Dave; Maclaughlin, Shane; Yarovsky, Irene

2015-01-01

The class I hydrophobin EAS is part of a family of small, amphiphilic fungal proteins best known for their ability to self-assemble into stable monolayers that modify the hydrophobicity of a surface to facilitate further microbial growth. These proteins have attracted increasing attention for industrial and biomedical applications, with the aim of designing surfaces that have the potential to maintain their clean state by resisting non-specific protein binding. To gain a better understanding of this process, we have employed all-atom molecular dynamics to study initial stages of the spontaneous adsorption of monomeric EAS hydrophobin on fully hydroxylated silica, a commonly used industrial and biomedical substrate. Particular interest has been paid to the Cys3-Cys4 loop, which has been shown to exhibit disruptive behavior in solution, and the Cys7-Cys8 loop, which is believed to be involved in the aggregation of EAS hydrophobin at interfaces. Specific and water mediated interactions with the surface were also analyzed. We have identified two possible binding motifs, one which allows unfolding of the Cys7-Cys8 loop due to the surfactant-like behavior of the Cys3-Cys4 loop, and another which has limited unfolding due to the Cys3-Cys4 loop remaining disordered in solution. We have also identified intermittent interactions with water which mediate the protein adsorption to the surface, as well as longer lasting interactions which control the diffusion of water around the adsorption site. These results have shown that EAS behaves in a similar way at the air-water and surface-water interfaces, and have also highlighted the need for hydrophilic ligand functionalization of the silica surface in order to prevent the adsorption of EAS hydrophobin.

Ordered adsorption of coagulation factor XII on negatively charged polymer surfaces probed by sum frequency generation vibrational spectroscopy.

PubMed

Chen, Xiaoyun; Wang, Jie; Paszti, Zoltan; Wang, Fulin; Schrauben, Joel N; Tarabara, Volodymyr V; Schmaier, Alvin H; Chen, Zhan

2007-05-01

Electrostatic interactions between negatively charged polymer surfaces and factor XII (FXII), a blood coagulation factor, were investigated by sum frequency generation (SFG) vibrational spectroscopy, supplemented by several analytical techniques including attenuated total reflection Fourier transform infrared spectroscopy (ATR-FTIR), quartz crystal microbalance (QCM), zeta-potential measurement, and chromogenic assay. A series of sulfonated polystyrenes (sPS) with different sulfonation levels were synthesized as model surfaces with different surface charge densities. SFG spectra collected from FXII adsorbed onto PS and sPS surfaces with different surface charge densities showed remarkable differences in spectral features and especially in spectral intensity. Chromogenic assay experiments showed that highly charged sPS surfaces induced FXII autoactivation. ATR-FTIR and QCM results indicated that adsorption amounts on the PS and sPS surfaces were similar even though the surface charge densities were different. No significant conformational change was observed from FXII adsorbed onto surfaces studied. Using theoretical calculations, the possible contribution from the third-order nonlinear optical effect induced by the surface electric field was evaluated, and it was found to be unable to yield the SFG signal enhancement observed. Therefore it was concluded that the adsorbed FXII orientation and ordering were the main reasons for the remarkable SFG amide I signal increase on sPS surfaces. These investigations indicate that negatively charged surfaces facilitate or induce FXII autoactivation on the molecular level by imposing specific orientation and ordering on the adsorbed protein molecules.

Activation of G-proteins by receptor-stimulated nucleoside diphosphate kinase in Dictyostelium.

PubMed Central

Bominaar, A A; Molijn, A C; Pestel, M; Veron, M; Van Haastert, P J

1993-01-01

Recently, interest in the enzyme nucleoside diphosphate kinase (EC2.7.4.6) has increased as a result of its possible involvement in cell proliferation and development. Since NDP kinase is one of the major sources of GTP in cells, it has been suggested that the effects of an altered NDP kinase activity on cellular processes might be the result of altered transmembrane signal transduction via guanine nucleotide-binding proteins (G-proteins). In the cellular slime mould Dictyostelium discoideum, extracellular cAMP induces an increase of phospholipase C activity via a surface cAMP receptor and G-proteins. In this paper it is demonstrated that part of the cellular NDP kinase is associated with the membrane and stimulated by cell surface cAMP receptors. The GTP produced by the action of NDP kinase is capable of activating G-proteins as monitored by altered G-protein-receptor interaction and the activation of the effector enzyme phospholipase C. Furthermore, specific monoclonal antibodies inhibit the effect of NDP kinase on G-protein activation. These results suggest that receptor-stimulated NDP kinase contributes to the mediation of hormone action by producing GTP for the activation of GTP-binding proteins. Images PMID:8389692

Water dynamics in protein hydration shells: the molecular origins of the dynamical perturbation.

PubMed

Fogarty, Aoife C; Laage, Damien

2014-07-17

Protein hydration shell dynamics play an important role in biochemical processes including protein folding, enzyme function, and molecular recognition. We present here a comparison of the reorientation dynamics of individual water molecules within the hydration shell of a series of globular proteins: acetylcholinesterase, subtilisin Carlsberg, lysozyme, and ubiquitin. Molecular dynamics simulations and analytical models are used to access site-resolved information on hydration shell dynamics and to elucidate the molecular origins of the dynamical perturbation of hydration shell water relative to bulk water. We show that all four proteins have very similar hydration shell dynamics, despite their wide range of sizes and functions, and differing secondary structures. We demonstrate that this arises from the similar local surface topology and surface chemical composition of the four proteins, and that such local factors alone are sufficient to rationalize the hydration shell dynamics. We propose that these conclusions can be generalized to a wide range of globular proteins. We also show that protein conformational fluctuations induce a dynamical heterogeneity within the hydration layer. We finally address the effect of confinement on hydration shell dynamics via a site-resolved analysis and connect our results to experiments via the calculation of two-dimensional infrared spectra.

Water Dynamics in Protein Hydration Shells: The Molecular Origins of the Dynamical Perturbation

PubMed Central

2014-01-01

Protein hydration shell dynamics play an important role in biochemical processes including protein folding, enzyme function, and molecular recognition. We present here a comparison of the reorientation dynamics of individual water molecules within the hydration shell of a series of globular proteins: acetylcholinesterase, subtilisin Carlsberg, lysozyme, and ubiquitin. Molecular dynamics simulations and analytical models are used to access site-resolved information on hydration shell dynamics and to elucidate the molecular origins of the dynamical perturbation of hydration shell water relative to bulk water. We show that all four proteins have very similar hydration shell dynamics, despite their wide range of sizes and functions, and differing secondary structures. We demonstrate that this arises from the similar local surface topology and surface chemical composition of the four proteins, and that such local factors alone are sufficient to rationalize the hydration shell dynamics. We propose that these conclusions can be generalized to a wide range of globular proteins. We also show that protein conformational fluctuations induce a dynamical heterogeneity within the hydration layer. We finally address the effect of confinement on hydration shell dynamics via a site-resolved analysis and connect our results to experiments via the calculation of two-dimensional infrared spectra. PMID:24479585

Protein Corona Prevents TiO2 Phototoxicity

PubMed Central

Garvas, Maja; Testen, Anze; Umek, Polona; Gloter, Alexandre; Koklic, Tilen; Strancar, Janez

2015-01-01

Background & Aim TiO2 nanoparticles have generally low toxicity in the in vitro systems although some toxicity is expected to originate in the TiO2-associated photo-generated radical production, which can however be modulated by the radical trapping ability of the serum proteins. To explore the role of serum proteins in the phototoxicity of the TiO2 nanoparticles we measure viability of the exposed cells depending on the nanoparticle and serum protein concentrations. Methods & Results Fluorescence and spin trapping EPR spectroscopy reveal that the ratio between the nanoparticle and protein concentrations determines the amount of the nanoparticles’ surface which is not covered by the serum proteins and is proportional to the amount of photo-induced radicals. Phototoxicity thus becomes substantial only at the protein concentration being too low to completely coat the nanotubes’ surface. Conclusion These results imply that TiO2 nanoparticles should be applied with ligands such as proteins when phototoxic effects are not desired - for example in cosmetics industry. On the other hand, the nanoparticles should be used in serum free medium or any other ligand free medium, when phototoxic effects are desired – as for efficient photodynamic cancer therapy. PMID:26083725

Tip Effect of the Tapping Mode of Atomic Force Microscope in Viscous Fluid Environments.

PubMed

Shih, Hua-Ju; Shih, Po-Jen

2015-07-28

Atomic force microscope with applicable types of operation in a liquid environment is widely used to scan the contours of biological specimens. The contact mode of operation allows a tip to touch a specimen directly but sometimes it damages the specimen; thus, a tapping mode of operation may replace the contact mode. The tapping mode triggers the cantilever of the microscope approximately at resonance frequencies, and so the tip periodically knocks the specimen. It is well known that the cantilever induces extra liquid pressure that leads to drift in the resonance frequency. Studies have noted that the heights of protein surfaces measured via the tapping mode of an atomic force microscope are ~25% smaller than those measured by other methods. This discrepancy may be attributable to the induced superficial hydrodynamic pressure, which is worth investigating. In this paper, we introduce a semi-analytical method to analyze the pressure distribution of various tip geometries. According to our analysis, the maximum hydrodynamic pressure on the specimen caused by a cone-shaped tip is ~0.5 Pa, which can, for example, pre-deform a cell by several nanometers in compression before the tip taps it. Moreover, the pressure calculated on the surface of the specimen is 20 times larger than the pressure without considering the tip effect; these results have not been motioned in other papers. Dominating factors, such as surface heights of protein surface, mechanical stiffness of protein increasing with loading velocity, and radius of tip affecting the local pressure of specimen, are also addressed in this study.

Deciphering the shape and deformation of secondary structures through local conformation analysis

PubMed Central

2011-01-01

Background Protein deformation has been extensively analysed through global methods based on RMSD, torsion angles and Principal Components Analysis calculations. Here we use a local approach, able to distinguish among the different backbone conformations within loops, α-helices and β-strands, to address the question of secondary structures' shape variation within proteins and deformation at interface upon complexation. Results Using a structural alphabet, we translated the 3 D structures of large sets of protein-protein complexes into sequences of structural letters. The shape of the secondary structures can be assessed by the structural letters that modeled them in the structural sequences. The distribution analysis of the structural letters in the three protein compartments (surface, core and interface) reveals that secondary structures tend to adopt preferential conformations that differ among the compartments. The local description of secondary structures highlights that curved conformations are preferred on the surface while straight ones are preferred in the core. Interfaces display a mixture of local conformations either preferred in core or surface. The analysis of the structural letters transition occurring between protein-bound and unbound conformations shows that the deformation of secondary structure is tightly linked to the compartment preference of the local conformations. Conclusion The conformation of secondary structures can be further analysed and detailed thanks to a structural alphabet which allows a better description of protein surface, core and interface in terms of secondary structures' shape and deformation. Induced-fit modification tendencies described here should be valuable information to identify and characterize regions under strong structural constraints for functional reasons. PMID:21284872

Deciphering the shape and deformation of secondary structures through local conformation analysis.

PubMed

Baussand, Julie; Camproux, Anne-Claude

2011-02-01

Protein deformation has been extensively analysed through global methods based on RMSD, torsion angles and Principal Components Analysis calculations. Here we use a local approach, able to distinguish among the different backbone conformations within loops, α-helices and β-strands, to address the question of secondary structures' shape variation within proteins and deformation at interface upon complexation. Using a structural alphabet, we translated the 3 D structures of large sets of protein-protein complexes into sequences of structural letters. The shape of the secondary structures can be assessed by the structural letters that modeled them in the structural sequences. The distribution analysis of the structural letters in the three protein compartments (surface, core and interface) reveals that secondary structures tend to adopt preferential conformations that differ among the compartments. The local description of secondary structures highlights that curved conformations are preferred on the surface while straight ones are preferred in the core. Interfaces display a mixture of local conformations either preferred in core or surface. The analysis of the structural letters transition occurring between protein-bound and unbound conformations shows that the deformation of secondary structure is tightly linked to the compartment preference of the local conformations. The conformation of secondary structures can be further analysed and detailed thanks to a structural alphabet which allows a better description of protein surface, core and interface in terms of secondary structures' shape and deformation. Induced-fit modification tendencies described here should be valuable information to identify and characterize regions under strong structural constraints for functional reasons.

Heat shock protein 70 surface-positive tumor exosomes stimulate migratory and cytolytic activity of natural killer cells.

PubMed

Gastpar, Robert; Gehrmann, Mathias; Bausero, Maria A; Asea, Alexzander; Gross, Catharina; Schroeder, Josef A; Multhoff, Gabriele

2005-06-15

Detergent-soluble membrane vesicles are actively released by human pancreas (Colo-/Colo+) and colon (CX-/CX+) carcinoma sublines, differing in their capacity to present heat shock protein 70 (Hsp70)/Bag-4 on their plasma membranes. Floating properties, acetylcholine esterase activity, and protein composition characterized them as exosomes. An enrichment of Rab-4 documented their intracellular transport route from early endosomes to the plasma membrane. After solubilization, comparable amounts of cytosolic proteins, including tubulin, Hsp70, Hsc70, and Bag-4, but not ER-residing Grp94 and calnexin, were detectable in tumor-derived exosomes. However, with respect to the exosomal surface, only Colo+/CX+ but not Colo-/CX- derived exosomes were Hsp70 membrane positive. Therefore, concomitant with an up-regulated cell surface density of activation markers, migration and Hsp70 reactivity of natural killer (NK) cells was stimulated selectively by Hsp70/Bag-4 surface-positive exosomes, but not by their negative counterparts and tumor cell lysates. Moreover, the exosome-mediated lytic activity of NK cells was blockable by Hsp70-specific antibody. As already shown for TKD stimulation, NK cells preincubated with Hsp70 surface-positive exosomes initiated apoptosis in tumors through granzyme B release. In summary, our data provide an explanation how Hsp70 reactivity in NK cells is induced by tumor-derived exosomes.

Heat Shock Protein 70 Surface-Positive Tumor Exosomes Stimulate Migratory and Cytolytic Activity of Natural Killer Cells

PubMed Central

Gastpar, Robert; Gehrmann, Mathias; Bausero, Maria A.; Asea, Alexzander; Gross, Catharina; Schroeder, Josef A.

2006-01-01

Detergent-soluble membrane vesicles are actively released by human pancreas (Colo−/Colo+) and colon (CX−/CX+) carcinoma sublines, differing in their capacity to present heat shock protein 70 (Hsp70)/Bag-4 on their plasma membranes. Floating properties, acetylcholine esterase activity, and protein composition characterized them as exosomes. An enrichment of Rab-4 documented their intracellular transport route from early endosomes to the plasma membrane. After solubilization, comparable amounts of cytosolic proteins, including tubulin, Hsp70, Hsc70, and Bag-4, but not ER-residing Grp94 and calnexin, were detectable in tumor-derived exosomes. However, with respect to the exosomal surface, only Colo+/CX+ but not Colo−/CX exosomes were Hsp70 membrane derived positive. Therefore, concomitant with an up-regulated cell surface density of activation markers, migration and Hsp70 reactivity of natural killer (NK) cells was stimulated selectively by Hsp70/Bag-4 surface-positive exosomes, but not by their negative counterparts and tumor cell lysates. Moreover, the exosome-mediated lytic activity of NK cells was blockable by Hsp70-specific antibody. As already shown for TKD stimulation, NK cells preincubated with Hsp70 surface-positive exosomes initiated apoptosis in tumors through granzyme B release. In summary, our data provide an explanation how Hsp70 reactivity in NK cells is induced by tumor-derived exosomes. PMID:15958569

[Cell entry mechanisms of coronaviruses].

PubMed

Taguchi, Fumihiro; Matsuyama, Shutoku

2009-12-01

Enveloped viruses enter into cells via fusion of their envelope and cellular membrane. Spike (S) protein of coronavirus (CoV) is responsible for entry events. We studied the cell entry mechanisms of two different CoVs, murine coronavirus mouse hepatitis virus (MHV) and severe acute respiratory syndrome coronavirus (SARS-CoV). MHV-JHM that induces syncytia in infected cells entered directly from cell surface, i.e., fusion of envelope and plasma membrane, whereas SARS-CoV and MHV-2 that fail to induce syncytia entered via endosome in a protease-dependent fashion, i.e., fusion of envelope and endosomal membrane. The latter viruses entered directly from cell surface, when receptor-bound viruses were treated with proteases that activate fusion activity of their S proteins. The entry pathway of SARS-CoV could influence the severity of the disease. It was also reveled that a highly neurovirulent JHM spread in a receptor-independent fashion, which could result in a high neuropathogenicity of the virus.

Functional improvements in dried egg white through the Maillard reaction.

PubMed

Handa, A; Kuroda, N

1999-05-01

The effects of the Maillard reaction on the functional properties of dried egg white (DEW) were investigated. Maillard-reacted DEW (M-DEW) was prepared by storing sugar-preserved DEW (SP-DEW) at 55 degrees C and 35% relative humidity for 0-12 days. The M-DEW developed an excellent gelling property, and hydrogen sulfide production from heat-induced M-DEW gels decreased. Surface sulfhydryl (SH) group content of M-DEW increased while total SH group and alpha-helix contents decreased with increasing heating time in the dry state. Breaking strength, breaking strain, water-holding capacity, and hydrogen sulfide of heat-induced M-DEW gels significantly correlated with surface and total SH group contents in M-DEW. SDS-PAGE revealed that M-DEW proteins were polymerized in which covalent bonds were involved. The present study demonstrated that the Maillard reaction partially unfolds and polymerizes proteins of SP-DEW and, consequently, improved gelling property of SP-DEW under certain controlled conditions.

Inducible recruitment of Cdc42 or WASP to a cell-surface receptor triggers actin polymerization and filopodium formation.

PubMed

Castellano, F; Montcourrier, P; Guillemot, J C; Gouin, E; Machesky, L; Cossart, P; Chavrier, P

1999-04-08

Cdc42, a GTP-binding protein of the Rho family, controls actin cytoskeletal organization and helps to generate actin-based protruding structures, such as filopodia. In vitro, Cdc42 regulates actin polymerization by facilitating the creation of free barbed ends - the more rapidly growing ends of actin filaments - and subsequent elongation at these ends. The Wiskott- Aldrich syndrome protein, WASP, which has a pleckstrin-homology domain and a Cdc42/Rac-binding motif, has been implicated in cell signaling and cytoskeleton reorganization. We have investigated the consequences of local recruitment of activated Cdc42 or WASP to the plasma membrane. We used an activated Cdc42 protein that could be recruited to an engineered membrane receptor by adding rapamycin as a bridge, and added antibody-coupled beads to aggregate these receptors. Inducible recruitment of Cdc42 to clusters of receptors stimulated actin polymerization, resulting in the formation of membrane protrusions. Cdc42-induced protrusions were enriched in the vasodilator-stimulated phosphoprotein VASP and the focal-adhesion-associated proteins zyxin and ezrin. The Cdc42 effector WASP could also induce the formation of protrusions, albeit of different morphology. This is the first demonstration that the local recruitment of activated Cdc42 or its downstream effector, WASP, to a membrane receptor in whole cells is sufficient to trigger actin polymerization that results in the formation of membrane protrusions. Our data suggest that Cdc42-induced actin-based protrusions result from the local and serial recruitment of cytoskeletal proteins including zyxin, VASP, and ezrin.

The role of heat shock protein 70 (Hsp70) in radiation-induced immunomodulation.

PubMed

Multhoff, Gabriele; Pockley, Alan G; Schmid, Thomas E; Schilling, Daniela

2015-11-28

Despite enormous progress in radiation technologies (high precision image-guided irradiation, proton irradiation, heavy ion irradiation) and radiotherapeutic concepts (hypofractionated irradiation schemes), the clinical outcome of radiotherapy in locally advanced and metastasized tumors and in hypoxic tumors which are radiation-resistant remains unsatisfactory. Given their key influence on a number of biological and immunological parameters, this article considers the influence of irradiation-induced stress proteins on radiation-induced immunomodulation. Depending on its location, the major stress-inducible Heat shock protein 70 (Hsp70) has been found to fulfill multiple roles. On the one hand, increased intracellular Hsp70 levels have been found to play a key role in the recovery from stress such as radio(chemo)therapy, and on the other hand extracellular Hsp70 proteins are potent stimulators of the innate immune system and mediators of anti-tumor immunity. Furthermore, if loaded with tumor-derived peptides, members of the Heat Shock Protein 70 (HSP70) and 90 (HSP90) families can stimulate the adaptive immune system via antigen cross-presentation. An irradiation-induced enhancement of the selective expression of a membrane form of Hsp70 on the surface of tumor cells which can act as a recognition structure for activated NK cells might have significant clinical relevance, in that the outcome of irradiation therapy for advanced tumors could be improved by combining it with cell-based and other immunotherapies that target this membrane form of Hsp70. Copyright © 2015 The Authors. Published by Elsevier Ireland Ltd.. All rights reserved.

Human Diversity in a Cell Surface Receptor that Inhibits Autophagy.

PubMed

Chaudhary, Anu; Leite, Mara; Kulasekara, Bridget R; Altura, Melissa A; Ogahara, Cassandra; Weiss, Eli; Fu, Wenqing; Blanc, Marie-Pierre; O'Keeffe, Michael; Terhorst, Cox; Akey, Joshua M; Miller, Samuel I

2016-07-25

Mutations in genes encoding autophagy proteins have been associated with human autoimmune diseases, suggesting that diversity in autophagy responses could be associated with disease susceptibility or severity. A cellular genome-wide association study (GWAS) screen was performed to explore normal human diversity in responses to rapamycin, a microbial product that induces autophagy. Cells from several human populations demonstrated variability in expression of a cell surface receptor, CD244 (SlamF4, 2B4), that correlated with changes in rapamycin-induced autophagy. High expression of CD244 and receptor activation with its endogenous ligand CD48 inhibited starvation- and rapamycin-induced autophagy by promoting association of CD244 with the autophagy complex proteins Vps34 and Beclin-1. The association of CD244 with this complex reduced Vps34 lipid kinase activity. Lack of CD244 is associated with auto-antibody production in mice, and lower expression of human CD244 has previously been implicated in severity of human rheumatoid arthritis and systemic lupus erythematosus, indicating that increased autophagy as a result of low levels of CD244 may alter disease outcomes. Copyright © 2016 Elsevier Ltd. All rights reserved.

Cigarette Smoke–Induced CXCR3 Receptor Up-Regulation Mediates Endothelial Apoptosis

PubMed Central

Green, Linden A.; Petrusca, Daniela; Rajashekhar, Gangaraju; Gianaris, Tom; Schweitzer, Kelly S.; Wang, Liang; Justice, Matthew J.; Petrache, Irina

2012-01-01

Endothelial monocyte–activating polypeptide II (EMAP II) and interferon-inducible protein (IP)–10 are proinflammatory mediators, which in addition to their chemokine activities, selectively induce apoptosis in endothelial cells and are up-regulated in the lungs of cigarette smoke–exposed humans. Previously, we showed that EMAP II is an essential mediator of cigarette smoke–induced lung emphysema in mice linking endothelial cell apoptosis with inflammation. Here we addressed the role of the CXCR3 receptor in EMAP II–induced and IP-10–induced apoptosis in endothelial cells and its regulation by cigarette smoke. We found that both neutralizing antibodies and small inhibitory RNA to CXCR3 abrogated EMAP II–induced and IP-10–induced endothelial caspase-3 activation and DNA fragmentation. CXCR3 receptor surface expression in human lung microvascular endothelial cells and in lung tissue endothelium was up-regulated by exposure to cigarette smoke. In tissue culture conditions, EMAP II–induced and IP-10–induced apoptosis was enhanced by preincubation with cigarette smoke extract. Interestingly, serum starvation also induced CXCR3 up-regulation and enhanced EMAP II–induced endothelial apoptosis. Signal transduction via p38 mitogen-activated protein kinase activation was essential for CXCR3-induced cell death, but not for CXCR3 receptor up-regulation by cigarette smoke. In turn, protein nitration was required for CXCR3 receptor up-regulation by cigarette smoke and consequently for subsequent CXCR3-induced cell death. In conclusion, the concerted up-regulation of proinflammatory EMAP II, IP-10, and CXCR3 by cigarette smoke could sustain a cascade of cell death that may promote the alveolar tissue loss noted in human emphysema. PMID:22936405

Activation of classical protein kinase C reduces the expression of human cationic amino acid transporter 3 (hCAT-3) in the plasma membrane.

PubMed

Rotmann, Alexander; Vékony, Nicole; Gassner, Davina; Niegisch, Günter; Strand, Dennis; Martiné, Ursula; Closs, Ellen I

2006-04-01

We have previously shown that activation of PKC (protein kinase C) results in internalization of hCAT-1 [human CAT-1 (cationic amino acid transporter 1)] and a decrease in arginine transport [Rotmann, Strand, Martiné and Closs (2004) J. Biol. Chem. 279, 54185-54192]. However, others found increased transport rates for arginine in response to PKC activation, suggesting a differential effect of PKC on different CAT isoforms. Therefore we investigated the effect of PKC on hCAT-3, an isoform expressed in thymus, brain, ovary, uterus and mammary gland. In Xenopus laevis oocytes and human U373MG glioblastoma cells, hCAT-3-mediated L-arginine transport was significantly reduced upon treatment with compounds that activate classical PKC. In contrast, inactive phorbol esters and an activator of novel PKC isoforms had no effect. PKC inhibitors (including the PKCalpha-preferring Ro 31-8280) reduced the inhibitory effect of the PKC-activating compounds. Microscopic analyses revealed a PMA-induced reduction in the cell-surface expression of fusion proteins between hCAT-3 and enhanced green fluorescent protein expressed in X. laevis oocytes and glioblastoma cells. Western-blot analysis of biotinylated surface proteins demonstrated a PMA-induced decrease in hCAT-3 in the plasma membrane, but not in total protein lysates. Pretreatment with a PKC inhibitor also reduced this PMA effect. It is concluded that similar to hCAT-1, hCAT-3 activity is decreased by PKC via reduction of transporter molecules in the plasma membrane. Classical PKC isoforms seem to be responsible for this effect.

Surface modification of poly (styrene-b-(ethylene-co-butylene)-b-styrene) elastomer and its plasma protein adsorption by QCM-D

NASA Astrophysics Data System (ADS)

Li, Rui; Jin, Jing; Sun, Yingchun

2014-05-01

Protein adsorption is a dynamic process and plays a major role in determining the hemocompatibility of biomaterials. We have obtained different poly (ethylene glycol) (PEG) graft concentrations of SEBS-g-PEG and the surface chemical compositions are confirmed by X-ray photoelectron spectroscopy (XPS). Graft concentration is defined by peak-area ratio of [C--O]/[C] on modified SEBS surface. With increasing graft concentration, water contact angles of the modified SEBS have significantly decreased. The platelet adhesion and static protein adsorption demonstrate that the hemocompatibility of copolymers films are improved effectively and SEBS-g-PEG-2 with larger graft concentration has more superior anticoagulation than that of SEBS-g-PEG-1. Moreover, we have quantitatively investigated the adsorption process of bovine serum albumin (BSA) and fibrinogen (Fib) on the surfaces of pristine SEBS and modified SEBS using quartz crystal microbalance with dissipation (QCM-D) in real time. The results indicate that the inactivated BSA on the pristine SEBS can continuously induce the subsequent Fib adsorption. The hemocompatibility of SEBS-g-PEG-2 with the graft concentration of 0.207 has excellent anti-protein property and the bio-inert BSA layer on the film can resist the subsequent Fib adsorption.

GPI-anchored GFP signals Ca2+ but is homogeneously distributed on the cell surface.

PubMed

Hiscox, Stephen; Hallett, Maurice B; Morgan, B Paul; van den Berg, Carmen W

2002-05-03

Glycosyl-phosphatidylinositol (GPI)-anchored proteins are unique in that they penetrate only the outer leaflet of the plasma membrane but are still able to mediate intracellular signalling events following antibody-induced ligation. Detergent solubilisation studies suggest that microdomains exist at the cell surface within which are sequestered GPI-linked proteins. Here we report the construction and expression of a fluorescent GPI anchor on the surface of CHO, EL4, and U937 cells by fusing green fluorescent protein (GFP) to the GPI-attachment site of CD59. The resultant GFP-GPI has properties comparable to that of endogenously expressed GPI-anchored molecules as shown by Triton X-114 partitioning. However, sucrose gradient floatation showed that GFP-GPI was only partially resistant to detergent solubilisation. Furthermore confocal scanning laser microscopy revealed a homogeneous distribution of GFP-GPI at the cell surface, which only became clustered following cross-linking of the GPI anchor via an anti-GFP antibody. Surprisingly, GFP-GPI signalled Ca2+ change upon cross-linking demonstrating its signalling competence. Our results suggest that the GPI-anchor itself does not confer a clustered distribution to molecules but that clustering occurs following ligation with antibody, which allows the protein to become Ca2+ signalling competent. Copyright 2002 Elsevier Science (USA).

Monomeric ephrinB2 binding induces allosteric changes in Nipah virus G that precede its full activation.

PubMed

Wong, Joyce J W; Young, Tracy A; Zhang, Jiayan; Liu, Shiheng; Leser, George P; Komives, Elizabeth A; Lamb, Robert A; Zhou, Z Hong; Salafsky, Joshua; Jardetzky, Theodore S

2017-10-03

Nipah virus is an emergent paramyxovirus that causes deadly encephalitis and respiratory infections in humans. Two glycoproteins coordinate the infection of host cells, an attachment protein (G), which binds to cell surface receptors, and a fusion (F) protein, which carries out the process of virus-cell membrane fusion. The G protein binds to ephrin B2/3 receptors, inducing G conformational changes that trigger F protein refolding. Using an optical approach based on second harmonic generation, we show that monomeric and dimeric receptors activate distinct conformational changes in G. The monomeric receptor-induced changes are not detected by conformation-sensitive monoclonal antibodies or through electron microscopy analysis of G:ephrinB2 complexes. However, hydrogen/deuterium exchange experiments confirm the second harmonic generation observations and reveal allosteric changes in the G receptor binding and F-activating stalk domains, providing insights into the pathway of receptor-activated virus entry.Nipah virus causes encephalitis in humans. Here the authors use a multidisciplinary approach to study the binding of the viral attachment protein G to its host receptor ephrinB2 and show that monomeric and dimeric receptors activate distinct conformational changes in G and discuss implications for receptor-activated virus entry.

Target cell cyclophilins facilitate human papillomavirus type 16 infection.

PubMed

Bienkowska-Haba, Malgorzata; Patel, Hetalkumar D; Sapp, Martin

2009-07-01

Following attachment to primary receptor heparan sulfate proteoglycans (HSPG), human papillomavirus type 16 (HPV16) particles undergo conformational changes affecting the major and minor capsid proteins, L1 and L2, respectively. This results in exposure of the L2 N-terminus, transfer to uptake receptors, and infectious internalization. Here, we report that target cell cyclophilins, peptidyl-prolyl cis/trans isomerases, are required for efficient HPV16 infection. Cell surface cyclophilin B (CyPB) facilitates conformational changes in capsid proteins, resulting in exposure of the L2 N-terminus. Inhibition of CyPB blocked HPV16 infection by inducing noninfectious internalization. Mutation of a putative CyP binding site present in HPV16 L2 yielded exposed L2 N-terminus in the absence of active CyP and bypassed the need for cell surface CyPB. However, this mutant was still sensitive to CyP inhibition and required CyP for completion of infection, probably after internalization. Taken together, these data suggest that CyP is required during two distinct steps of HPV16 infection. Identification of cell surface CyPB will facilitate the study of the complex events preceding internalization and adds a putative drug target for prevention of HPV-induced diseases.

Live attenuated influenza vaccine (LAIV) impacts innate and adaptive immune responses.

PubMed

Lanthier, Paula A; Huston, Gail E; Moquin, Amy; Eaton, Sheri M; Szaba, Frank M; Kummer, Lawrence W; Tighe, Micheal P; Kohlmeier, Jacob E; Blair, Patrick J; Broderick, Michael; Smiley, Stephen T; Haynes, Laura

2011-10-13

Influenza A infection induces a massive inflammatory response in the lungs that leads to significant illness and increases the susceptibility to secondary bacterial pneumonia. The most efficient way to prevent influenza infection is through vaccination. While inactivated vaccines induce protective levels of serum antibodies to influenza hemaglutinin (HA) and neuraminidase (NA) surface proteins, these are strain specific and offer little protection against heterosubtypic influenza viruses. In contrast, live attenuated influenza vaccines (LAIVs) induce a T cell response in addition to antibody responses against HA and NA surface proteins. Importantly, LAIV vaccination induces a response in a mouse model that protects against illness due to heterosubtypic influenza strains. While it is not completely clear what is the mechanism of action of LAIV heterosubtypic protection in humans, it has been shown that LAIV induces heterosubtypic protection in mice that is dependent upon a Type 1 immune response and requires CD8 T cells. In this study, we show that LAIV-induced immunity leads to significantly reduced viral titers and inflammatory responses in the lungs of mice following heterosubtypic infection. Not only are viral titers reduced in LAIV vaccinated mice, the amounts of inflammatory cytokines and chemokines in lung tissue are significantly lower. Additionally, we show that LAIV vaccination of healthy adults also induces a robust Type 1 memory response including the production of chemokines and cytokines involved in T cell activation and recruitment. Thus, our results indicate that LAIV vaccination functions by inducing immune memory which can act to modulate the immune response to subsequent heterosubtypic challenge by influencing both innate and adaptive responses. Copyright © 2011 Elsevier Ltd. All rights reserved.

Nonlinear Surface Dilatational Rheology and Foaming Behavior of Protein and Protein Fibrillar Aggregates in the Presence of Natural Surfactant.

PubMed

Wan, Zhili; Yang, Xiaoquan; Sagis, Leonard M C

2016-04-19

The surface and foaming properties of native soy glycinin (11S) and its heat-induced fibrillar aggregates, in the presence of natural surfactant steviol glycoside (STE), were investigated and compared at pH 7.0 to determine the impact of protein structure modification on protein-surfactant interfacial interactions. The adsorption at, and nonlinear dilatational rheological behavior of, the air-water interface were studied by combining drop shape analysis tensiometry, ellipsometry, and large-amplitude oscillatory dilatational rheology. Lissajous plots of surface pressure versus deformation were used to analyze the surface rheological response in terms of interfacial microstructure. The heat treatment generates a mixture of long fibrils and unconverted peptides. The presence of small peptides in 11S fibril samples resulted in a faster adsorption kinetics than that of native 11S. The addition of STE affected the adsorption of 11S significantly, whereas no apparent effect on the adsorption of the 11S fibril-peptide system was observed. The rheological response of interfaces stabilized by 11S-STE mixtures also differed significantly from the response for 11S fibril-peptide-STE mixtures. For 11S, the STE reduces the degree of strain hardening in extension and increases strain hardening in compression, suggesting the interfacial structure may change from a surface gel to a mixed phase of protein patches and STE domains. The foams generated from the mixtures displayed comparable foam stability to that of pure 11S. For 11S fibril-peptide mixtures STE only significantly affects the response in extension, where the degree of strain softening is decreased compared to the pure fibril-peptide system. The foam stability of the fibril-peptide system was significantly reduced by STE. These findings indicate that fibrillization of globular proteins could be a potential strategy to modify the complex surface and foaming behaviors of protein-surfactant mixtures.

Salting effects on protein components in aqueous NaCl and urea solutions: toward understanding of urea-induced protein denaturation.

PubMed

Li, Weifeng; Zhou, Ruhong; Mu, Yuguang

2012-02-02

The mechanism of urea-induced protein denaturation is explored through studying the salting effect of urea on 14 amino acid side chain analogues, and N-methylacetamide (NMA) which mimics the protein backbone. The solvation free energies of the 15 molecules were calculated in pure water, aqueous urea, and NaCl solutions. Our results show that NaCl displays strong capability to salt out all 15 molecules, while urea facilitates the solvation (salting-in) of all the 15 molecules on the other hand. The salting effect is found to be largely enthalpy-driven for both NaCl and urea. Our observations can explain the higher stability of protein's secondary and tertiary structures in typical salt solutions than that in pure water. Meanwhile, urea's capability to better solvate protein backbone and side-chain components can be extrapolated to explain protein's denaturation in aqueous urea solution. Urea salts in molecules through direct binding to solute surface, and the strength is linearly dependent on the number of heavy atoms of solute molecules. The van der Waals interactions are found to be the dominant force, which challenges a hydrogen-bonding-driven mechanism proposed previously.

Glial-released proteins in clonal cultures and their modulation by hydrocortisone

DOE Office of Scientific and Technical Information (OSTI.GOV)

Arenander, A.T.; de Vellis, J.

Rat glial C6 cells release into the culture medium a reproducible spectrum of soluble proteins of 12 major peaks over a broad molecular weight range as determined by fractionation on SDS-gel electrophoresis. Exposing C6 monolayers to hydrocortisone (HC) results in a selective alteration in the pattern of glial-released protein (GRP). The selective HC-induced increase or decrease in GRP peaks is specific to HC in that 17 ..beta..-estradiol, dibutyryl cyclic AMP, isoproterenol, and melatonin exert either no detectable or a qualitatively different influence on the GRP pattern. The HC influence is dose dependent over a physiological range of concentrations from 10/supmore » -9/ to 10/sup -6/ M. Differences in culture age and in subclones of C6 can influence both the normal and the HC-induced pattern of GRP. The origin of the GRP is unknown, but pattern reproducibility, viability tests, surface labelling studies, and metabolic labelling studies of soluble and particulate compartment proteins and glycoproteins support the position that cell lysis is not an important source of GRP. More importantly, these studies indicate that GRP and HC-induced changes in GRP pattern are physiologically significant aspects of glial cell behavior.« less

Subcritical Water Induced Complexation of Soy Protein and Rutin: Improved Interfacial Properties and Emulsion Stability.

PubMed

Chen, Xiao-Wei; Wang, Jin-Mei; Yang, Xiao-Quan; Qi, Jun-Ru; Hou, Jun-Jie

2016-09-01

Rutin is a common dietary flavonoid with important antioxidant and pharmacological activities. However, its application in the food industry is limited mainly because of its poor water solubility. The subcritical water (SW) treatment provides an efficient technique to solubilize and achieve the enrichment of rutin in soy protein isolate (SPI) by inducing their complexation. The physicochemical, interfacial, and emulsifying properties of the complex were investigated and compared to the mixtures. SW treatment had much enhanced rutin-combined capacity of SPI than that of conventional method, ascribing to the well-contacted for higher water solubility of rutin with stronger collision-induced hydrophobic interactions. Compared to the mixtures of rutin with proteins, the complex exhibited an excellent surface activity and improved the physical and oxidative stability of its stabilized emulsions. This improving effect could be attributed to the targeted accumulation of rutin at the oil-water interface accompanied by the adsorption of SPI resulting in the thicker interfacial layer, as evidenced by higher interfacial protein and rutin concentrations. This study provides a novel strategy for the design and enrichment of nanovehicle providing water-insoluble hydrophobic polyphenols for interfacial delivery in food emulsified systems. © 2016 Institute of Food Technologists®

Identification of a G protein coupled receptor induced in activated T cells.

PubMed

Kaplan, M H; Smith, D I; Sundick, R S

1993-07-15

Many genes are induced after T cell activation to make a cell competent for proliferation and ultimately, function. Many of these genes encode surface receptors for growth factors that signal a cell to proliferate. We have cloned a novel gene (clone 6H1) that codes for a member of the G protein-coupled receptor superfamily. This gene was isolated from a chicken activated T cell cDNA library by low level hybridization to mammalian IL-2 cDNA probes. The 308 amino acid open reading frame has seven hydrophobic, presumably transmembrane domains and a consensus site for interaction with G proteins. Tissue distribution studies suggest that gene expression is restricted to activated T cells. The message appears by 1 h after activation and is maintained for at least 45 h. Transcription of 6H1 is induced by a number of T cell stimuli and is inhibited by cyclosporin A, but not by cycloheximide. This is the first description of a member of this superfamily expressed specifically in activated T cells. The gene product may provide a link between T cell growth factors and G protein activation.

Protein adsorption at the electrified air-water interface: implications on foam stability.

PubMed

Engelhardt, Kathrin; Rumpel, Armin; Walter, Johannes; Dombrowski, Jannika; Kulozik, Ulrich; Braunschweig, Björn; Peukert, Wolfgang

2012-05-22

The surface chemistry of ions, water molecules, and proteins as well as their ability to form stable networks in foams can influence and control macroscopic properties such as taste and texture of dairy products considerably. Despite the significant relevance of protein adsorption at liquid interfaces, a molecular level understanding on the arrangement of proteins at interfaces and their interactions has been elusive. Therefore, we have addressed the adsorption of the model protein bovine serum albumin (BSA) at the air-water interface with vibrational sum-frequency generation (SFG) and ellipsometry. SFG provides specific information on the composition and average orientation of molecules at interfaces, while complementary information on the thickness of the adsorbed layer can be obtained with ellipsometry. Adsorption of charged BSA proteins at the water surface leads to an electrified interface, pH dependent charging, and electric field-induced polar ordering of interfacial H(2)O and BSA. Varying the bulk pH of protein solutions changes the intensities of the protein related vibrational bands substantially, while dramatic changes in vibrational bands of interfacial H(2)O are simultaneously observed. These observations have allowed us to determine the isoelectric point of BSA directly at the electrolyte-air interface for the first time. BSA covered air-water interfaces with a pH near the isoelectric point form an amorphous network of possibly agglomerated BSA proteins. Finally, we provide a direct correlation of the molecular structure of BSA interfaces with foam stability and new information on the link between microscopic properties of BSA at water surfaces and macroscopic properties such as the stability of protein foams.

Simple, benign, aqueous-based amination of polycarbonate surfaces

DOE PAGES

VanDelinder, Virginia; Wheeler, David R.; Small, Leo J.; ...

2015-03-18

Here we report a simple, safe, environmentally-friendly aqueous method that uses diamines to functionalize a polycarbonate surface with amino groups. We demonstrate the ability of this facile method to serve as a foundation upon which other functionalities may be attached, including anti-fouling coatings and oriented membrane proteins. The use of water as the solvent for the functionalization ensures that solvent induced swelling does not affect the optical or mechanical properties of the polycarbonate.

Global proteome profiling of dental cementum under experimentally-induced apposition.

PubMed

Salmon, Cristiane R; Giorgetti, Ana Paula O; Paes Leme, Adriana Franco; Domingues, Romênia R; Sallum, Enilson Antonio; Alves, Marcelo C; Kolli, Tamara N; Foster, Brian L; Nociti, Francisco H

2016-06-01

Dental cementum (DC) covers the tooth root and has important functions in tooth attachment and position. DC can be lost to disease, and regeneration is currently unpredictable due to limited understanding of DC formation. This study used a model of experimentally-induced apposition (EIA) in mice to identify proteins associated with new DC formation. Mandibular first molars were induced to super-erupt for 6 and 21days after extracting opposing maxillary molars. Decalcified and formalin-fixed paraffin-embedded mandible sections were prepared for laser capture microdissection. Microdissected protein extracts were analyzed by liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS), and the data submitted to repeated measure ANOVA test (RM-ANOVA, alpha=5%). A total of 519 proteins were identified, with 97 (18.6%) proteins found exclusively in EIA sites and 50 (9.6%) proteins exclusively expressed in control sites. Fifty six (10.7%) proteins were differentially regulated by RM-ANOVA (p<0.05), with 24 regulated by the exclusive effect of EIA (12 proteins) or the interaction between EIA and time (12 proteins), including serpin 1a, procollagen C-endopeptidase enhancer, tenascin X (TNX), and asporin (ASPN). In conclusion, proteomic analysis demonstrated significantly altered protein profile in DC under EIA, providing new insights on DC biology and potential candidates for tissue engineering applications. Dental cementum (DC) is a mineralized tissue that covers the tooth root surface and has important functions in tooth attachment and position. DC and other periodontal tissues can be lost to disease, and regeneration is currently unpredictable due to lack of understanding of DC formation. This study used a model of experimentally-induced apposition (EIA) in mice to promote new cementum formation, followed by laser capture microdissection (LCM) and liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) proteomic analysis. This approach identified proteins associated with new cementum formation that may be targets for promoting cementum regeneration. Copyright © 2016 Elsevier B.V. All rights reserved.

Effect of Surface Curvature and Chemistry on Protein Stability, Adsorption and Aggregation

NASA Astrophysics Data System (ADS)

Radhakrishna, Mithun

Enzyme immobilization has been of great industrial importance because of its use in various applications like bio-fuel cells, bio-sensors, drug delivery and bio-catalytic films. Although research on enzyme immobilization dates back to the 1970's, it has been only in the past decade that scientists have started to address the problems involved systematically. Most of the previous works on enzyme immobilization have been retrospective in nature i.e enzymes were immobilized on widely used substrates without a compatibility study between the enzyme and the substrate. Consequently, most of the enzymes lost their activity upon immobilization onto these substrates due to many governing factors like protein-surface and inter-protein interactions. These interactions also play a major role biologically in cell signaling, cell adhesion and inter-protein interactions specifically is believed to be the major cause for neurodegenerative diseases like Alzheimer's and Parkinson's disease. Therefore understanding the role of these forces on proteins is the need of the hour. In my current research, I have mainly focused on two factors a) Surface Curvature b) Surface Chemistry as both of these play a pivotal role in influencing the activity of the enzymes upon immobilization. I study the effect of these factors computationally using a stochastic method known as Monte Carlo simulations. My research work carried out in the frame work of a Hydrophobic-Polar (HP) lattice model for the protein shows that immobilizing enzymes inside moderately hydrophilic or hydrophobic pores results in an enhancement of the enzymatic activity compared to that in the bulk. Our results also indicate that there is an optimal value of surface curvature and hydrophobicity/hydrophilicity where this enhancement of enzymatic activity is highest. Further, our results also show that immobilization of enzymes inside hydrophobic pores of optimal sizes are most effective in mitigating protein-aggregation. These results provide us a rationale to understand the role of chaperonins in protein folding and disaggregation. Our results indicate that strong protein-surface interactions and confinement inducement stability inside pores makes it best suitable for enzyme immobilization.

Group A Streptococcus tissue invasion by CD44-mediated cell signalling

NASA Astrophysics Data System (ADS)

Cywes, Colette; Wessels, Michael R.

2001-12-01

Streptococcus pyogenes (also known as group A Streptococcus, GAS), the agent of streptococcal sore throat and invasive soft-tissue infections, attaches to human pharyngeal or skin epithelial cells through specific recognition of its hyaluronic acid capsular polysaccharide by the hyaluronic-acid-binding protein CD44 (refs 1, 2). Because ligation of CD44 by hyaluronic acid can induce epithelial cell movement on extracellular matrix, we investigated whether molecular mimicry by the GAS hyaluronic acid capsule might induce similar cellular responses. Here we show that CD44-dependent GAS binding to polarized monolayers of human keratinocytes induced marked cytoskeletal rearrangements manifested by membrane ruffling and disruption of intercellular junctions. Transduction of the signal induced by GAS binding to CD44 on the keratinocyte surface involved Rac1 and the cytoskeleton linker protein ezrin, as well as tyrosine phosphorylation of cellular proteins. Studies of bacterial translocation in two models of human skin indicated that cell signalling triggered by interaction of the GAS capsule with CD44 opened intercellular junctions and promoted tissue penetration by GAS through a paracellular route. These results support a model of host cytoskeleton manipulation and tissue invasion by an extracellular bacterial pathogen.

Soybean agglutinin binding to corneal endothelial cell surfaces disrupts in situ monolayer integrity and actin organization and interferes with wound repair.

PubMed

Gordon, Sheldon R; Wood, Meredith

2009-03-01

Rat corneal endothelium demonstrates cell-surface soybean agglutinin (SBA) binding during organ-culture or injury. When organ-cultured in medium containing SBA, the endothelial monolayer is disrupted because of cell-cell and cell-matrix alterations. SBA binding disorganizes the circumferential microfilament bundles (CMBs), an effect that is partially prevented by phallacidin preincubation. This disruption is reversible if tissues are returned to standard culture medium. Serum heightens SBA binding, whereas puromycin prevents it. Neither actinomycin D nor alpha-amanitin inhibits SBA binding, suggesting that SBA-binding protein(s) may be post-transcriptionally regulated. During injury-induced cell migration in the presence of SBA, cellular processes are blunted and fail to extend significantly outward. By 72 h post-injury, cells of SBA-treated tissues repopulate the wound but demonstrate little association with neighboring cells. Cells migrating in the presence of N-acetylgalactosamine appear normal but also fail to reassociate with other cells in the jury zone. Immunofluorescent staining for ZO-1 reveals punctuate patterns in cells of control tissues, whereas neither SBA- nor N-acetylgalactosamine-treated tissues exhibit ZO-1 staining. Terminal N-acetylgalactosamine removal fails to affect cell morphology, actin organization, or migration but prevents lectin binding. Our results suggest that SBA binding reflects the synthesis of a stress-induced protein(s) that may play a role in reestablishing cell-cell relationships during monolayer reorganization following injury.

Serum Proteins Enhance Dispersion Stability and Influence the Cytotoxicity and Dosimetry of ZnO Nanoparticles in Suspension and Adherent Cancer Cell Models.

PubMed

Anders, Catherine B; Chess, Jordan J; Wingett, Denise G; Punnoose, Alex

2015-12-01

Agglomeration and sedimentation of nanoparticles (NPs) within biological solutions is a major limitation in their use in many downstream applications. It has been proposed that serum proteins associate with the NP surface to form a protein corona that limits agglomeration and sedimentation. Here, we investigate the effect of fetal bovine serum (FBS) proteins on the dispersion stability, dosimetry, and NP-induced cytotoxicity of cationic zinc oxide nanoparticles (nZnO) synthesized via forced hydrolysis with a core size of 10 nm. Two different in vitro cell culture models, suspension and adherent, were evaluated by comparing a phosphate buffered saline (PBS) nZnO dispersion (nZnO/PBS) and an FBS-stabilized PBS nZnO dispersion (nZnO - FBS/PBS). Surface interactions of FBS on nZnO were analyzed via spectroscopic and optical techniques. Fourier transformed infrared spectroscopy (FTIR) confirmed the adsorption of negatively charged protein components on the cationic nZnO surface through the disappearance of surfaced-adsorbed carboxyl functional groups and the subsequent detection of vibrational modes associated with the protein backbone of FBS-associated proteins. Further confirmation of these interactions was noted in the isoelectric point shift of the nZnO from the characteristic pH of 9.5 to a pH of 6.1. In nZnO - FBS/PBS dispersions, the FBS reduced agglomeration and sedimentation behaviors to impart long-term improvements (>24 h) to the nZnO dispersion stability. Furthermore, mathematical dosimetry models indicate that nZnO - FBS/PBS dispersions had consistent NP deposition patterns over time unlike unstable nZnO/PBS dispersions. In suspension cell models, the stable nZnO - FBS/PBS dispersion resulted in a ~33 % increase in the NP-induced cytotoxicity for both Jurkat leukemic and Hut-78 lymphoma cancer cells. In contrast, the nZnO - FBS/PBS dispersion resulted in 49 and 71 % reductions in the cytotoxicity observed towards the adherent breast (T-47D) and prostate (LNCaP) cancer cell lines, respectively. Presence of FBS in the NP dispersions also increased the reactive oxygen species generation. These observations indicate that the improved dispersion stability leads to increased NP bioavailability for suspension cell models and reduced NP sedimentation onto adherent cell layers resulting in more accurate in vitro toxicity assessments.

Serum Proteins Enhance Dispersion Stability and Influence the Cytotoxicity and Dosimetry of ZnO Nanoparticles in Suspension and Adherent Cancer Cell Models

NASA Astrophysics Data System (ADS)

Anders, Catherine B.; Chess, Jordan J.; Wingett, Denise G.; Punnoose, Alex

2015-11-01

Agglomeration and sedimentation of nanoparticles (NPs) within biological solutions is a major limitation in their use in many downstream applications. It has been proposed that serum proteins associate with the NP surface to form a protein corona that limits agglomeration and sedimentation. Here, we investigate the effect of fetal bovine serum (FBS) proteins on the dispersion stability, dosimetry, and NP-induced cytotoxicity of cationic zinc oxide nanoparticles (nZnO) synthesized via forced hydrolysis with a core size of 10 nm. Two different in vitro cell culture models, suspension and adherent, were evaluated by comparing a phosphate buffered saline (PBS) nZnO dispersion (nZnO/PBS) and an FBS-stabilized PBS nZnO dispersion (nZnO - FBS/PBS). Surface interactions of FBS on nZnO were analyzed via spectroscopic and optical techniques. Fourier transformed infrared spectroscopy (FTIR) confirmed the adsorption of negatively charged protein components on the cationic nZnO surface through the disappearance of surfaced-adsorbed carboxyl functional groups and the subsequent detection of vibrational modes associated with the protein backbone of FBS-associated proteins. Further confirmation of these interactions was noted in the isoelectric point shift of the nZnO from the characteristic pH of 9.5 to a pH of 6.1. In nZnO - FBS/PBS dispersions, the FBS reduced agglomeration and sedimentation behaviors to impart long-term improvements (>24 h) to the nZnO dispersion stability. Furthermore, mathematical dosimetry models indicate that nZnO - FBS/PBS dispersions had consistent NP deposition patterns over time unlike unstable nZnO/PBS dispersions. In suspension cell models, the stable nZnO - FBS/PBS dispersion resulted in a ~33 % increase in the NP-induced cytotoxicity for both Jurkat leukemic and Hut-78 lymphoma cancer cells. In contrast, the nZnO - FBS/PBS dispersion resulted in 49 and 71 % reductions in the cytotoxicity observed towards the adherent breast (T-47D) and prostate (LNCaP) cancer cell lines, respectively. Presence of FBS in the NP dispersions also increased the reactive oxygen species generation. These observations indicate that the improved dispersion stability leads to increased NP bioavailability for suspension cell models and reduced NP sedimentation onto adherent cell layers resulting in more accurate in vitro toxicity assessments.

Molecular characterization of Trypanosoma cruzi SAP proteins with host-cell lysosome exocytosis-inducing activity required for parasite invasion.

PubMed

Zanforlin, Tamiris; Bayer-Santos, Ethel; Cortez, Cristian; Almeida, Igor C; Yoshida, Nobuko; da Silveira, José Franco

2013-01-01

To invade target cells, Trypanosoma cruzi metacyclic forms engage distinct sets of surface and secreted molecules that interact with host components. Serine-, alanine-, and proline-rich proteins (SAP) comprise a multigene family constituted of molecules with a high serine, alanine and proline residue content. SAP proteins have a central domain (SAP-CD) responsible for interaction with and invasion of mammalian cells by metacyclic forms. Using a 513 bp sequence from SAP-CD in blastn analysis, we identified 39 full-length SAP genes in the genome of T. cruzi. Although most of these genes were mapped in the T. cruzi in silico chromosome TcChr41, several SAP sequences were spread out across the genome. The level of SAP transcripts was twice as high in metacyclic forms as in epimastigotes. Monoclonal (MAb-SAP) and polyclonal (anti-SAP) antibodies produced against the recombinant protein SAP-CD were used to investigate the expression and localization of SAP proteins. MAb-SAP reacted with a 55 kDa SAP protein released by epimastigotes and metacyclic forms and with distinct sets of SAP variants expressed in amastigotes and tissue culture-derived trypomastigotes (TCTs). Anti-SAP antibodies reacted with components located in the anterior region of epimastigotes and between the nucleus and the kinetoplast in metacyclic trypomastigotes. In contrast, anti-SAP recognized surface components of amastigotes and TCTs, suggesting that SAP proteins are directed to different cellular compartments. Ten SAP peptides were identified by mass spectrometry in vesicle and soluble-protein fractions obtained from parasite conditioned medium. Using overlapping sequences from SAP-CD, we identified a 54-aa peptide (SAP-CE) that was able to induce host-cell lysosome exocytosis and inhibit parasite internalization by 52%. This study provides novel information about the genomic organization, expression and cellular localization of SAP proteins and proposes a triggering role for extracellular SAP proteins in host-cell lysosome exocytosis during metacyclic internalization.

Molecular Characterization of Trypanosoma cruzi SAP Proteins with Host-Cell Lysosome Exocytosis-Inducing Activity Required for Parasite Invasion

PubMed Central

Zanforlin, Tamiris; Bayer-Santos, Ethel; Cortez, Cristian; Almeida, Igor C.; Yoshida, Nobuko; da Silveira, José Franco

2013-01-01

Background To invade target cells, Trypanosoma cruzi metacyclic forms engage distinct sets of surface and secreted molecules that interact with host components. Serine-, alanine-, and proline-rich proteins (SAP) comprise a multigene family constituted of molecules with a high serine, alanine and proline residue content. SAP proteins have a central domain (SAP-CD) responsible for interaction with and invasion of mammalian cells by metacyclic forms. Methods and Findings Using a 513 bp sequence from SAP-CD in blastn analysis, we identified 39 full-length SAP genes in the genome of T. cruzi. Although most of these genes were mapped in the T. cruzi in silico chromosome TcChr41, several SAP sequences were spread out across the genome. The level of SAP transcripts was twice as high in metacyclic forms as in epimastigotes. Monoclonal (MAb-SAP) and polyclonal (anti-SAP) antibodies produced against the recombinant protein SAP-CD were used to investigate the expression and localization of SAP proteins. MAb-SAP reacted with a 55 kDa SAP protein released by epimastigotes and metacyclic forms and with distinct sets of SAP variants expressed in amastigotes and tissue culture-derived trypomastigotes (TCTs). Anti-SAP antibodies reacted with components located in the anterior region of epimastigotes and between the nucleus and the kinetoplast in metacyclic trypomastigotes. In contrast, anti-SAP recognized surface components of amastigotes and TCTs, suggesting that SAP proteins are directed to different cellular compartments. Ten SAP peptides were identified by mass spectrometry in vesicle and soluble-protein fractions obtained from parasite conditioned medium. Using overlapping sequences from SAP-CD, we identified a 54-aa peptide (SAP-CE) that was able to induce host-cell lysosome exocytosis and inhibit parasite internalization by 52%. Conclusions This study provides novel information about the genomic organization, expression and cellular localization of SAP proteins and proposes a triggering role for extracellular SAP proteins in host-cell lysosome exocytosis during metacyclic internalization. PMID:24391838

Induction of neutralizing antibodies specific for the envelope proteins of the koala retrovirus by immunization with recombinant proteins or with DNA.

PubMed

Fiebig, Uwe; Dieckhoff, Britta; Wurzbacher, Christian; Möller, Annekathrin; Kurth, Reinhard; Denner, Joachim

2015-04-30

The koala retrovirus (KoRV) is the result of a transspecies transmission of a gammaretrovirus with fatal consequences for the new host. Like many retroviruses, KoRV induces lymphoma, leukemia and an immunodeficiency that is associated with opportunistic infections in the virus-infected animals. We recently reported the induction of neutralizing antibodies by immunization with the recombinant ectodomain of the transmembrane envelope protein p15E of KoRV. Since the neutralization titers of the p15E-specific sera were only moderate, we investigated the use of the surface envelope protein gp70 to induce neutralizing antibodies. We immunized rats and goats with the recombinant gp70 protein of the KoRV, an unglycosylated protein of 52kD (rgp70/p52) or with the corresponding DNA. In parallel we immunized with recombinant rp15E or with a combination of rp15E and rgp70/p52. In all cases binding and neutralizing antibodies were induced. The gp70-specific sera had titers of neutralizing antibodies that were 15-fold higher than the p15E-specific sera. Combining rp15E and rgp70/p52 did not significantly increase neutralizing titers compared to rgp70/p52 alone. High titers of neutralizing antibodies specific for gp70 were also induced by immunization with DNA. Since KoRV and PERV are closely related, we investigated cross-neutralization of the antisera. The antisera against p15E and gp70 of PERV and KoRV inhibited infection by both viruses. The envelope proteins of the KoRV may therefore form the basis of an effective preventive vaccine to protect uninfected koalas from infection and possibly an immunotherapeutic treatment for those already infected.

Organic crystal-binding peptides: morphology control and one-pot formation of protein-displaying organic crystals

NASA Astrophysics Data System (ADS)

Niide, Teppei; Ozawa, Kyohei; Nakazawa, Hikaru; Oliveira, Daniel; Kasai, Hitoshi; Onodera, Mari; Asano, Ryutaro; Kumagai, Izumi; Umetsu, Mitsuo

2015-11-01

Crystalline assemblies of fluorescent molecules have different functional properties than the constituent monomers, as well as unique optical characteristics that depend on the structure, size, and morphological homogeneity of the crystal particles. In this study, we selected peptides with affinity for the surface of perylene crystal particles by exposing a peptide-displaying phage library in aqueous solution to perylene crystals, eluting the surface-bound phages by means of acidic desorption or liquid-liquid extraction, and amplifying the obtained phages in Escherichia coli. One of the perylene-binding peptides, PeryBPb1: VQHNTKYSVVIR, selected by this biopanning procedure induced perylene molecules to form homogenous planar crystal nanoparticles by means of a poor solvent method, and fusion of the peptide to a fluorescent protein enabled one-pot formation of protein-immobilized crystalline nanoparticles. The nanoparticles were well-dispersed in aqueous solution, and Förster resonance energy transfer from the perylene crystals to the fluorescent protein was observed. Our results show that the crystal-binding peptide could be used for simultaneous control of perylene crystal morphology and dispersion and protein immobilization on the crystals.Crystalline assemblies of fluorescent molecules have different functional properties than the constituent monomers, as well as unique optical characteristics that depend on the structure, size, and morphological homogeneity of the crystal particles. In this study, we selected peptides with affinity for the surface of perylene crystal particles by exposing a peptide-displaying phage library in aqueous solution to perylene crystals, eluting the surface-bound phages by means of acidic desorption or liquid-liquid extraction, and amplifying the obtained phages in Escherichia coli. One of the perylene-binding peptides, PeryBPb1: VQHNTKYSVVIR, selected by this biopanning procedure induced perylene molecules to form homogenous planar crystal nanoparticles by means of a poor solvent method, and fusion of the peptide to a fluorescent protein enabled one-pot formation of protein-immobilized crystalline nanoparticles. The nanoparticles were well-dispersed in aqueous solution, and Förster resonance energy transfer from the perylene crystals to the fluorescent protein was observed. Our results show that the crystal-binding peptide could be used for simultaneous control of perylene crystal morphology and dispersion and protein immobilization on the crystals. Electronic supplementary information (ESI) available: Schematic representation of PeryBPb1-fused DsRed-Monomer, fluorescence spectra of perylene crystals and DsRed-Monomer, and emission spectra of DsRed-Monomer at various excitation wavelengths. See DOI: 10.1039/c5nr06471f

The Glycolytic Enzyme Triosephosphate Isomerase of Trichomonas vaginalis Is a Surface-Associated Protein Induced by Glucose That Functions as a Laminin- and Fibronectin-Binding Protein

PubMed Central

Miranda-Ozuna, Jesús F. T.; Hernández-García, Mar S.; Brieba, Luis G.; Benítez-Cardoza, Claudia G.; Ortega-López, Jaime; González-Robles, Arturo

2016-01-01

Triosephosphate isomerase of Trichomonas vaginalis (TvTIM) is a 27-kDa cytoplasmic protein encoded by two genes, tvtim1 and tvtim2, that participates in glucose metabolism. TvTIM is also localized to the parasite surface. Thus, the goal of this study was to identify the novel functions of the surface-associated TvTIM in T. vaginalis and to assess the effect of glucose as an environmental factor that regulates its expression and localization. Reverse transcription-PCR (RT-PCR) showed that the tvtim genes were differentially expressed in response to glucose concentration. tvtim1 was overexpressed under glucose-restricted (GR) conditions, whereas tvtim2 was overexpressed under glucose-rich, or high-glucose (HG), conditions. Western blot and indirect immunofluorescence assays also showed that glucose positively affected the amount and surface localization of TvTIM in T. vaginalis. Affinity ligand assays demonstrated that the recombinant TvTIM1 and TvTIM2 proteins bound to laminin (Lm) and fibronectin (Fn) but not to plasminogen. Moreover, higher levels of adherence to Lm and Fn were detected in parasites grown under HG conditions than in those grown under GR conditions. Furthermore, pretreatment of trichomonads with an anti-TvTIMr polyclonal antibody or pretreatment of Lm- or Fn-coated wells with both recombinant proteins (TvTIM1r and TvTIM2r) specifically reduced the binding of live parasites to Lm and Fn in a concentration-dependent manner. Moreover, T. vaginalis was exposed to different glucose concentrations during vaginal infection of women with trichomoniasis. Our data indicate that TvTIM is a surface-associated protein under HG conditions that mediates specific binding to Lm and Fn as a novel virulence factor of T. vaginalis. PMID:27481251

Surface modification of a gold-coated microcantilever and application in biomarker detection

NASA Astrophysics Data System (ADS)

Binh Pham, Van; Nhat Khoa Phan, Thanh; Nguyen, Thanh Trung; Pham, Xuan Thanh Tung; Thanh Tuyen Le, Thi; Chien Dang, Mau

2015-12-01

Biosensors have been rapidly developed recently. Biological receptors, such as antibodies, must be immobilized on these sensors’ surfaces to make the sensor capable of capturing a target analyte. In this research we studied how to modify a gold-coated surface of a microcantilever, a sensor with high potential in biological and medical applications. Thiol chemistry was adapted to create a cysteamine layer on a gold surface, and subsequently glutaraldehyde was used as a cross-linking agent to react with amine groups in receptors. In order to evaluate the efficiency of immobilizing protein on an Au surface and also whether the protein retains its biological activity, horseradish peroxidase enzyme (HRP) with its activity to catalyze a reaction between 2,2‧-azino-bis [3-ethylbenzothiazoline-6-sulphonic acid] (ABTS) and {{{H}}}2{{{{O}}}2}- was used as a testing protein. The result showed that HRP was immobilized successfully on cysteamine and glutaraldehyde layers and retained its activity. The cantilever’s tip deflection was also measured, and results showed that each layer created surface stress and made the cantilever bend—in particular, the cysteamine layer induced bending as high as 6 μm. An antibody of alpha-fetoprotein (AFP) was immobilized on the cantilever surface, and the measurement deflection showed that the sensor responded to solution containing AFP with concentration from 100 to 500 ng ml-1.

Multistage adsorption of diffusing macromolecules and viruses

NASA Astrophysics Data System (ADS)

Chou, Tom; D'Orsogna, Maria R.

2007-09-01

We derive the equations that describe adsorption of diffusing particles onto a surface followed by additional surface kinetic steps before being transported across the interface. Multistage surface kinetics occurs during membrane protein insertion, cell signaling, and the infection of cells by virus particles. For example, viral entry into healthy cells is possible only after a series of receptor and coreceptor binding events occurs at the cellular surface. We couple the diffusion of particles in the bulk phase with the multistage surface kinetics and derive an effective, integrodifferential boundary condition that contains a memory kernel embodying the delay induced by the surface reactions. This boundary condition takes the form of a singular perturbation problem in the limit where particle-surface interactions are short ranged. Moreover, depending on the surface kinetics, the delay kernel induces a nonmonotonic, transient replenishment of the bulk particle concentration near the interface. The approach generalizes that of Ward and Tordai [J. Chem. Phys. 14, 453 (1946)] and Diamant and Andelman [Colloids Surf. A 183-185, 259 (2001)] to include surface kinetics, giving rise to qualitatively new behaviors. Our analysis also suggests a simple scheme by which stochastic surface reactions may be coupled to deterministic bulk diffusion.

Brain region-selective cellular redistribution of mGlu5 but not GABA(B) receptors following methamphetamine-induced associative learning.

PubMed

Herrold, Amy A; Voigt, Robin M; Napier, T Celeste

2011-12-01

Alterations in receptor expression and distribution between cell surface and cytoplasm are means by which psychostimulants regulate neurotransmission. Metabotropic glutamate receptor group I, subtype 5 (mGluR5) and GABA(B) receptors (GABA(B) R) are critically involved in the development and expression of stimulant-induced behaviors, including conditioned place preference (CPP), an index of drug-seeking. However, it is not known if psychostimulant-induced CPP alters the trafficking of these receptors. To fill this gap, this study used methamphetamine (Meth)-induced CPP in rats to ascertain if receptor changes occur in limbic brain regions that regulate drug-seeking, the medial prefrontal cortex (mPFC), nucleus accumbens (NAc), and ventral pallidum (VP). To do so, ex vivo tissue was assessed for changes in expression and surface vs. intracellular distribution of mGluR5 and GABA(B) Rs. There was a decrease in the surface to intracellular ratio of mGluR5 in the mPFC in Meth-conditioned rats, commensurate with an increase in intracellular levels. mGluR5 levels in the NAc or the VP were unaltered. There were no changes for GABA(B) R in any brain region assayed. This ex vivo snapshot of metabotropic glutamate and GABA receptor cellular distribution following induction of Meth-induced CPP is the first report to determine if these receptors are differentially altered after Meth-induced CPP. The results suggest that this Meth treatment paradigm likely induced a compensatory change in mGluR5 surface to intracellular ratio such that the surface remains unaltered while an increase in intracellular protein occurred. Copyright © 2011 Wiley-Liss, Inc.

17beta-estradiol stimulates the growth of human keratinocytes by inducing cyclin D2 expression.

PubMed

Kanda, Naoko; Watanabe, Shinichi

2004-08-01

Estrogen is reported to prevent age-associated epidermal thinning in the skin. We examined if 17beta-estradiol (E2) may enhance the growth of human keratinocytes, focusing on its effects on the expression of cell cycle-regulatory proteins. E2 enhanced proliferation, bromodeoxyuridine incorporation of keratinocytes, and increased the proportion of cells in the S phase. The E2-induced stimulation of proliferation and bromodeoxyuridine incorporation was suppressed by antisense oligonucleotide against cyclin D2, which induces G1 to S phase progression. E2 increased protein and mRNA levels of cyclin D2, and resultantly enhanced assembly and kinase activities of cyclin D2-cyclin-dependent kinases 4 or 6 complexes. E2 enhanced cyclin D2 promoter activity, and the element homologous to cAMP response element (CRE) on the promoter was responsible for the effect. Cyclin D2 expression was enhanced by antiestrogens, ICI 182,780 and 4-hydroxytamoxifen, and membrane-impermeable bovine serum albumin-conjugated E2, indicating the effects via membrane E2-binding sites. E2 increased the enhancer activity of CRE-like element and the amount of phosphorylated cAMP response element binding protein (CREB) binding this element, and the increases were suppressed by H-89, an inhibitor of cAMP-dependent protein kinase A. H-89 also suppressed E2-induced cyclin D2 expression, proliferation, and bromodeoxyuridine incorporation in keratinocytes. Antisense oligonucleotide against G-protein-coupled receptor GPR30 suppressed the E2-induced increases of phosphorylated CREB, cyclin D2 level, proliferation, and bromodeoxyuridine incorporation in keratinocytes. These results suggest that E2 may stimulate the growth of keratinocytes by inducing cyclin D2 expression via CREB phosphorylation by protein kinase A, dependent on cAMP. These effects of E2 may be mediated via cell surface GPR30.

Lysozyme Photochemistry as a Function of Temperature. The Protective Effect of Nanoparticles on Lysozyme Photostability

PubMed Central

Oliveira Silva, Catarina; Petersen, Steffen B.; Pinto Reis, Catarina; Rijo, Patrícia; Molpeceres, Jesús; Vorum, Henrik; Neves-Petersen, Maria Teresa

2015-01-01

The presence of aromatic residues and their close spatial proximity to disulphide bridges makes hen egg white lysozyme labile to UV excitation. UVB induced photo-oxidation of tryptophan and tyrosine residues leads to photochemical products, such as, kynurenine, N–formylkynurenine and dityrosine and to the disruption of disulphide bridges in proteins. We here report that lysozyme UV induced photochemistry is modulated by temperature, excitation power, illumination time, excitation wavelength and by the presence of plasmonic quencher surfaces, such as gold, and by the presence of natural fluorescence quenchers, such as hyaluronic acid and oleic acid. We show evidence that the photo-oxidation effects triggered by 295 nm at 20°C are reversible and non-reversible at 10°C, 25°C and 30°C. This paper provides evidence that the 295 nm damage threshold of lysozyme lies between 0.1 μW and 0.3 μW. Protein conformational changes induced by temperature and UV light have been detected upon monitoring changes in the fluorescence emission spectra of lysozyme tryptophan residues and SYPRO® Orange. Lysozyme has been conjugated onto gold nanoparticles, coated with hyaluronic acid and oleic acid (HAOA). Steady state and time resolved fluorescence studies of free and conjugated lysozyme onto HAOA gold nanoparticles reveals that the presence of the polymer decreased the rate of the observed photochemical reactions and induced a preference for short fluorescence decay lifetimes. Size and surface charge of the HAOA gold nanoparticles have been determined by dynamic light scattering and zeta potential measurements. TEM analysis of the particles confirms the presence of a gold core surrounded by a HAOA matrix. We conclude that HAOA gold nanoparticles may efficiently protect lysozyme from the photochemical effects of UVB light and this nanocarrier could be potentially applied to other proteins with clinical relevance. In addition, this study confirms that the temperature plays a critical role in the photochemical pathways a protein enters upon UV excitation. PMID:26656259

Lysozyme Photochemistry as a Function of Temperature. The Protective Effect of Nanoparticles on Lysozyme Photostability.

PubMed

Oliveira Silva, Catarina; Petersen, Steffen B; Pinto Reis, Catarina; Rijo, Patrícia; Molpeceres, Jesús; Vorum, Henrik; Neves-Petersen, Maria Teresa

2015-01-01

The presence of aromatic residues and their close spatial proximity to disulphide bridges makes hen egg white lysozyme labile to UV excitation. UVB induced photo-oxidation of tryptophan and tyrosine residues leads to photochemical products, such as, kynurenine, N-formylkynurenine and dityrosine and to the disruption of disulphide bridges in proteins. We here report that lysozyme UV induced photochemistry is modulated by temperature, excitation power, illumination time, excitation wavelength and by the presence of plasmonic quencher surfaces, such as gold, and by the presence of natural fluorescence quenchers, such as hyaluronic acid and oleic acid. We show evidence that the photo-oxidation effects triggered by 295 nm at 20°C are reversible and non-reversible at 10°C, 25°C and 30°C. This paper provides evidence that the 295 nm damage threshold of lysozyme lies between 0.1 μW and 0.3 μW. Protein conformational changes induced by temperature and UV light have been detected upon monitoring changes in the fluorescence emission spectra of lysozyme tryptophan residues and SYPRO® Orange. Lysozyme has been conjugated onto gold nanoparticles, coated with hyaluronic acid and oleic acid (HAOA). Steady state and time resolved fluorescence studies of free and conjugated lysozyme onto HAOA gold nanoparticles reveals that the presence of the polymer decreased the rate of the observed photochemical reactions and induced a preference for short fluorescence decay lifetimes. Size and surface charge of the HAOA gold nanoparticles have been determined by dynamic light scattering and zeta potential measurements. TEM analysis of the particles confirms the presence of a gold core surrounded by a HAOA matrix. We conclude that HAOA gold nanoparticles may efficiently protect lysozyme from the photochemical effects of UVB light and this nanocarrier could be potentially applied to other proteins with clinical relevance. In addition, this study confirms that the temperature plays a critical role in the photochemical pathways a protein enters upon UV excitation.

Nardosinone protects H9c2 cardiac cells from angiotensin II-induced hypertrophy.

PubMed

Du, Meng; Huang, Kun; Gao, Lu; Yang, Liu; Wang, Wen-Shuo; Wang, Bo; Huang, Kai; Huang, Dan

2013-12-01

Pathological cardiac hypertrophy induced by angiotensin II (AngII) can subsequently give rise to heart failure, a leading cause of mortality. Nardosinone is a pharmacologically active compound extracted from the roots of Nardostachys chinensis, a well-known traditional Chinese medicine. In order to investigate the effects of nardosinone on AngII-induced cardiac cell hypertrophy and the related mechanisms, the myoblast cell line H9c2, derived from embryonic rat heart, was treated with nardosinone (25, 50, 100, and 200 μmol/L) or AngII (1 μmol/L). Then cell surface area and mRNA expression of classical markers of hypertrophy were detected. The related protein levels in PI3K/Akt/mTOR and MEK/ERK signaling pathways were examined by Western blotting. It was found that pretreatment with nardosinone could significantly inhibit the enlargement of cell surface area induced by AngII. The mRNA expression of ANP, BNP and β-MHC was obviously elevated in AngII-treated H9c2 cells, which could be effectively blocked by nardosinone at the concentration of 100 μmol/L. Further study revealed that the protective effects of nardosinone might be mediated by repressing the phosphorylation of related proteins in PI3K/Akt and MEK/ERK signaling pathways. It was suggested that the inhibitory effect of nardosinone on Ang II-induced hypertrophy in H9c2 cells might be mediated by targeting PI3K/Akt and MEK/ERK signaling pathways.

A polyvalent hybrid protein elicits antibodies against the diverse allelic types of block 2 in Plasmodium falciparum merozoite surface protein 1.

PubMed

Tetteh, Kevin K A; Conway, David J

2011-10-13

Merozoite surface protein 1 (MSP1) of Plasmodium falciparum has been implicated as an important target of acquired immunity, and candidate components for a vaccine include polymorphic epitopes in the N-terminal polymorphic block 2 region. We designed a polyvalent hybrid recombinant protein incorporating sequences of the three major allelic types of block 2 together with a composite repeat sequence of one of the types and N-terminal flanking T cell epitopes, and compared this with a series of recombinant proteins containing modular sub-components and similarly expressed in Escherichia coli. Immunogenicity of the full polyvalent hybrid protein was tested in both mice and rabbits, and comparative immunogenicity studies of the sub-component modules were performed in mice. The full hybrid protein induced high titre antibodies against each of the major block 2 allelic types expressed as separate recombinant proteins and against a wide range of allelic types naturally expressed by a panel of diverse P. falciparum isolates, while the sub-component modules had partial antigenic coverage as expected. This encourages further development and evaluation of the full MSP1 block 2 polyvalent hybrid protein as a candidate blood-stage component of a malaria vaccine. Copyright © 2011 Elsevier Ltd. All rights reserved.

Dexamethasone enhances interaction of endogenous annexin 1 with L-selectin and triggers shedding of L-selectin in the monocytic cell line U-937.

PubMed

de Coupade, Catherine; Solito, Egle; Levine, Jon D

2003-09-01

(1) L-selectin, constitutively expressed by leukocytes, is involved in the initial binding of leukocytes to activated endothelium. Anti-inflammatory drugs like glucocorticoids can induce shedding of L-selectin, but the mechanism is still unknown. Annexin 1, a protein whose synthesis and externalization/secretion are induced during the inflammatory response, has been proposed as a mediator of the anti-inflammatory actions of glucocorticoids. (2) The monocytic cell line U-937 strongly expresses Annexin 1 after 24 h of phorbol 12-myristate 13-acetate (PMA, 1 nm) treatment and externalizes/releases the protein after additional 16 h of dexamethasone (1 microm) treatment. (3) This study investigated the possible regulation of cell surface L-selectin shedding by endogenous Annexin 1, and its role in glucocorticoid-induced L-selectin shedding in the U-937 cell line. (4) PMA- and dexamethasone treatment-induced L-selectin shedding was potentially mediated by Annexin 1, since neutralizing antibodies against Annexin 1 reduced dexamethasone- and Annexin 1-induced shedding. (5) Immunoprecipitation and binding assays provided support for the suggestion that this effect could be mediated by an interaction between externalized Annexin 1 and L-selectin. Such interaction involved the N-terminal domain of Annexin 1 and was calcium-dependent. Confocal microscopy studies demonstrated increased colocalization of Annexin 1 and L-selectin on the cell surface. (6) Overall, our study provides new insights into the potential role of endogenous ANXA1 as a mediator of dexamethasone-induced L-selectin shedding, which may contribute to the anti-inflammatory activity of glucocorticoids.

Nanoscale Surface Plasmonics Sensor With Nanofluidic Control

NASA Technical Reports Server (NTRS)

Wei, Jianjun; Singhal, Sameer; Waldeck, David H.; Kofke, Matthew

2013-01-01

Conventional quantitative protein assays of bodily fluids typically involve multiple steps to obtain desired measurements. Such methods are not well suited for fast and accurate assay measurements in austere environments such as spaceflight and in the aftermath of disasters. Consequently, there is a need for a protein assay technology capable of routinely monitoring proteins in austere environments. For example, there is an immediate need for a urine protein assay to assess astronaut renal health during spaceflight. The disclosed nanoscale surface plasmonics sensor provides a core detection method that can be integrated to a lab-on-chip device that satisfies the unmet need for such a protein assay technology. Assays based upon combinations of nanoholes, nanorings, and nanoslits with transmission surface plasmon resonance (SPR) are used for assays requiring extreme sensitivity, and are capable of detecting specific analytes at concentrations as low as picomole to femtomole level in well-controlled environments. The device operates in a transmission mode configuration in which light is directed at one planar surface of the array, which functions as an optical aperture. The incident light induces surface plasmon light transmission from the opposite surface of the array. The presence of a target analyte is detected by changes in the spectrum of light transmitted by the array when a target analyte induces a change in the refractive index of the fluid within the nanochannels. This occurs, for example, when a target analyte binds to a receptor fixed to the walls of the nanochannels in the array. Independent fluid handling capability for individual nanoarrays on a nanofluidic chip containing a plurality of nanochannel arrays allows each array to be used to sense a different target analyte and/or for paired arrays to analyze control and test samples simultaneously in parallel. The present invention incorporates transmission mode nanoplasmonics and nanofluidics into a single, microfluidically controlled device. The device comprises one or more arrays of aligned nanochannels that are in fluid communication with inflowing and outflowing fluid handling manifolds that control the flow of fluid through the arrays. The array acts as an aperture in a plasmonic sensor. Fluid, in the form of a liquid or a gas and comprising a sample for analysis, is moved from an inlet manifold through the nanochannel array, and out through an exit manifold. The fluid may also contain a reagent used to modify the interior surfaces of the nanochannels, and/or a reagent required for the detection of an analyte.

Regulated internalization of NMDA receptors drives PKD1-mediated suppression of the activity of residual cell-surface NMDA receptors.

PubMed

Fang, Xiao-Qian; Qiao, Haifa; Groveman, Bradley R; Feng, Shuang; Pflueger, Melissa; Xin, Wen-Kuan; Ali, Mohammad K; Lin, Shuang-Xiu; Xu, Jindong; Duclot, Florian; Kabbaj, Mohamed; Wang, Wei; Ding, Xin-Sheng; Santiago-Sim, Teresa; Jiang, Xing-Hong; Salter, Michael W; Yu, Xian-Min

2015-11-19

Constitutive and regulated internalization of cell surface proteins has been extensively investigated. The regulated internalization has been characterized as a principal mechanism for removing cell-surface receptors from the plasma membrane, and signaling to downstream targets of receptors. However, so far it is still not known whether the functional properties of remaining (non-internalized) receptor/channels may be regulated by internalization of the same class of receptor/channels. The N-methyl-D-aspartate receptor (NMDAR) is a principal subtype of glutamate-gated ion channel and plays key roles in neuronal plasticity and memory functions. NMDARs are well-known to undergo two types of regulated internalization - homologous and heterologous, which can be induced by high NMDA/glycine and DHPG, respectively. In the present work, we investigated effects of regulated NMDAR internalization on the activity of residual cell-surface NMDARs and neuronal functions. In electrophysiological experiments we discovered that the regulated internalization of NMDARs not only reduced the number of cell surface NMDARs but also caused an inhibition of the activity of remaining (non-internalized) surface NMDARs. In biochemical experiments we identified that this functional inhibition of remaining surface NMDARs was mediated by increased serine phosphorylation of surface NMDARs, resulting from the activation of protein kinase D1 (PKD1). Knockdown of PKD1 did not affect NMDAR internalization but prevented the phosphorylation and inhibition of remaining surface NMDARs and NMDAR-mediated synaptic functions. These data demonstrate a novel concept that regulated internalization of cell surface NMDARs not only reduces the number of NMDARs on the cell surface but also causes an inhibition of the activity of remaining surface NMDARs through intracellular signaling pathway(s). Furthermore, modulating the activity of remaining surface receptors may be an effective approach for treating receptor internalization-induced changes in neuronal functions of the CNS.

Potentiation of Tumor Necrosis Factor-α-induced Tumor Cell Apoptosis by a Small Molecule Inhibitor for Anti-apoptotic Protein hPEBP4

PubMed Central

Qiu, Jianming; Xiao, Jianfeng; Han, Chaofeng; Li, Nan; Shen, Xu; Jiang, Hualiang; Cao, Xuetao

2010-01-01

hPEBP4 (human phosphatidylethanolamine-binding protein 4) has been identified to be able to potentiate the resistance of breast, prostate, and ovarian cancers, with the preferential expression of hPEBP4, to tumor necrosis factor-α (TNF-α) or tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced apoptosis, suggesting that inhibitors targeting the anti-apoptotic protein hPEBP4 may be useful to increase the sensitivity of hPEBP4-expressing cancer cells to TNF-α or TRAIL-induced apoptosis. By structure-based virtual screening and following surface plasmon resonance-based binding assay, seven small compounds were found to potently bind with hPEBP4. The hit compounds were further functionally screened for their ability to inhibit cancer cell growth, and one small compound, IOI-42, was identified to be able to promote TNF-α-mediated growth inhibition of MCF-7 breast cancer cells. IOI-42 could potentiate TNF-α-induced apoptosis of MCF-7 cells by inhibiting hPEBP4 and could suppress anchorage-independent cell growth of MCF-7 cells. We further demonstrated that IOI-42 could reduce the endogenous association of hPEBP4 with Raf-1/MEK1 and enhance the activation of ERK1/2 and JNK while inhibiting Akt activation. Furthermore, IOI-42 also promoted TRAIL-induced cell apoptosis of prostate cancer cells. Taken together, our data suggest that IOI-42, as the first chemical inhibitor of anti-apoptotic protein hPEBP4, may serve as a potential anti-tumor drug by sensitizing tumor cells to apoptotic inducers. PMID:20177075

Photo-Induced Click Chemistry for DNA Surface Structuring by Direct Laser Writing.

PubMed

Kerbs, Antonina; Mueller, Patrick; Kaupp, Michael; Ahmed, Ishtiaq; Quick, Alexander S; Abt, Doris; Wegener, Martin; Niemeyer, Christof M; Barner-Kowollik, Christopher; Fruk, Ljiljana

2017-04-11

Oligonucleotides containing photo-caged dienes were prepared and shown to react quantitatively in a light-induced Diels-Alder cycloaddition with functional maleimides in aqueous solution within minutes. Due to its high yield and fast rate, the reaction was exploited for DNA surface patterning with sub-micrometer resolution employing direct laser writing (DLW). Functional DNA arrays were written by direct laser writing (DLW) in variable patterns, which were further encoded with fluorophores and proteins through DNA directed immobilization. This mild and efficient light-driven platform technology holds promise for the fabrication of complex bioarrays with sub-micron resolution. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Local ice melting by an antifreeze protein.

PubMed

Calvaresi, Matteo; Höfinger, Siegfried; Zerbetto, Francesco

2012-07-09

Antifreeze proteins, AFP, impede freezing of bodily fluids and damaging of cellular tissues by low temperatures. Adsorption-inhibition mechanisms have been developed to explain their functioning. Using in silico Molecular Dynamics, we show that type I AFP can also induce melting of the local ice surface. Simulations of antifreeze-positive and antifreeze-negative mutants show a clear correlation between melting induction and antifreeze activity. The presence of local melting adds a function to type I AFPs that is unique to these proteins. It may also explain some apparently conflicting experimental results where binding to ice appears both quasipermanent and reversible.

Pressure effects on collective density fluctuations in water and protein solutions

PubMed Central

Russo, Daniela; Laloni, Alessio; Filabozzi, Alessandra; Heyden, Matthias

2017-01-01

Neutron Brillouin scattering and molecular dynamics simulations have been used to investigate protein hydration water density fluctuations as a function of pressure. Our results show significant differences between the pressure and density dependence of collective dynamics in bulk water and in concentrated protein solutions. Pressure-induced changes in the tetrahedral order of the water HB network have direct consequences for the high-frequency sound velocity and damping coefficients, which we find to be a sensitive probe for changes in the HB network structure as well as the wetting of biomolecular surfaces. PMID:29073065

Effects of high hydrostatic pressure on the functional and rheological properties of the protein fraction extracted from pine nuts.

PubMed

Cao, Baiying; Fang, Li; Liu, Chunlei; Min, Weihong; Liu, Jingsheng

2018-01-01

High hydrostatic pressure treatments could increase the protein solubility (200 MPa), water holding capacity (400 MPa), and oil holding capacity (400 MPa) of pine nuts protein fractions, respectively. The exposed sufhydryl content for albumin was highest at 100 MPa while for other fractions it was 400 MPa, contrary for total sufhydryl content-generally it was at 100 MPa, except glutelin (400 MPa). Pine nuts protein fractions demonstrated the typical behavior of weak gels (G' > G″). After the treatments of high hydrostatic pressure the specific surface area of pine nuts protein particle was increased upon pressure, and the surface of protein became rough which increased the particle size. The functional groups of protein were found to be unchanged, but the characteristic peaks of pine nuts protein moved to a low-band displacement and the value of peaks was amplified accordingly to the pressure. The high hydrostatic pressure treatments were found to improve the functional properties of pine nuts protein isolates by enhancing the heat-induced gel strength of pine nuts protein isolates which make proteins more stretchable. These results suggest that high hydrostatic pressure treatments can increase the functional properties and alter the rheological properties of pine nuts protein fractions which will broaden its applications in food industry.

Dual-mode fluorophore-doped nickel nitrilotriacetic acid-modified silica nanoparticles combine histidine-tagged protein purification with site-specific fluorophore labeling.

PubMed

Kim, Sung Hoon; Jeyakumar, M; Katzenellenbogen, John A

2007-10-31

We present the first example of a fluorophore-doped nickel chelate surface-modified silica nanoparticle that functions in a dual mode, combining histidine-tagged protein purification with site-specific fluorophore labeling. Tetramethylrhodamine (TMR)-doped silica nanoparticles, estimated to contain 700-900 TMRs per ca. 23 nm particle, were surface modified with nitrilotriacetic acid (NTA), producing TMR-SiO2-NTA-Ni2+. Silica-embedded TMR retains very high quantum yield, is resistant to quenching by buffer components, and is modestly quenched and only to a certain depth (ca. 2 nm) by surface-attached Ni2+. When exposed to a bacterial lysate containing estrogen receptor alpha ligand binding domain (ERalpha) as a minor component, these beads showed very high specificity binding, enabling protein purification in one step. The capacity and specificity of these beads for binding a his-tagged protein were characterized by electrophoresis, radiometric counting, and MALDI-TOF MS. ERalpha, bound to TMR-SiO2-NTA-Ni++ beads in a site-specific manner, exhibited good activity for ligand binding and for ligand-induced binding to coactivators in solution FRET experiments and protein microarray fluorometric and FRET assays. This dual-mode type TMR-SiO2-NTA-Ni2+ system represents a powerful combination of one-step histidine-tagged protein purification and site-specific labeling with multiple fluorophore species.

Quantum mechanical/molecular mechanical/continuum style solvation model: time-dependent density functional theory.

PubMed

Thellamurege, Nandun M; Cui, Fengchao; Li, Hui

2013-08-28

A combined quantum mechanical/molecular mechanical/continuum (QM/MMpol/C) style method is developed for time-dependent density functional theory (TDDFT, including long-range corrected TDDFT) method, induced dipole polarizable force field, and induced surface charge continuum model. Induced dipoles and induced charges are included in the TDDFT equations to solve for the transition energies, relaxed density, and transition density. Analytic gradient is derived and implemented for geometry optimization and molecular dynamics simulation. QM/MMpol/C style DFT and TDDFT methods are used to study the hydrogen bonding of the photoactive yellow protein chromopore in ground state and excited state.

A small antigenic determinant of the Chikungunya virus E2 protein is sufficient to induce neutralizing antibodies which are partially protective in mice.

PubMed

Weber, Christopher; Büchner, Sarah M; Schnierle, Barbara S

2015-04-01

The mosquito-borne Chikungunya virus (CHIKV) causes high fever and severe joint pain in humans. It is expected to spread in the future to Europe and has recently reached the USA due to globalization, climate change and vector switch. Despite this, little is known about the virus life cycle and, so far, there is no specific treatment or vaccination against Chikungunya infections. We aimed here to identify small antigenic determinants of the CHIKV E2 protein able to induce neutralizing immune responses. E2 enables attachment of the virus to target cells and a humoral immune response against E2 should protect from CHIKV infections. Seven recombinant proteins derived from E2 and consisting of linear and/or structural antigens were created, and were expressed in and purified from E. coli. BALB/c mice were vaccinated with these recombinant proteins and the mouse sera were screened for neutralizing antibodies. Whereas a linear N-terminally exposed peptide (L) and surface-exposed parts of the E2 domain A (sA) alone did not induce neutralizing antibodies, a construct containing domain B and a part of the β-ribbon (called B+) was sufficient to induce neutralizing antibodies. Furthermore, domain sA fused to B+ (sAB+) induced the highest amount of neutralizing antibodies. Therefore, the construct sAB+ was used to generate a recombinant modified vaccinia virus Ankara (MVA), MVA-CHIKV-sAB+. Mice were vaccinated with MVA-CHIKV-sAB+ and/or the recombinant protein sAB+ and were subsequently challenged with wild-type CHIKV. Whereas four vaccinations with MVA-CHIKV-sAB+ were not sufficient to protect mice from a CHIKV infection, protein vaccination with sAB+ markedly reduced the viral titers of vaccinated mice. The recombinant protein sAB+ contains important structural antigens for a neutralizing antibody response in mice and its formulation with appropriate adjuvants might lead to a future CHIKV vaccine.

The type II cGMP dependent protein kinase regulates GluA1 levels at the plasma membrane of developing cerebellar granule cells

PubMed Central

Incontro, Salvatore; Ciruela, Francisco; Ziff, Edward; Hofmann, Franz; Sánchez-Prieto, José; Torres, Magdalena

2014-01-01

Trafficking of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (AMPARs) is regulated by specific interactions with other proteins and by post-translational mechanisms, such as phosphorylation. We have found that the type II cGMP-dependent protein kinase (cGKII) phosphorylates GluA1 (formerly GluR1) at S845, augmenting the surface expression of AMPARs at both synaptic and extrasynaptic sites. Activation of cGKII by 8-Br-cGMP enhances the surface expression of GluA1, whereas its inhibition or suppression effectively diminished the expression of this protein at the cell surface. In granule cells, NMDA receptor activation (NMDAR) stimulates nitric oxide and cGMP production, which in turn activates cGKII and induces the phosphorylation of GluA1, promoting its accumulation in the plasma membrane. GluA1 is mainly incorporated into calcium permeable AMPARs as exposure to 8-Br-cGMP or NMDA activation enhanced AMPA-elicited calcium responses that are sensitive to NASPM inhibition. We summarize evidence for an increase of calcium permeable AMPA receptors downstream of NMDA receptor activation that might be relevant for granule cell development and plasticity. PMID:23545413

DOE Office of Scientific and Technical Information (OSTI.GOV)

Purvina, Maija; Hoste, Astrid; Rossignol, Jean-Michel

Highlights: Black-Right-Pointing-Pointer P20, precursor of the HBeAg, interacts with the cellular protein gC1qR. Black-Right-Pointing-Pointer HBeAg and P20 bind to T cell surface and inhibit mitogen-induced T cell division. Black-Right-Pointing-Pointer HBeAg and P20 inhibition of T cell proliferation is gC1qR and IL-1RAcP-independent. -- Abstract: The hepatitis B virus (HBV) Precore protein is processed through the secretory pathway directly as HBeAg or with the generation of an intermediate (P20). Precore gene has been shown to be implicated in viral persistence, but the functions of HBeAg and its precursors have not been fully elucidated. We show that the secreted proteins HBeAg and P20more » interact with T cell surface and alter Kit-225 and primary T cells proliferation, a process which may facilitate the establishment of HBV persistence. Our data indicate that the N-terminal end of Precore is important for these inhibitory effects and exclude that they are dependent on the association of HBeAg and P20 with two characterized cell surface ligands, the Interleukin-1 Receptor Accessory Protein and gC1qR (present study).« less

Adsorption of fibrinogen on a biomedical-grade stainless steel 316LVM surface: a PM-IRRAS study of the adsorption thermodynamics, kinetics and secondary structure changes.

PubMed

Desroches, Marie-Josee; Omanovic, Sasha

2008-05-14

Polarization-modulation infrared reflection-absorption spectroscopy (PM-IRRAS) was employed to investigate the interaction of serum protein fibrinogen with a biomedical-grade 316LVM stainless steel surface, in terms of the adsorption thermodynamics, kinetics and secondary structure changes of the protein. Apparent Gibbs energy of adsorption values indicated a highly spontaneous and strong adsorption of fibrinogen onto the surface. The kinetics of fibrinogen adsorption were successfully modeled using a pseudo first-order kinetic model. Deconvolution of the amide I bands indicated that the adsorption of fibrinogen on 316LVM results in significant changes in the protein's secondary structure that occur predominantly within the first minute of adsorption. Among the investigated structures, the alpha-helix structure undergoes the smallest changes, while the beta-sheet and beta-turns structures undergo significant changes. It was shown that lateral interactions between the adsorbed molecules do not play a role in controlling the secondary structure changes. An increase in temperature induced changes in the secondary structure of the protein, characterized by a loss of the alpha-helical content and its transformation into the beta-turns structure.

27-Hydroxycholesterol upregulates the production of heat shock protein 60 of monocytic cells.

PubMed

Kim, Bo-Young; Son, Yonghae; Choi, Jeongyoon; Eo, Seong-Kug; Park, Young Chul; Kim, Koanhoi

2017-09-01

Investigating differentially expressed proteins in a milieu rich in cholesterol oxidation products, we found via mass spectrometry-based proteomics that surface levels of heat shock protein 60 (HSP60) were upregulated on monocytic cells in the presence of 27-hydroxycholesterol (27OHChol). The elevated levels of cytoplasmic membrane HSP60 were verified via Western blot analysis and visualized by confocal microscopy. Treatment with 27OHChol also resulted in increased levels of cellular HSP60 without altering its transcription. Cholesterol, however, did not affect cell-surface levels and cellular amount of HSP60. GSK 2033, an LXR antagonist, inhibited expression of live X receptor α, but not of HSP60, induced by 27OHChol. Treatment with 27OHChol also resulted in increased release of HSP60 from monocytic cells, but the release was significantly reduced by inhibitors of endoplasmic reticulum-Golgi protein trafficking, brefeldin A and monensin. Results of the current study indicate that 27OHChol upregulates not only cell-surface and cellular levels of HSP60 but also its release from monocytic cells, thereby contributing to activation of the immune system. Copyright © 2017 Elsevier Ltd. All rights reserved.

Selective protein adsorption modulates platelet adhesion and activation to oligo(ethylene glycol)-terminated self-assembled monolayers with C18 ligands.

PubMed

Gonçalves, Inês C; Martins, M Cristina L; Barbosa, Mário A; Naeemi, Esmaeel; Ratner, Buddy D

2009-06-01

This study focuses on the selective binding of albumin to a nanostructured surfaces to inhibit other blood proteins from adsorbing thereby reducing platelet adhesion and activation. Tetra (ethylene-glycol)-terminated self-assembled monolayers (EG4 SAMs) with different percentages of C18 ligands on the surface were characterized by contact angle measurements, X-ray photoelectron microscopy, infrared reflection-absorption spectroscopy, and ellipsometry. A specific surface (2.5% C18 SAM) was found to be selective for human serum albumin (HSA) in the presence of both albumin and fibrinogen (HFG). The importance of this concentration of C18 ligands was stressed in reversibility studies since that surface exchanged almost all the preadsorbed HSA by HSA in solution, but not by HFG. The effect of protein adsorption in the subsequent adhesion and activation of platelets was studied by pre-immersing the surfaces in albumin and plasma before contact with platelets. Scanning electron microscopy and glutaraldehyde induced fluorescence technique images showed that as surfaces got more hydrophobic due to the immobilization of C18 ligands, the number of adherent platelets increased and their morphology changed from round to fully spread. Pre-immersion in HSA led to an 80% decrease in platelet adhesion and reduction of activation. Pre-immersion in 1% plasma was only relevant in 2.5% C18 SAMs since this was the only surface that demonstrated less adhesion of platelets comparing with buffer pre-immersion. However, they still adsorb more platelets then when HSA was preadsorbed. This was confirmed in competition studies between HSA and plasma that suggested that other plasma proteins were also adsorbing to this surface. 2008 Wiley Periodicals, Inc.

Identification of a novel mechanism of action of bovine IgG antibodies specific for Staphylococcus aureus.

PubMed

Furukawa, Mutsumi; Yoneyama, Hiroshi; Hata, Eiji; Iwano, Hidetomo; Higuchi, Hidetoshi; Ando, Tasuke; Sato, Mika; Hayashi, Tomohito; Kiku, Yoshio; Nagasawa, Yuya; Niimi, Kanae; Usami, Katsuki; Ito, Kumiko; Watanabe, Kouichi; Nochi, Tomonori; Aso, Hisashi

2018-02-26

Staphylococcus aureus is a major pathogen that causes subclinical mastitis associated with huge economic losses to the dairy industry. A few vaccines for bovine mastitis are available, and they are expected to induce the production of S. aureus-specific antibodies that prevent bacterial adherence to host cells or promote opsonization by phagocytes. However, the efficacy of such vaccines are still under debate; therefore, further research focusing on improving the current vaccines by seeking additional mechanisms of action is required to reduce economic losses due to mastitis in the dairy industry. Here, we generated S. aureus-specific bovine IgG antibodies (anti-S. aureus) that directly inhibited bacterial growth in vitro. Inhibition depended on specificity for anti-S. aureus, not the interaction between Protein A and the fragment crystallizable region of the IgG antibodies or bacterial agglutination. An in vitro culture study using S. aureus strain JE2 and its deletion mutant JE2ΔSrtA, which lacks the gene encoding sortase A, revealed that the effect of anti-S. aureus was sortase-A-independent. Sortase A is involved in the synthesis of cell-wall-associated proteins. Thus, other surface molecules, such as membrane proteins, cell surface polysaccharides, or both, may trigger the inhibition of bacterial growth by anti-S. aureus. Together, our findings contribute insights into developing new strategies to further improve the available mastitis vaccine by designing a novel antigen on the surface of S. aureus to induce inhibitory signals that prevent bacterial growth.

TNFα induces co-trafficking of TRPV1/TRPA1 in VAMP1-containing vesicles to the plasmalemma via Munc18–1/syntaxin1/SNAP-25 mediated fusion

PubMed Central

Meng, Jianghui; Wang, Jiafu; Steinhoff, Martin; Dolly, James Oliver

2016-01-01

Transient receptor potential (TRP) A1 and V1 channels relay sensory signals, yet little is known about their transport to the plasmalemma during inflammation. Herein, TRPA1 and TRPV1 were found on vesicles containing calcitonin gene-related peptide (CGRP), accumulated at sites of exo- and endo-cytosis, and co-localised on fibres and cell bodies of cultured sensory neurons expressing both. A proinflammatory cytokine, TNFα, elevated their surface content, and both resided in close proximity, indicating co-trafficking. Syntaxin 1–interacting protein, Munc18–1, proved necessary for the response to TNFα, and for TRPV1-triggered CGRP release. TNFα-induced surface trafficking of TRPV1 and TRPA1 required a synaptic vesicle membrane protein VAMP1 (but not 2/3), which is essential for CGRP exocytosis from large dense-core vesicles. Inactivation of two proteins on the presynaptic plasma membrane, syntaxin-1 or SNAP-25, by botulinum neurotoxin (BoNT)/C1 or /A inhibited the TNFα-elevated delivery. Accordingly, enhancement by TNFα of Ca2+ influx through the upregulated surface-expressed TRPV1 and TRPA1 channels was abolished by BoNT/A. Thus, in addition, the neurotoxins’ known inhibition of the release of pain transmitters, their therapeutic potential is augmented by lowering the exocytotic delivery of transducing channels and the resultant hyper-sensitisation in inflammation. PMID:26888187

Antibody against Surface-Bound C5a Peptidase Is Opsonic and Initiates Macrophage Killing of Group B Streptococci

PubMed Central

Cheng, Qi; Carlson, Brian; Pillai, Sub; Eby, Ron; Edwards, Lorri; Olmsted, Stephen B.; Cleary, Patrick

2001-01-01

The capsular polysaccharides of group B streptococci (GBS) are a primary focus of vaccine development. Immunogenicity and long-lasting protection are best achieved by conjugating polysaccharides to a T-cell-dependent protein antigen. Streptococcal C5a peptidase (SCPB) is a conserved surface protein that is expressed by all streptococcal serotypes tested to date, and it is a possible carrier protein that could itself induce a protective immune response. Clearance of GBS from lungs, mucosal surfaces, or blood probably depends on the opsonophagocytic response of tissue-specific macrophages and polymorphonuclear leukocytes (PMNs). In this study, we examined the potential of antibody directed against SCPB from a serotype II strain to enhance the capacity of mouse bone marrow macrophages (from primary cultures) and human PMNs in whole blood to kill GBS in vitro. Our experiments demonstrated that Streptococcus serotypes Ia, Ib, II, III, and V, preopsonized with anti-SCPB antibody, were killed more rapidly by cultured macrophages and PMNs in whole blood than were nonopsonized GBS. The increased rate of killing was accompanied by an increased macrophage oxidative burst. Furthermore, opsonization was serotype transparent. Immunization with SCPB conjugated to capsular polysaccharide type III produced polysaccharide-specific antibodies. It is interesting that this antiserum promoted serotype-independent killing of streptococci. These data support the use of SCPB in a GBS polysaccharide conjugate vaccine. SCPB not only enhanced the immunogenicity of polysaccharide components of the vaccine, but it might also induce additional serotype-independent protective antibodies. PMID:11254587

Antibody against surface-bound C5a peptidase is opsonic and initiates macrophage killing of group B streptococci.

PubMed

Cheng, Q; Carlson, B; Pillai, S; Eby, R; Edwards, L; Olmsted, S B; Cleary, P

2001-04-01

The capsular polysaccharides of group B streptococci (GBS) are a primary focus of vaccine development. Immunogenicity and long-lasting protection are best achieved by conjugating polysaccharides to a T-cell-dependent protein antigen. Streptococcal C5a peptidase (SCPB) is a conserved surface protein that is expressed by all streptococcal serotypes tested to date, and it is a possible carrier protein that could itself induce a protective immune response. Clearance of GBS from lungs, mucosal surfaces, or blood probably depends on the opsonophagocytic response of tissue-specific macrophages and polymorphonuclear leukocytes (PMNs). In this study, we examined the potential of antibody directed against SCPB from a serotype II strain to enhance the capacity of mouse bone marrow macrophages (from primary cultures) and human PMNs in whole blood to kill GBS in vitro. Our experiments demonstrated that Streptococcus serotypes Ia, Ib, II, III, and V, preopsonized with anti-SCPB antibody, were killed more rapidly by cultured macrophages and PMNs in whole blood than were nonopsonized GBS. The increased rate of killing was accompanied by an increased macrophage oxidative burst. Furthermore, opsonization was serotype transparent. Immunization with SCPB conjugated to capsular polysaccharide type III produced polysaccharide-specific antibodies. It is interesting that this antiserum promoted serotype-independent killing of streptococci. These data support the use of SCPB in a GBS polysaccharide conjugate vaccine. SCPB not only enhanced the immunogenicity of polysaccharide components of the vaccine, but it might also induce additional serotype-independent protective antibodies.

High-pressure-induced water penetration into 3-­isopropylmalate dehydrogenase

PubMed Central

Nagae, Takayuki; Kawamura, Takashi; Chavas, Leonard M. G.; Niwa, Ken; Hasegawa, Masashi; Kato, Chiaki; Watanabe, Nobuhisa

2012-01-01

Hydrostatic pressure induces structural changes in proteins, including denaturation, the mechanism of which has been attributed to water penetration into the protein interior. In this study, structures of 3-isopropylmalate dehydrogenase (IPMDH) from Shewanella oneidensis MR-1 were determined at about 2 Å resolution under pressures ranging from 0.1 to 650 MPa using a diamond anvil cell (DAC). Although most of the protein cavities are monotonically compressed as the pressure increases, the volume of one particular cavity at the dimer interface increases at pressures over 340 MPa. In parallel with this volume increase, water penetration into the cavity could be observed at pressures over 410 MPa. In addition, the generation of a new cleft on the molecular surface accompanied by water penetration could also be observed at pressures over 580 MPa. These water-penetration phenomena are considered to be initial steps in the pressure-denaturation process of IPMDH. PMID:22349232

Protein structural development of threadfin bream ( Nemipterus spp.) surimi gels induced by glucose oxidase.

PubMed

Wang, Lei; Fan, Daming; Fu, Lulu; Jiao, Xidong; Huang, Jianlian; Zhao, Jianxin; Yan, Bowen; Zhou, Wenguo; Zhang, Wenhai; Ye, Weijian; Zhang, Hao

2018-01-01

This study investigated the effect of glucose oxidase on the gel properties of threadfin bream surimi. The gel strength of surimi increased with the addition of 0.5‰ glucose oxidase after two-step heating. Based on the results of the chemical interactions, the hydrophobic interaction and disulfide bond of glucose oxidase-treated surimi samples increased compared with the control samples at the gelation temperature and gel modori temperature. The surface hydrophobicity of samples with glucose oxidase and glucose increased significantly ( p < 0.05) and total sulfhydryl groups decreased significantly ( p < 0.05). The analysis of Raman spectroscopy shows that the addition of glucose oxidase induced more α-helixes to turn into a more elongated random and flocculent structure. Glucose oxidase changes the secondary structure of the surimi protein, making more proteins depolarize and stretch and causing actomyosin to accumulate to each other, resulting in the formation of surimi gel.

Fluid Mechanical Properties of Silkworm Fibroin Solutions

NASA Astrophysics Data System (ADS)

Matsumoto, Akira

2005-11-01

The aqueous solution behavior of silk fibroin is of interest due to the assembly and processing of this protein related to the spinning of protein fibers that exhibit remarkable mechanical properties. To gain insight into the origins of this functional feature, it is desired to determine how the protein behaves under a range of solution conditions. Pure fibroin at different concentrations in water was studied for surface tension, as a measure of surfactancy. In addition, shear induced changes on these solutions in terms of structure and morphology was also determined. Fibroin solutions exhibited shear rate-sensitive viscosity changes and precipitated at a critical shear rate where a dramatic increase of 75-150% of the initial value was observed along with a decrease in viscosity. In surface tension measurements, critical micelle concentrations were in the range of 3-4% w/v. The influence of additional factors, such as sericin protein, divalent and monovalent cations, and pH on the solution behavior in relation to structural and morphological features will also be described.

Why do gelatinized starch granules not dissolve completely? Roles for amylose, protein, and lipid in granule "ghost" integrity.

PubMed

Debet, Martine R; Gidley, Michael J

2007-06-13

After gelatinization in water, starch granules persist in swollen hydrated forms known as ghosts. Three potential mechanisms for ghost formation are tested. Proteins and lipids on the granule surface are found to be a determinant of ghost robustness, but not ghost formation. Proteins inside pre-made maize or wheat starch ghosts are degraded extensively by proteases without any apparent change in ghost properties, making an internal protein cross-linking mechanism unlikely. Waxy maize mutants with a range of amylose contents have ghost integrities that correlate with (low) apparent amylose levels. It is hypothesized that ghost formation is due to cross-linking of polysaccharide chains within swollen granules, most likely involving double helices formed from polymer chains that become free to move following heat-induced granule swelling. The size and robustness of granule ghosts is proposed to be determined by the relative rates of swelling and cross-linking, modulated by surface non-polysaccharide components.

Design of Stomach Acid-Stable and Mucin-Binding Enzyme Polymer Conjugates.

PubMed

Cummings, Chad S; Campbell, Alan S; Baker, Stefanie L; Carmali, Sheiliza; Murata, Hironobu; Russell, Alan J

2017-02-13

The reduced immunogenicity and increased stability of protein-polymer conjugates has made their use in therapeutic applications particularly attractive. However, the physicochemical interactions between polymer and protein, as well as the effect of this interaction on protein activity and stability, are still not fully understood. In this work, polymer-based protein engineering was used to examine the role of polymer physicochemical properties on the activity and stability of the chymotrypsin-polymer conjugates and their degree of binding to intestinal mucin. Four different chymotrypsin-polymer conjugates, each with the same polymer density, were synthesized using "grafting-from" atom transfer radical polymerization. The influence of polymer charge on chymotrypsin-polymer conjugate mucin binding, bioactivity, and stability in stomach acid was determined. Cationic polymers covalently attached to chymotrypsin showed high mucin binding, while zwitterionic, uncharged, and anionic polymers showed no mucin binding. Cationic polymers also increased chymotrypsin activity from pH 6-8, while zwitterionic polymers had no effect, and uncharged and anionic polymers decreased enzyme activity. Lastly, cationic polymers decreased the tendency of chymotrypsin to structurally unfold at extremely low pH, while uncharged and anionic polymers induced unfolding more quickly. We hypothesized that when polymers are covalently attached to the surface of a protein, the degree to which those polymers interact with the protein surface is the predominant determinant of whether the polymer will stabilize or inactivate the protein. Preferential interactions between the polymer and the protein lead to removal of water from the surface of the protein, and this, we believe, inactivates the enzyme.

Tissue Inhibitor of Metalloproteinase-3 (TIMP-3) induces FAS dependent apoptosis in human vascular smooth muscle cells.

PubMed

English, William R; Ireland-Zecchini, Heather; Baker, Andrew H; Littlewood, Trevor D; Bennett, Martin R; Murphy, Gillian

2018-01-01

Over expression of Tissue Inhibitor of Metalloproteinases-3 (TIMP-3) in vascular smooth muscle cells (VSMCs) induces apoptosis and reduces neointima formation occurring after saphenous vein interposition grafting or coronary stenting. In studies to address the mechanism of TIMP-3-driven apoptosis in human VSMCs we find that TIMP-3 increased activation of caspase-8 and apoptosis was inhibited by expression of Cytokine response modifier A (CrmA) and dominant negative FAS-Associated protein with Death Domain (FADD). TIMP-3 induced apoptosis did not cause mitochondrial depolarisation, increase activation of caspase-9 and was not inhibited by over-expression of B-cell Lymphoma 2 (Bcl2), indicating a mitochondrial independent/type-I death receptor pathway. TIMP-3 increased levels of the First Apoptosis Signal receptor (FAS) and depletion of FAS with shRNA showed TIMP-3-induced apoptosis was FAS dependent. TIMP-3 induced formation of the Death-Inducing Signalling Complex (DISC), as detected by immunoprecipitation and by immunofluorescence. Cellular-FADD-like IL-1 converting enzyme-Like Inhibitory Protein (c-FLIP) localised with FAS at the cell periphery in the absence of TIMP-3 and this localisation was lost on TIMP-3 expression with c-FLIP adopting a perinuclear localisation. Although TIMP-3 inhibited FAS shedding, this did not increase total surface levels of FAS but instead increased FAS levels within localised regions at the cell surface. A Disintegrin And Metalloproteinase 17 (ADAM17) is inhibited by TIMP-3 and depletion of ADAM17 with shRNA significantly decreased FAS shedding. However ADAM17 depletion did not induce apoptosis or replicate the effects of TIMP-3 by increasing localised clustering of cell surface FAS. ADAM17-depleted cells could activate caspase-3 when expressing levels of TIMP-3 that were otherwise sub-apoptotic, suggesting a partial role for ADAM17 mediated ectodomain shedding in TIMP-3 mediated apoptosis. We conclude that TIMP-3 induced apoptosis in VSMCs is highly dependent on FAS and is associated with changes in FAS and c-FLIP localisation, but is not solely dependent on shedding of the FAS ectodomain.

Tissue Inhibitor of Metalloproteinase–3 (TIMP-3) induces FAS dependent apoptosis in human vascular smooth muscle cells

PubMed Central

Ireland-Zecchini, Heather; Baker, Andrew H.; Littlewood, Trevor D.; Bennett, Martin R.; Murphy, Gillian

2018-01-01

Over expression of Tissue Inhibitor of Metalloproteinases-3 (TIMP-3) in vascular smooth muscle cells (VSMCs) induces apoptosis and reduces neointima formation occurring after saphenous vein interposition grafting or coronary stenting. In studies to address the mechanism of TIMP-3-driven apoptosis in human VSMCs we find that TIMP-3 increased activation of caspase-8 and apoptosis was inhibited by expression of Cytokine response modifier A (CrmA) and dominant negative FAS-Associated protein with Death Domain (FADD). TIMP-3 induced apoptosis did not cause mitochondrial depolarisation, increase activation of caspase-9 and was not inhibited by over-expression of B-cell Lymphoma 2 (Bcl2), indicating a mitochondrial independent/type-I death receptor pathway. TIMP-3 increased levels of the First Apoptosis Signal receptor (FAS) and depletion of FAS with shRNA showed TIMP-3-induced apoptosis was FAS dependent. TIMP-3 induced formation of the Death-Inducing Signalling Complex (DISC), as detected by immunoprecipitation and by immunofluorescence. Cellular-FADD-like IL-1 converting enzyme-Like Inhibitory Protein (c-FLIP) localised with FAS at the cell periphery in the absence of TIMP-3 and this localisation was lost on TIMP-3 expression with c-FLIP adopting a perinuclear localisation. Although TIMP-3 inhibited FAS shedding, this did not increase total surface levels of FAS but instead increased FAS levels within localised regions at the cell surface. A Disintegrin And Metalloproteinase 17 (ADAM17) is inhibited by TIMP-3 and depletion of ADAM17 with shRNA significantly decreased FAS shedding. However ADAM17 depletion did not induce apoptosis or replicate the effects of TIMP-3 by increasing localised clustering of cell surface FAS. ADAM17-depleted cells could activate caspase-3 when expressing levels of TIMP-3 that were otherwise sub-apoptotic, suggesting a partial role for ADAM17 mediated ectodomain shedding in TIMP-3 mediated apoptosis. We conclude that TIMP-3 induced apoptosis in VSMCs is highly dependent on FAS and is associated with changes in FAS and c-FLIP localisation, but is not solely dependent on shedding of the FAS ectodomain. PMID:29617412

Neu1 Sialidase and Matrix Metalloproteinase-9 Cross-talk Is Essential for Toll-like Receptor Activation and Cellular Signaling*

PubMed Central

Abdulkhalek, Samar; Amith, Schammim Ray; Franchuk, Susan L.; Jayanth, Preethi; Guo, Merry; Finlay, Trisha; Gilmour, Alanna; Guzzo, Christina; Gee, Katrina; Beyaert, Rudi; Szewczuk, Myron R.

2011-01-01

The signaling pathways of mammalian Toll-like receptors (TLRs) are well characterized, but the precise mechanism(s) by which TLRs are activated upon ligand binding remains poorly defined. Recently, we reported a novel membrane sialidase-controlling mechanism that depends on ligand binding to its TLR to induce mammalian neuraminidase-1 (Neu1) activity, to influence receptor desialylation, and subsequently to induce TLR receptor activation and the production of nitric oxide and proinflammatory cytokines in dendritic and macrophage cells. The α-2,3-sialyl residue of TLR was identified as the specific target for hydrolysis by Neu1. Here, we report a membrane signaling paradigm initiated by endotoxin lipopolysaccharide (LPS) binding to TLR4 to potentiate G protein-coupled receptor (GPCR) signaling via membrane Gαi subunit proteins and matrix metalloproteinase-9 (MMP9) activation to induce Neu1. Central to this process is that a Neu1-MMP9 complex is bound to TLR4 on the cell surface of naive macrophage cells. Specific inhibition of MMP9 and GPCR Gαi-signaling proteins blocks LPS-induced Neu1 activity and NFκB activation. Silencing MMP9 mRNA using lentivirus MMP9 shRNA transduction or siRNA transfection of macrophage cells and MMP9 knock-out primary macrophage cells significantly reduced Neu1 activity and NFκB activation associated with LPS-treated cells. These findings uncover a molecular organizational signaling platform of a novel Neu1 and MMP9 cross-talk in alliance with TLR4 on the cell surface that is essential for ligand activation of TLRs and subsequent cellular signaling. PMID:21873432

Electrostatics of lipid bilayer bending.

PubMed Central

Chou, T; Jarić, M V; Siggia, E D

1997-01-01

The electrostatic contribution to spontaneous membrane curvature is calculated within Poisson-Boltzmann theory under a variety of assumptions and emphasizing parameters in the physiological range. Asymmetrical surface charges can be fixed with respect to bilayer midplane area or with respect to the lipid-water area, but induce curvatures of opposite signs. Unequal screening layers on the two sides of a vesicle (e.g., multivalent cationic proteins on one side and monovalent salt on the other) also induce bending. For reasonable parameters, tubules formed by electrostatically induced bending can have radii in the 50-100-nm range, often seen in many intracellular organelles. Thus membrane associated proteins may induce curvature and subsequent budding, without themselves being intrinsically curved. Furthermore, we derive the previously unexplored effects of respecting the strict conservation of charge within the interior of a vesicle. The electrostatic component of the bending modulus is small under most of our conditions and is left as an experimental parameter. The large parameter space of conditions is surveyed in an array of graphs. Images FIGURE 1 FIGURE 10 PMID:9129807

Acid gelation properties of heated skim milk as a result of enzymatically induced changes in the micelle/serum distribution of the whey protein/kappa-casein aggregates.

PubMed

Guyomarc'h, Fanny; Renan, Marie; Chatriot, Marc; Gamerre, Valérie; Famelart, Marie-Hélène

2007-12-26

Changes in the acid gelation properties of skim milk as a result of variations in the micelle/serum distribution of the heat-induced whey protein/kappa-casein aggregates, induced by the combination of heat treatment and limited renneting, were investigated. No dramatic change in the zeta potential or the isoelectric point of the casein micelles was suggested, whether the aggregates were all attached to the casein micelle or not. Fluorescence intensity measurement using 8-anilino-1-naphthalenesulfonic acid (ANS) showed that the heat-induced aggregates were highly hydrophobic. Dynamic oscillation viscosimetry showed that acid gelation using glucono-delta-lactone (GDL) started at a higher pH value in prerenneted milk. However, no change in the gelation profile of skim milk could be related to the proportion of aggregates bound to the surface of the casein micelles. The results support the idea of an early interaction between the serum aggregates and the casein micelles on acidification.

Isthmin targets cell-surface GRP78 and triggers apoptosis via induction of mitochondrial dysfunction.

PubMed

Chen, M; Zhang, Y; Yu, V C; Chong, Y-S; Yoshioka, T; Ge, R

2014-05-01

Isthmin (ISM) is a secreted 60-kDa protein that potently induces endothelial cell (EC) apoptosis. It suppresses tumor growth and angiogenesis in mice when stably overexpressed in cancer cells. Although αvβ5 integrin serves as a low-affinity receptor for ISM, the mechanism by which ISM mediates antiangiogenesis and apoptosis in ECs remain to be fully resolved. In this work, we report the identification of cell-surface glucose-regulated protein 78 kDa (GRP78) as a high-affinity receptor for ISM (Kd=8.6 nM). We demonstrated that ISM-GRP78 interaction triggers apoptosis not only in activated ECs but also in cancer cells expressing high level of cell-surface GRP78. Normal cells and benign tumor cells tend to express low level of cell-surface GRP78 and are resistant to ISM-induced apoptosis. Upon binding to GRP78, ISM is internalized into ECs through clathrin-dependent endocytosis that is essential for its proapoptotic activity. Once inside the cell, ISM co-targets with GRP78 to mitochondria where it interacts with ADP/ATP carriers on the inner membrane and blocks ATP transport from mitochondria to cytosol, thereby causing apoptosis. Hence, ISM is a novel proapoptotic ligand that targets cell-surface GRP78 to trigger apoptosis by inducing mitochondrial dysfunction. The restricted and high-level expression of cell-surface GRP78 on cancer cells and cancer ECs make them uniquely susceptible to ISM-targeted apoptosis. Indeed, systemic delivery of recombinant ISM potently suppressed subcutaneous 4T1 breast carcinoma and B16 melanoma growth in mice by eliciting apoptosis selectively in the cancer cells and cancer ECs. Together, this work reveals a novel ISM-GRP78 apoptosis pathway and demonstrates the potential of ISM as a cancer-specific and dual-targeting anticancer agent.

Isthmin targets cell-surface GRP78 and triggers apoptosis via induction of mitochondrial dysfunction

PubMed Central

Chen, M; Zhang, Y; Yu, V C; Chong, Y-S; Yoshioka, T; Ge, R

2014-01-01

Isthmin (ISM) is a secreted 60-kDa protein that potently induces endothelial cell (EC) apoptosis. It suppresses tumor growth and angiogenesis in mice when stably overexpressed in cancer cells. Although αvβ5 integrin serves as a low-affinity receptor for ISM, the mechanism by which ISM mediates antiangiogenesis and apoptosis in ECs remain to be fully resolved. In this work, we report the identification of cell-surface glucose-regulated protein 78 kDa (GRP78) as a high-affinity receptor for ISM (Kd=8.6 nM). We demonstrated that ISM-GRP78 interaction triggers apoptosis not only in activated ECs but also in cancer cells expressing high level of cell-surface GRP78. Normal cells and benign tumor cells tend to express low level of cell-surface GRP78 and are resistant to ISM-induced apoptosis. Upon binding to GRP78, ISM is internalized into ECs through clathrin-dependent endocytosis that is essential for its proapoptotic activity. Once inside the cell, ISM co-targets with GRP78 to mitochondria where it interacts with ADP/ATP carriers on the inner membrane and blocks ATP transport from mitochondria to cytosol, thereby causing apoptosis. Hence, ISM is a novel proapoptotic ligand that targets cell-surface GRP78 to trigger apoptosis by inducing mitochondrial dysfunction. The restricted and high-level expression of cell-surface GRP78 on cancer cells and cancer ECs make them uniquely susceptible to ISM-targeted apoptosis. Indeed, systemic delivery of recombinant ISM potently suppressed subcutaneous 4T1 breast carcinoma and B16 melanoma growth in mice by eliciting apoptosis selectively in the cancer cells and cancer ECs. Together, this work reveals a novel ISM-GRP78 apoptosis pathway and demonstrates the potential of ISM as a cancer-specific and dual-targeting anticancer agent. PMID:24464222

Effects of copper ions on the characteristics of egg white gel induced by strong alkali.

PubMed

Shao, Yaoyao; Zhao, Yan; Xu, Mingsheng; Chen, Zhangyi; Wang, Shuzhen; Tu, Yonggang

2017-09-01

This study investigated the effects of copper ions on egg white (EW) gel induced by strong alkali. Changes in gel characteristics were examined through texture profile analysis, scanning electron microscopy (SEM), and chemical methods. The value of gel strength reached its maximum when 0.1% copper ions was added. However, the lowest cohesiveness values were observed at 0.1%. The springiness of gel without copper ions was significantly greater than the gel with copper ions added. SEM results illustrated that the low concentration of copper ions contributes to a dense and uniform gel network, and an open matrix was formed at 0.4%. The free and total sulphhydryl group content in the egg white protein gel significantly decreased with the increased copper. The increase of copper ions left the contents of ionic and hydrogen bonds basically unchanged, hydrophobic interaction presented an increasing trend, and the disulfide bond exhibited a completely opposite change. The change of surface hydrophobicity proved that the main binding force of copper induced gel was hydrophobic interaction. However, copper ions had no effect on the protein component of the gels. Generally, a low level of copper ions facilitates protein-protein association, which is involved in the characteristics of gels. Instead, high ionic strength had a negative effect on gels induced by strong alkali. © 2017 Poultry Science Association Inc.

Effect of pro-inflammatory mediators on membrane-associated mucins expressed by human ocular surface epithelial cells.

PubMed

Albertsmeyer, Ann-Christin; Kakkassery, Vinodh; Spurr-Michaud, Sandra; Beeks, Olivia; Gipson, Ilene K

2010-03-01

Membrane-associated mucins are altered on the ocular surface in non-Sjögren's dry eye. This study sought to determine if inflammatory mediators, present in tears of dry eye patients, regulate membrane-associated mucins MUC1 and -16 at the level of gene expression, protein biosynthesis and/or ectodomain release. A human corneal limbal epithelial cell line (HCLE), which produces membrane-associated mucins, was used. Cells were treated with interleukin (IL)-6, -8, or -17, tumor necrosis factor-alpha (TNF-alpha), and Interferon-gamma (IFN-gamma), or a combination of TNF-alpha and IFN-gamma, or IFN-gamma and IL-17, for 1, 6, 24, or 48 h. Presence of receptors for these mediators was verified by RT-PCR. Effects of the cytokines on expression levels of MUC1 and -16 were determined by real-time PCR, and on mucin protein biosynthesis and ectodomain release in cell lysates and culture media, respectively, by immunoblot analysis. TNF-alpha and IFN-gamma each significantly induced MUC1 expression, cellular protein content and ectodomain release over time. Combined treatment with the two cytokines was not additive. By comparison, one of the inflammatory mediators, IFN-gamma, affected all three parameters-gene expression, cellular protein, and ectodomain release-for MUC16. Combined treatment with TNF-alpha and IFN-gamma showed effects similar to IFN-gamma alone, except that ectodomain release followed that of TNF-alpha, which induced MUC16 ectodomain release. In conclusion, inflammatory mediators present in tears of dry eye patients can affect MUC1 and -16 on corneal epithelial cells and may be responsible for alterations of surface mucins in dry eye.

Roles of Fragile X Mental Retardation Protein in Dopaminergic Stimulation-induced Synapse-associated Protein Synthesis and Subsequent α-Amino-3-hydroxyl-5-methyl-4-isoxazole-4-propionate (AMPA) Receptor Internalization*

PubMed Central

Wang, Hansen; Kim, Susan S.; Zhuo, Min

2010-01-01

Fragile X syndrome, the most common form of inherited mental retardation, is caused by the absence of the RNA-binding protein fragile X mental retardation protein (FMRP). FMRP regulates local protein synthesis in dendritic spines. Dopamine (DA) is involved in the modulation of synaptic plasticity. Activation of DA receptors can regulate higher brain functions in a protein synthesis-dependent manner. Our recent study has shown that FMRP acts as a key messenger for DA modulation in forebrain neurons. Here, we demonstrate that FMRP is critical for DA D1 receptor-mediated synthesis of synapse-associated protein 90/PSD-95-associated protein 3 (SAPAP3) in the prefrontal cortex (PFC). DA D1 receptor stimulation induced dynamic changes of FMRP phosphorylation. The changes in FMRP phosphorylation temporally correspond with the expression of SAPAP3 after D1 receptor stimulation. Protein phosphatase 2A, ribosomal protein S6 kinase, and mammalian target of rapamycin are the key signaling molecules for FMRP linking DA D1 receptors to SAPAP3. Knockdown of SAPAP3 did not affect surface expression of α-amino-3-hydroxyl-5-methyl-4-isoxazole-4-propionate (AMPA) GluR1 receptors induced by D1 receptor activation but impaired their subsequent internalization in cultured PFC neurons; the subsequent internalization of GluR1 was also impaired in Fmr1 knock-out PFC neurons, suggesting that FMRP may be involved in subsequent internalization of GluR1 through regulating the abundance of SAPAP3 after DA D1 receptor stimulation. Our study thus provides further insights into FMRP involvement in DA modulation and may help to reveal the molecular mechanisms underlying impaired learning and memory in fragile X syndrome. PMID:20457613

Roles of fragile X mental retardation protein in dopaminergic stimulation-induced synapse-associated protein synthesis and subsequent alpha-amino-3-hydroxyl-5-methyl-4-isoxazole-4-propionate (AMPA) receptor internalization.

PubMed

Wang, Hansen; Kim, Susan S; Zhuo, Min

2010-07-09

Fragile X syndrome, the most common form of inherited mental retardation, is caused by the absence of the RNA-binding protein fragile X mental retardation protein (FMRP). FMRP regulates local protein synthesis in dendritic spines. Dopamine (DA) is involved in the modulation of synaptic plasticity. Activation of DA receptors can regulate higher brain functions in a protein synthesis-dependent manner. Our recent study has shown that FMRP acts as a key messenger for DA modulation in forebrain neurons. Here, we demonstrate that FMRP is critical for DA D1 receptor-mediated synthesis of synapse-associated protein 90/PSD-95-associated protein 3 (SAPAP3) in the prefrontal cortex (PFC). DA D1 receptor stimulation induced dynamic changes of FMRP phosphorylation. The changes in FMRP phosphorylation temporally correspond with the expression of SAPAP3 after D1 receptor stimulation. Protein phosphatase 2A, ribosomal protein S6 kinase, and mammalian target of rapamycin are the key signaling molecules for FMRP linking DA D1 receptors to SAPAP3. Knockdown of SAPAP3 did not affect surface expression of alpha-amino-3-hydroxyl-5-methyl-4-isoxazole-4-propionate (AMPA) GluR1 receptors induced by D1 receptor activation but impaired their subsequent internalization in cultured PFC neurons; the subsequent internalization of GluR1 was also impaired in Fmr1 knock-out PFC neurons, suggesting that FMRP may be involved in subsequent internalization of GluR1 through regulating the abundance of SAPAP3 after DA D1 receptor stimulation. Our study thus provides further insights into FMRP involvement in DA modulation and may help to reveal the molecular mechanisms underlying impaired learning and memory in fragile X syndrome.

Recombinant Protein Truncation Strategy for Inducing Bactericidal Antibodies to the Macrophage Infectivity Potentiator Protein of Neisseria meningitidis and Circumventing Potential Cross-Reactivity with Human FK506-Binding Proteins

PubMed Central

Bielecka, Magdalena K.; Devos, Nathalie; Gilbert, Mélanie; Hung, Miao-Chiu; Weynants, Vincent; Heckels, John E.

2014-01-01

A recombinant macrophage infectivity potentiator (rMIP) protein of Neisseria meningitidis induces significant serum bactericidal antibody production in mice and is a candidate meningococcal vaccine antigen. However, bioinformatics analysis of MIP showed some amino acid sequence similarity to human FK506-binding proteins (FKBPs) in residues 166 to 252 located in the globular domain of the protein. To circumvent the potential concern over generating antibodies that could recognize human proteins, we immunized mice with recombinant truncated type I rMIP proteins that lacked the globular domain and the signal leader peptide (LP) signal sequence (amino acids 1 to 22) and contained the His purification tag at either the N or C terminus (C-term). The immunogenicity of truncated rMIP proteins was compared to that of full (i.e., full-length) rMIP proteins (containing the globular domain) with either an N- or C-terminal His tag and with or without the LP sequence. By comparing the functional murine antibody responses to these various constructs, we determined that C-term His truncated rMIP (−LP) delivered in liposomes induced high levels of antibodies that bound to the surface of wild-type but not Δmip mutant meningococci and showed bactericidal activity against homologous type I MIP (median titers of 128 to 256) and heterologous type II and III (median titers of 256 to 512) strains, thereby providing at least 82% serogroup B strain coverage. In contrast, in constructs lacking the LP, placement of the His tag at the N terminus appeared to abrogate bactericidal activity. The strategy used in this study would obviate any potential concerns regarding the use of MIP antigens for inclusion in bacterial vaccines. PMID:25452551

Effect of Microcystin-LR on human placental villous trophoblast differentiation in vitro

EPA Science Inventory

Microcystin-LR is a cyanobacterial toxin found in surface and recreational waters that inhibits protein phosphatases and may disrupt the cytoskeleton. Microcystins induce apoptosis in hepatocytes at ≤2.0 μM. Nothing is known about the effects of microcystins on human placental tr...

Quantitative characterization of conformational-specific protein-DNA binding using a dual-spectral interferometric imaging biosensor.

PubMed

Zhang, Xirui; Daaboul, George G; Spuhler, Philipp S; Dröge, Peter; Ünlü, M Selim

2016-03-14

DNA-binding proteins play crucial roles in the maintenance and functions of the genome and yet, their specific binding mechanisms are not fully understood. Recently, it was discovered that DNA-binding proteins recognize specific binding sites to carry out their functions through an indirect readout mechanism by recognizing and capturing DNA conformational flexibility and deformation. High-throughput DNA microarray-based methods that provide large-scale protein-DNA binding information have shown effective and comprehensive analysis of protein-DNA binding affinities, but do not provide information of DNA conformational changes in specific protein-DNA complexes. Building on the high-throughput capability of DNA microarrays, we demonstrate a quantitative approach that simultaneously measures the amount of protein binding to DNA and nanometer-scale DNA conformational change induced by protein binding in a microarray format. Both measurements rely on spectral interferometry on a layered substrate using a single optical instrument in two distinct modalities. In the first modality, we quantitate the amount of binding of protein to surface-immobilized DNA in each DNA spot using a label-free spectral reflectivity technique that accurately measures the surface densities of protein and DNA accumulated on the substrate. In the second modality, for each DNA spot, we simultaneously measure DNA conformational change using a fluorescence vertical sectioning technique that determines average axial height of fluorophores tagged to specific nucleotides of the surface-immobilized DNA. The approach presented in this paper, when combined with current high-throughput DNA microarray-based technologies, has the potential to serve as a rapid and simple method for quantitative and large-scale characterization of conformational specific protein-DNA interactions.

Engineering the cell surface display of cohesins for assembly of cellulosome-inspired enzyme complexes on Lactococcus lactis

PubMed Central

2010-01-01

Background The assembly and spatial organization of enzymes in naturally occurring multi-protein complexes is of paramount importance for the efficient degradation of complex polymers and biosynthesis of valuable products. The degradation of cellulose into fermentable sugars by Clostridium thermocellum is achieved by means of a multi-protein "cellulosome" complex. Assembled via dockerin-cohesin interactions, the cellulosome is associated with the cell surface during cellulose hydrolysis, forming ternary cellulose-enzyme-microbe complexes for enhanced activity and synergy. The assembly of recombinant cell surface displayed cellulosome-inspired complexes in surrogate microbes is highly desirable. The model organism Lactococcus lactis is of particular interest as it has been metabolically engineered to produce a variety of commodity chemicals including lactic acid and bioactive compounds, and can efficiently secrete an array of recombinant proteins and enzymes of varying sizes. Results Fragments of the scaffoldin protein CipA were functionally displayed on the cell surface of Lactococcus lactis. Scaffolds were engineered to contain a single cohesin module, two cohesin modules, one cohesin and a cellulose-binding module, or only a cellulose-binding module. Cell toxicity from over-expression of the proteins was circumvented by use of the nisA inducible promoter, and incorporation of the C-terminal anchor motif of the streptococcal M6 protein resulted in the successful surface-display of the scaffolds. The facilitated detection of successfully secreted scaffolds was achieved by fusion with the export-specific reporter staphylococcal nuclease (NucA). Scaffolds retained their ability to associate in vivo with an engineered hybrid reporter enzyme, E. coli β-glucuronidase fused to the type 1 dockerin motif of the cellulosomal enzyme CelS. Surface-anchored complexes exhibited dual enzyme activities (nuclease and β-glucuronidase), and were displayed with efficiencies approaching 104 complexes/cell. Conclusions We report the successful display of cellulosome-inspired recombinant complexes on the surface of Lactococcus lactis. Significant differences in display efficiency among constructs were observed and attributed to their structural characteristics including protein conformation and solubility, scaffold size, and the inclusion and exclusion of non-cohesin modules. The surface-display of functional scaffold proteins described here represents a key step in the development of recombinant microorganisms capable of carrying out a variety of metabolic processes including the direct conversion of cellulosic substrates into fuels and chemicals. PMID:20840763

Light-induced protein nitration and degradation with HONO emission

NASA Astrophysics Data System (ADS)

Meusel, Hannah; Elshorbany, Yasin; Kuhn, Uwe; Bartels-Rausch, Thorsten; Reinmuth-Selzle, Kathrin; Kampf, Christopher J.; Li, Guo; Wang, Xiaoxiang; Lelieveld, Jos; Pöschl, Ulrich; Hoffmann, Thorsten; Su, Hang; Ammann, Markus; Cheng, Yafang

2017-10-01

Proteins can be nitrated by air pollutants (NO2), enhancing their allergenic potential. This work provides insight into protein nitration and subsequent decomposition in the presence of solar radiation. We also investigated light-induced formation of nitrous acid (HONO) from protein surfaces that were nitrated either online with instantaneous gas-phase exposure to NO2 or offline by an efficient nitration agent (tetranitromethane, TNM). Bovine serum albumin (BSA) and ovalbumin (OVA) were used as model substances for proteins. Nitration degrees of about 1 % were derived applying NO2 concentrations of 100 ppb under VIS/UV illuminated conditions, while simultaneous decomposition of (nitrated) proteins was also found during long-term (20 h) irradiation exposure. Measurements of gas exchange on TNM-nitrated proteins revealed that HONO can be formed and released even without contribution of instantaneous heterogeneous NO2 conversion. NO2 exposure was found to increase HONO emissions substantially. In particular, a strong dependence of HONO emissions on light intensity, relative humidity, NO2 concentrations and the applied coating thickness was found. The 20 h long-term studies revealed sustained HONO formation, even when concentrations of the intact (nitrated) proteins were too low to be detected after the gas exchange measurements. A reaction mechanism for the NO2 conversion based on the Langmuir-Hinshelwood kinetics is proposed.

Engineering a Biocompatible Scaffold with Either Micrometre or Nanometre Scale Surface Topography for Promoting Protein Adsorption and Cellular Response

PubMed Central

Le, Xuan; Poinern, Gérrard Eddy Jai; Ali, Nurshahidah; Berry, Cassandra M.; Fawcett, Derek

2013-01-01

Surface topographical features on biomaterials, both at the submicrometre and nanometre scales, are known to influence the physicochemical interactions between biological processes involving proteins and cells. The nanometre-structured surface features tend to resemble the extracellular matrix, the natural environment in which cells live, communicate, and work together. It is believed that by engineering a well-defined nanometre scale surface topography, it should be possible to induce appropriate surface signals that can be used to manipulate cell function in a similar manner to the extracellular matrix. Therefore, there is a need to investigate, understand, and ultimately have the ability to produce tailor-made nanometre scale surface topographies with suitable surface chemistry to promote favourable biological interactions similar to those of the extracellular matrix. Recent advances in nanoscience and nanotechnology have produced many new nanomaterials and numerous manufacturing techniques that have the potential to significantly improve several fields such as biological sensing, cell culture technology, surgical implants, and medical devices. For these fields to progress, there is a definite need to develop a detailed understanding of the interaction between biological systems and fabricated surface structures at both the micrometre and nanometre scales. PMID:23533416

Kinesin motor protein as an electrostatic ratchet machine

NASA Astrophysics Data System (ADS)

Tsironis, George; Ciudad, Aleix; Sancho, Jose Maria

2008-03-01

Kinesin and related motor proteins utilize ATP fuel to propel themselves along the external surface of microtubules in a processive and directional fashion. We show that the observed step-like motion is possible through time varying charge distributions furnished by the ATP hydrolysis circle while the static charge configuration on the microtuble provides the guide for motion. Thus, while the chemical hydrolysis energy induces appropriate local conformational changes, the motor translational energy is fundamentally electrostatic. Numerical simulations of the mechanical equations of motion show that processivity and directionality are direct consequences of the ATP-dependent electrostatic interaction between the different charge distributions of kinesin and microtubule. Treating proterins as continuous dielectric media and using a Green's function formalism we find analytical expressions for the electrostatic energy in the vicinity of the protein surfaces. We calculate the Bjerrum length in the interior of the protein and analyze its dependence on the charge proximity to the protein interface. We apply these results to kinesin and estimate the pure electrostatic ATP-ADP interaction to be larger than 2k T.

Differential Expression of Immunogenic Proteins on Virulent Mycobacterium tuberculosis Clinical Isolates

PubMed Central

Klepp, Laura; Vazquez, Camila; Rocha, Roxana Valeria; Blanco, Federico Carlos; López, Beatriz; Bigi, Fabiana; Sasiain, María del Carmen

2014-01-01

Molecular epidemiology has revealed that Mycobacterium tuberculosis (Mtb), formerly regarded as highly conserved species, displays a considerable degree of genetic variability that can influence the outcome of the disease as well as the innate and adaptive immune response. Recent studies have demonstrated that Mtb families found worldwide today differ in pathology, transmissibility, virulence, and development of immune response. By proteomic approaches seven proteins that were differentially expressed between a local clinical isolate from Latin-American-Mediterranean (LAM) and from Haarlem (H) lineages were identified. In order to analyze the immunogenic ability, recombinant Rv2241, Rv0009, Rv0407, and Rv2624c proteins were produced for testing specific antibody responses. We found that these proteins induced humoral immune responses in patients with drug-sensitive and drug-resistant tuberculosis with substantial cross-reactivity among the four proteins. Moreover, such reactivity was also correlated with anti-Mtb-cell surface IgM, but not with anti-ManLAM, anti-PPD, or anti-Mtb-surface IgG antibodies. Therefore, the present results describe new Mtb antigens with potential application as biomarkers of TB. PMID:25105140

Quantum dots induce charge-specific amyloid-like fibrillation of insulin at physiological conditions

NASA Astrophysics Data System (ADS)

Sukhanova, Alyona; Poly, Simon; Shemetov, Anton; Nabiev, Igor R.

2012-10-01

Agglomeration of some proteins may give rise to aggregates that have been identified as the main cause of amyloid diseases. For example, fibrillation of insulin is related to diabetes mellitus. Quantum dots (QDs) are of special interest as tagging agents for diagnostic and therapeutic studies due to their broad absorption spectra, narrow emission spectra, and high photostability. In this study, PEGylated CdSe/ZnS QDs have been shown to induce the formation of amyloid-like fibrils of human insulin under physiological conditions, this process being dependent on the variation of the surface charge of the nanoparticles (NPs) used. Circular dichroism (CD), protein secondary structure analysis, thioflavin T (ThT) fluorescence assay, and the dynamic light scattering (DLS) technique have been used for comparative analysis of different stages of the fibrillation process. In particular, insulin secondary structure remodelling accompanied by a considerable increase in the rate of amyloid fiber formation have been observed after insulin was mixed with PEGylated QDs. Nanoparticles may significantly influence the rate of protein fibrillation and induce new mechanisms of amyloid diseases, as well as offer opportunities for their treatment.

Aerosolization properties, surface composition and physical state of spray-dried protein powders.

PubMed

Bosquillon, Cynthia; Rouxhet, Paul G; Ahimou, François; Simon, Denis; Culot, Christine; Préat, Véronique; Vanbever, Rita

2004-10-19

Powder aerosols made of albumin, dipalmitoylphosphatidylcholine (DPPC) and a protein stabilizer (lactose, trehalose or mannitol) were prepared by spray-drying and analyzed for aerodynamic behavior, surface composition and physical state. The powders exited a Spinhaler inhaler as particle aggregates, the size of which depending on composition, spray-drying parameters and airflow rate. However, due to low bulk powder tap density (<0.15 g/cm3), the aerodynamic size of a large fraction of aggregates remained respirable (<5 microm). Fine particle fractions ranged between 21% and 41% in an Andersen cascade impactor operated at 28.3 l/min, with mannitol and lactose providing the most cohesive and free-flowing powders, respectively. Particle surface analysis by X-ray photoelectron spectroscopy (XPS) revealed a surface enrichment with DPPC relative to albumin for powders prepared under certain spray-drying conditions. DPPC self-organized in a gel phase in the particle and no sugar or mannitol crystals were detected by X-ray diffraction. Water sorption isotherms showed that albumin protected lactose from moisture-induced crystallization. In conclusion, a proper combination of composition and spray-drying parameters allowed to obtain dry powders with elevated fine particle fractions (FPFs) and a physical environment favorable to protein stability.

Engineered Lentivector Targeting of Dendritic Cells for In Vivo Immunization

PubMed Central

Yang, Lili; Yang, Haiguang; Rideout, Kendra; Cho, Taehoon; Joo, Kye il; Ziegler, Leslie; Elliot, Abigail; Walls, Anthony; Yu, Dongzi; Baltimore, David; Wang, Pin

2008-01-01

We report a method of inducing antigen production in dendritic cells (DCs) by in vivo targeting with lentiviral vectors that specifically bind to the DC surface protein, DC-SIGN. To target the DCs, the lentivector was enveloped with a viral glycoprotein from Sindbis virus, engineered to be DC-SIGN-specific. In vitro, this lentivector specifically transduced DCs and induced DC maturation. A remarkable frequency (up to 12%) of ovalbumin (OVA)-specific CD8+ T cells and a significant antibody response were observed 2 weeks following injection of a targeted lentiviral vector encoding an OVA transgene into naïve mice. These mice were solidly protected against the growth of the OVA-expressing E.G7 tumor and this methodology could even induce regression of an established tumor. Thus, lentiviral vectors targeting DCs provide a simple method of producing effective immunity and may provide an alternative route for immunization with protein antigens. PMID:18297056

New Insights from Sum Frequency Generation Vibrational Spectroscopy into the Interactions of Islet Amyloid Polypeptides with Lipid Membranes

PubMed Central

Wang, Zhuguang; Batista, Victor S.; Yan, Elsa C. Y.

2016-01-01

Studies of amyloid polypeptides on membrane surfaces have gained increasing attention in recent years. Several studies have revealed that membranes can catalyze protein aggregation and that the early products of amyloid aggregation can disrupt membrane integrity, increasing water permeability and inducing ion cytotoxicity. Nonetheless, probing aggregation of amyloid proteins on membrane surfaces is challenging. Surface-specific methods are required to discriminate contributions of aggregates at the membrane interface from those in the bulk phase and to characterize protein secondary structures in situ and in real time without the use of perturbing spectroscopic labels. Here, we review the most recent applications of sum frequency generation (SFG) vibrational spectroscopy applied in conjunction with computational modeling techniques, a joint experimental and computational methodology that has provided valuable insights into the aggregation of islet amyloid polypeptide (IAPP) on membrane surfaces. These applications show that SFG can provide detailed information about structures, kinetics, and orientation of IAPP during interfacial aggregation, relevant to the molecular mechanisms of type II diabetes. These recent advances demonstrate the promise of SFG as a new approach for studying amyloid diseases at the molecular level and for the rational drug design targeting early aggregation products on membrane surfaces. PMID:26697504

Salt- and pH-induced desorption: Comparison between non-aggregated and aggregated mussel adhesive protein, Mefp-1, and a synthetic cationic polyelectrolyte.

PubMed

Krivosheeva, Olga; Dedinaite, Andra; Claesson, Per M

2013-10-15

Mussel adhesive proteins are of great interest in many applications due to their ability to bind strongly to many types of surfaces under water. Effective use such proteins, for instance the Mytilus edulis foot protein - Mefp-1, for surface modification requires achievement of a large adsorbed amount and formation of a layer that is resistant towards desorption under changing conditions. In this work we compare the adsorbed amount and layer properties obtained by using a sample containing small Mefp-1 aggregates with that obtained by using a non-aggregated sample. We find that the use of the sample containing small aggregates leads to higher adsorbed amount, larger layer thickness and similar water content compared to what can be achieved with a non-aggregated sample. The layer formed by the aggregated Mefp-1 was, after removal of the protein from bulk solution, exposed to aqueous solutions with high ionic strength (up to 1M NaCl) and to solutions with low pH in order to reduce the electrostatic surface affinity. It was found that the preadsorbed Mefp-1 layer under all conditions explored was significantly more resistant towards desorption than a layer built by a synthetic cationic polyelectrolyte with similar charge density. These results suggest that the non-electrostatic surface affinity for Mefp-1 is larger than for the cationic polyelectrolyte. Copyright © 2013 The Authors. Published by Elsevier Inc. All rights reserved.

Aberrant expression of genes and proteins in pterygium and their implications in the pathogenesis

PubMed Central

Feng, Qing-Yang; Hu, Zi-Xuan; Song, Xi-Ling; Pan, Hong-Wei

2017-01-01

Pterygium is a common ocular surface disease induced by a variety of factors. The exact pathogenesis of pterygium remains unclear. Numbers of genes and proteins are discovered in pterygium and they function differently in the occurrence and development of this disease. We searched the Web of Science and PubMed throughout history for literatures about the subject. The keywords we used contain pterygium, gene, protein, angiogenesis, fibrosis, proliferation, inflammation, pathogenesis and therapy. In this review, we summarize the aberrant expression of a range of genes and proteins in pterygium compared with normal conjunctiva or cornea, including growth factors, matrix metalloproteinases and tissue inhibitors of metalloproteinases, interleukins, tumor suppressor genes, proliferation related proteins, apoptosis related proteins, cell adhesion molecules, extracellular matrix proteins, heat shock proteins and tight junction proteins. We illustrate their possible mechanisms in the pathogenesis of pterygium as well as the related intervention based on them for pterygium therapy. PMID:28730091

Insights into Jumonji C-domain containing protein 6 (JMJD6): a multifactorial role in foot-and-mouth disease virus replication in cells.

PubMed

Lawrence, Paul; Rieder, Elizabeth

2017-06-01

The Jumonji C-domain containing protein 6 (JMJD6) has had a convoluted history, and recent reports indicating a multifactorial role in foot-and-mouth disease virus (FMDV) infection have further complicated the functionality of this protein. It was first identified as the phosphatidylserine receptor on the cell surface responsible for recognizing phosphatidylserine on the surface of apoptotic cells resulting in their engulfment by phagocytic cells. Subsequent study revealed a nuclear subcellular localization, where JMJD6 participated in lysine hydroxylation and arginine demethylation of histone proteins and other non-histone proteins. Interestingly, to date, JMDJ6 remains the only known arginine demethylase with a growing list of known substrate molecules. These conflicting associations rendered the subcellular localization of JMJD6 to be quite nebulous. Further muddying this area, two different groups illustrated that JMJD6 could be induced to redistribute from the cell surface to the nucleus of a cell. More recently, JMJD6 was demonstrated to be a host factor contributing to the FMDV life cycle, where it was not only exploited for its arginine demethylase activity, but also served as an alternative virus receptor. This review attempts to coalesce these divergent roles for a single protein into one cohesive account. Given the diverse functionalities already characterized for JMJD6, it is likely to continue to be a confounding protein resulting in much contention going into the near future.

Mixed quantum-classical simulations of the vibrational relaxation of photolyzed carbon monoxide in a hemoprotein

NASA Astrophysics Data System (ADS)

Schubert, Alexander; Falvo, Cyril; Meier, Christoph

2016-08-01

We present mixed quantum-classical simulations on relaxation and dephasing of vibrationally excited carbon monoxide within a protein environment. The methodology is based on a vibrational surface hopping approach treating the vibrational states of CO quantum mechanically, while all remaining degrees of freedom are described by means of classical molecular dynamics. The CO vibrational states form the "surfaces" for the classical trajectories of protein and solvent atoms. In return, environmentally induced non-adiabatic couplings between these states cause transitions describing the vibrational relaxation from first principles. The molecular dynamics simulation yields a detailed atomistic picture of the energy relaxation pathways, taking the molecular structure and dynamics of the protein and its solvent fully into account. Using the ultrafast photolysis of CO in the hemoprotein FixL as an example, we study the relaxation of vibrationally excited CO and evaluate the role of each of the FixL residues forming the heme pocket.

Signaling induced by hop/STI-1 depends on endocytosis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Americo, Tatiana A.; Chiarini, Luciana B.; Linden, Rafael

The co-chaperone hop/STI-1 is a ligand of the cell surface prion protein (PrP{sup C}), and their interaction leads to signaling and biological effects. Among these, hop/STI-1 induces proliferation of A172 glioblastoma cells, dependent on both PrP{sup C} and activation of the Erk pathway. We tested whether clathrin-mediated endocytosis affects signaling induced by hop/STI-1. Both hyperosmolarity induced by sucrose and monodansyl-cadaverine blocked Erk activity induced by hop/STI-1, without affecting the high basal Akt activity typical of A172. The endocytosis inhibitors also affected the sub-cellular distribution of phosphorylated Erk, consistent with blockade of the latter's activity. The data indicate that signaling inducedmore » by hop/STI-1 depends on endocytosis. These findings are consistent with a role of sub-cellular trafficking in signal transduction following engagement by PrP{sup C} by ligands such as hop/STI-1, and may help help unravel both the functions of the prion protein, as well as possible loss-of-function components of prion diseases.« less

Antigen processing of glycoconjugate vaccines; the polysaccharide portion of the pneumococcal CRM(197) conjugate vaccine co-localizes with MHC II on the antigen processing cell surface.

PubMed

Lai, Zengzu; Schreiber, John R

2009-05-21

Pneumococcal (Pn) polysaccharides (PS) are T-independent (TI) antigens and do not induce immunological memory or antibodies in infants. Conjugation of PnPS to the carrier protein CRM(197) induces PS-specific antibody in infants, and memory similar to T-dependent (Td) antigens. Conjugates have improved immunogenicity via antigen processing and presentation of carrier protein with MHC II and recruitment of T cell help, but the fate of the PS attached to the carrier is unknown. To determine the location of the PS component of PnPS-CRM(197) in the APC, we separately labeled PS and protein and tracked their location. The PS of types 14-CRM(197) and 19F-CRM(197) was specifically labeled by Alexa Fluor 594 hydrazide (red). The CRM(197) was separately labeled red in a reaction that did not label PS. Labeled antigens were incubated with APC which were fixed, permeabilized and incubated with anti-MHC II antibody labeled green by Alexa Fluor 488, followed by confocal microscopy. Labeled CRM(197) was presented on APC surface and co-localized with MHC II (yellow). Labeled unconjugated 14 or 19F PS did not go to the APC surface, but PS labeled 14-CRM(197) and 19F-CRM(197) was internalized and co-localized with MHC II. Monoclonal antibody to type 14 PS bound to intracellular type 14 PS and PS-CRM(197). Brefeldin A and chloroquine blocked both CRM(197) and PS labeled 14-CRM(197) and 19F-CRM(197) from co-localizing with MHC II. These data suggest that the PS component of the CRM(197) glycoconjugate enters the endosome, travels with CRM(197) peptides to the APC surface and co-localizes with MHC II.

Selective albumin-binding surfaces modified with a thrombin-inhibiting peptide.

PubMed

Freitas, Sidónio C; Maia, Sílvia; Figueiredo, Ana C; Gomes, Paula; Pereira, Pedro J B; Barbosa, Mário A; Martins, M Cristina L

2014-03-01

Blood-contacting medical devices have been associated with severe clinical complications, such as thrombus formation, triggered by the activation of the coagulation cascade due to the adsorption of certain plasma proteins on the surface of biomaterials. Hence, the coating of such surfaces with antithrombotic agents has been used to increase biomaterial haemocompatibility. Biomaterial-induced clotting may also be decreased by albumin adsorption from blood plasma in a selective and reversible way, since this protein is not involved in the coagulation cascade. In this context, this paper reports that the immobilization of the thrombin inhibitor D-Phe-Pro-D-Arg-D-Thr-CONH2 (fPrt) onto nanostructured surfaces induces selective and reversible adsorption of albumin, delaying the clotting time when compared to peptide-free surfaces. fPrt, synthesized with two glycine residues attached to the N-terminus (GGfPrt), was covalently immobilized onto self-assembled monolayers (SAMs) having different ratios of carboxylate-hexa(ethylene glycol)- and tri(ethylene glycol)-terminated thiols (EG6-COOH/EG3) that were specifically designed to control GGfPrt orientation, exposure and density at the molecular level. In solution, GGfPrt was able to inactivate the enzymatic activity of thrombin and to delay plasma clotting time in a concentration-dependent way. After surface immobilization, and independently of its concentration, GGfPrt lost its selectivity to thrombin and its capacity to inhibit thrombin enzymatic activity against the chromogenic substrate n-p-tosyl-Gly-Pro-Arg-p-nitroanilide. Nevertheless, surfaces with low concentrations of GGfPrt could delay the capacity of adsorbed thrombin to cleave fibrinogen. In contrast, GGfPrt immobilized in high concentrations was found to induce the procoagulant activity of the adsorbed thrombin. However, all surfaces containing GGfPrt have a plasma clotting time similar to the negative control (empty polystyrene wells), showing resistance to coagulation, which is explained by its capacity to adsorb albumin in a selective and reversible way. This work opens new perspectives to the improvement of the haemocompatibility of blood-contacting medical devices. Copyright © 2013 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

Mucosal immunization with PspA (Pneumococcal surface protein A)-adsorbed nanoparticles targeting the lungs for protection against pneumococcal infection

PubMed Central

Rodrigues, Tasson C.; Oliveira, Maria Leonor S.; Soares-Schanoski, Alessandra; Chavez-Rico, Stefanni L.; Figueiredo, Douglas B.; Gonçalves, Viviane M.; Ferreira, Daniela M.; Kunda, Nitesh K.; Saleem, Imran Y.

2018-01-01

Burden of pneumonia caused by Streptococcus pneumoniae remains high despite the availability of conjugate vaccines. Mucosal immunization targeting the lungs is an attractive alternative for the induction of local immune responses to improve protection against pneumonia. Our group had previously described the development of poly(glycerol adipate-co-ω-pentadecalactone) (PGA-co-PDL) polymeric nanoparticles (NPs) adsorbed with Pneumococcal surface protein A from clade 4 (PspA4Pro) within L-leucine microcarriers (nanocomposite microparticles—NCMPs) for mucosal delivery targeting the lungs (NP/NCMP PspA4Pro). NP/NCMP PspA4Pro was now used for immunization of mice. Inoculation of this formulation induced anti-PspA4Pro IgG antibodies in serum and lungs. Analysis of binding of serum IgG to intact bacteria showed efficient binding to bacteria expressing PspA from clades 3, 4 and 5 (family 2), but no binding to bacteria expressing PspA from clades 1 and 2 (family 1) was observed. Both mucosal immunization with NP/NCMP PspA4Pro and subcutaneous injection of the protein elicited partial protection against intranasal lethal pneumococcal challenge with a serotype 3 strain expressing PspA from clade 5 (PspA5). Although similar survival levels were observed for mucosal immunization with NP/NCMP PspA4Pro and subcutaneous immunization with purified protein, NP/NCMP PspA4Pro induced earlier control of the infection. Conversely, neither immunization with NP/NCMP PspA4Pro nor subcutaneous immunization with purified protein reduced bacterial burden in the lungs after challenge with a serotype 19F strain expressing PspA from clade 1 (PspA1). Mucosal immunization with NP/NCMP PspA4Pro targeting the lungs is thus able to induce local and systemic antibodies, conferring protection only against a strain expressing PspA from the homologous family 2. PMID:29360883

Dengue virus induces increased activity of the complement alternative pathway in infected cells.

PubMed

Cabezas, Sheila; Bracho, Gustavo; L Aloia, Amanda; Adamson, Penelope J; Bonder, Claudine S; Smith, Justine R; Gordon, David L; Carr, Jillian M

2018-05-09

Severe dengue virus (DENV) infection is associated with overactivity of the complement alternative pathway (AP) in patient studies. Here, the molecular changes in components of the AP during DENV infection in vitro are investigated. mRNA for factor H (FH) a major negative regulator of the AP, is significantly increased in DENV-infected endothelial cells (EC) and macrophages but in contrast production of extracellular FH protein is not. This discord is not seen for the AP activator, factor B (FB), with DENV induction of both FB mRNA and protein, nor with Toll-like receptor 3 or 4 stimulation of EC and macrophages, which induces both FH and FB mRNA and protein. Surface bound and intracellular FH protein is however induced by DENV, but only in DENV antigen-positive cells, while in two other DENV-susceptible immortalised cell lines (ARPE-19 and HREC) FH protein is induced both intracellularly and extracellularly by DENV infection. Regardless of the cell type, there is an imbalance in AP components and an increase in markers of complement AP activity associated with DENV-infected cells - with lower FH relative to FB protein, increased ability to promote AP-mediated lytic activity and increased deposition of complement component C3b on the surface of DENV-infected cells. For EC in particular, these changes are predicted to result in higher complement activity in the local cellular microenvironment, with the potential to induce functional changes that may result in increased vascular permeability, a hallmark of dengue disease. IMPORTANCE Dengue virus (DENV) is a significant human viral pathogen with global medical and economic impact. DENV may cause serious and life-threatening disease with increased vascular permeability and plasma leakage. The pathogenic mechanisms underlying these features remain unclear; however overactivity of the complement alternative pathway has been suggested to play a role. In this study we investigate the molecular events that may be responsible for this observed alternative pathway overactivity and provide novel findings of changes in the complement system in response to DENV infection in primary cell types that are a major target for DENV infection (macrophages) and pathogenesis (endothelial cells) in vivo Our results suggest a new dimension of cellular events that may influence endothelial cell barrier function during DENV infection that could expand strategies for developing therapeutics to prevent or control DENV-mediated vascular disease. Copyright © 2018 American Society for Microbiology.

Dissociation, aggregation of sesame alpha-globulin in nonionic detergent solution.

PubMed

Lakshmi, T S; Nandi, P K

1978-10-01

Nonionic detergents Triton X-100 and Brij 36T induce dissociation and aggregation of the protein sesame alpha-globulin above the critical micelle concentrations (cmc) of the detergents. Spectrophotometric titration in Triton shows no change in the pKInt value of the tyrosyl groups at 1x10-3 M detergent where both dissociation and aggregation of the protein are observed. Fluorescence measurement does not indicate any change in the environment of the tryptophan groups of the protein in Brij. Viscosity measurements show no major conformational change of the protein in the detergent solution. Binding measurements suggest that perhaps micelles of the detergent predominantly bind to the protein. The detergent micelles preferentially bind to the exposed hydrophobic surfaces of the protein subunits. The association of the protein detergent complex through electrostatic interaction is probably responsible for the formation of the aggregates.

Regulation of CD93 cell surface expression by protein kinase C isoenzymes.

PubMed

Ikewaki, Nobunao; Kulski, Jerzy K; Inoko, Hidetoshi

2006-01-01

Human CD93, also known as complement protein 1, q subcomponent, receptor (C1qRp), is selectively expressed by cells with a myeloid lineage, endothelial cells, platelets, and microglia and was originally reported to be involved in the complement protein 1, q subcomponent (C1q)-mediated enhancement of phagocytosis. The intracellular molecular events responsible for the regulation of its expression on the cell surface, however, have not been determined. In this study, the effect of protein kinases in the regulation of CD93 expression on the cell surface of a human monocyte-like cell line (U937), a human NK-like cell line (KHYG-1), and a human umbilical vein endothelial cell line (HUV-EC-C) was investigated using four types of protein kinase inhibitors, the classical protein kinase C (cPKC) inhibitor Go6976, the novel PKC (nPKC) inhibitor Rottlerin, the protein kinase A (PKA) inhibitor H-89 and the protein tyrosine kinase (PTK) inhibitor herbimycin A at their optimum concentrations for 24 hr. CD93 expression was analyzed using flow cytometry and glutaraldehyde-fixed cellular enzyme-linked immunoassay (EIA) techniques utilizing a CD93 monoclonal antibody (mAb), mNI-11, that was originally established in our laboratory as a CD93 detection probe. The nPKC inhibitor Rottlerin strongly down-regulated CD93 expression on the U937 cells in a dose-dependent manner, whereas the other inhibitors had little or no effect. CD93 expression was down-regulated by Go6976, but not by Rottlerin, in the KHYG-1 cells and by both Rottlerin and Go6976 in the HUV-EC-C cells. The PKC stimulator, phorbol myristate acetate (PMA), strongly up-regulated CD93 expression on the cell surface of all three cell-lines and induced interleukin-8 (IL-8) production by the U937 cells and interferon-gamma (IFN-gamma) production by the KHYG-1 cells. In addition, both Go6976 and Rottlerin inhibited the up-regulation of CD93 expression induced by PMA and IL-8 or IFN-gamma production in the respective cell-lines. Whereas recombinant tumor necrosis factor-alpha (rTNF-alpha) slightly up-regulated CD93 expression on the U937 cells, recombinant interleukin-1beta (rIL-1beta), recombinant interleukin-2 (rIL-2), recombinant interferon-gamma (rIFN-gamma) and lipopolysaccharide (LPS) had no effect. Taken together, these findings indicate that the regulation of CD93 expression on these cells involves the PKC isoenzymes.

Release of Membrane-associated Mucins from Ocular Surface Epithelia

PubMed Central

Blalock, Timothy D.; Spurr-Michaud, Sandra J.; Tisdale, Ann S.; Gipson, Ilene K.

2008-01-01

Purpose Three membrane-associated mucins (MAMs)—MUC1, MUC4 and MUC16—are expressed at the ocular surface epithelium. Soluble forms of MAMs are detected in human tears, but the mechanisms of their release from the apical cells are unknown. The purpose of this study was to identify physiologic agents that induce ocular surface MAM release. Methods An immortalized human corneal-limbal epithelial cell line (HCLE) expressing the same MAMs as native tissue was used. An antibody specific to MUC16’s cytoplasmic tail was developed to confirm that only the extracellular domain is released into the tear fluid or culture media. Effects of agents that have been shown to be present in tears or are implicated in release/shedding of MAMs in other epithelia (neutrophil elastase, tumor necrosis factor (TNF), TNF-α-converting enzyme, and matrix metalloproteinases-7 and –9) were assessed on HCLE cells. HCLE cell surface proteins were biotinylated to measure efficiency of induced MAM release and surface restoration. Effects of induced release on surface barrier function were measured by rose bengal dye penetrance. Results MUC16 in tears and in HCLE-conditioned medium lacked the cytoplasmic tail. TNF induced release of MUC1, MUC4, and MUC16 from the HCLE surface. Matrix metalloproteinase-7 and neutrophil elastase induced release of MUC16 but not MUC1 or MUC4. Neutrophil elastase removed 68% of MUC16—78% of which was restored to the HCLE cell surface 24 hours after release. Neutrophil elastase-treated HCLE cells showed significantly reduced rose bengal dye exclusion. Conclusions Results suggest that extracellular domains of MUC1, 4, and 16 can be released from the ocular surface by agents present in tears. Neutrophil elastase and TNF present in higher amounts in dry eye patients’ tears may cause MAM release—allowing rose bengal staining. PMID:18436821

Basigin-2 Is a Cell Surface Receptor for Soluble Basigin Ligand*S⃞

PubMed Central

Belton, Robert J.; Chen, Li; Mesquita, Fernando S.; Nowak, Romana A.

2008-01-01

The metastatic spread of a tumor is dependent upon the ability of the tumor to stimulate surrounding stromal cells to express enzymes required for tissue remodeling. The immunoglobulin superfamily protein basigin (EMMPRIN/CD147) is a cell surface glycoprotein expressed by tumor cells that stimulates matrix metalloproteinase and vascular endothelial growth factor expression in stromal cells. The ability of basigin to stimulate expression of molecules involved in tissue remodeling and angiogenesis makes basigin a potential target for the development of strategies to block metastasis. However, the identity of the cell surface receptor for basigin remains controversial. The goal of this study was to determine the identity of the receptor for basigin. Using a novel recombinant basigin protein (rBSG) corresponding to the extracellular domain of basigin, it was demonstrated that the native, nonglycosylated rBSG protein forms dimers in solution. Furthermore, rBSG binds to the surface of uterine fibroblasts, activates the ERK1/2 signaling pathway, and induces expression of matrix metalloproteinases 1, 2, and 3. Proteins that interact with rBSG were isolated using a biotin label transfer technique and sequenced by matrix-assisted laser desorption ionization tandem mass spectrophotometry. The results demonstrate that rBSG interacts with basigin expressed on the surface of fibroblasts and is subsequently internalized. During internalization, rBSG associates with a novel form of human basigin (basigin-3). It was concluded that cell surface basigin functions as a membrane receptor for soluble basigin and this homophilic interaction is not dependent upon glycosylation of the basigin ligand. PMID:18434307

Analysis of hard protein corona composition on selective iron oxide nanoparticles by MALDI-TOF mass spectrometry: identification and amplification of a hidden mastitis biomarker in milk proteome.

PubMed

Magro, Massimiliano; Zaccarin, Mattia; Miotto, Giovanni; Da Dalt, Laura; Baratella, Davide; Fariselli, Piero; Gabai, Gianfranco; Vianello, Fabio

2018-05-01

Surface active maghemite nanoparticles (SAMNs) are able to recognize and bind selected proteins in complex biological systems, forming a hard protein corona. Upon a 5-min incubation in bovine whey from mastitis-affected cows, a significant enrichment of a single peptide characterized by a molecular weight at 4338 Da originated from the proteolysis of a S1 -casein was observed. Notably, among the large number of macromolecules in bovine milk, the detection of this specific peptide can hardly be accomplished by conventional analytical techniques. The selective formation of a stable binding between the peptide and SAMNs is due to the stability gained by adsorption-induced surface restructuration of the nanomaterial. We attributed the surface recognition properties of SAMNs to the chelation of iron(III) sites on their surface by sterically compatible carboxylic groups of the peptide. The specific peptide recognition by SAMNs allows its easy determination by MALDI-TOF mass spectrometry, and a threshold value of its normalized peak intensity was identified by a logistic regression approach and suggested for the rapid diagnosis of the pathology. Thus, the present report proposes the analysis of hard protein corona on nanomaterials as a perspective for developing fast analytical procedures for the diagnosis of mastitis in cows. Moreover, the huge simplification of proteome complexity by exploiting the selectivity derived by the peculiar SAMN surface topography, due to the iron(III) distribution pattern, could be of general interest, leading to competitive applications in food science and in biomedicine, allowing the rapid determination of hidden biomarkers by a cutting edge diagnostic strategy. Graphical abstract The topography of iron(III) sites on surface active maghemite nanoparticles (SAMNs) allows the recognition of sterically compatible carboxylic groups on proteins and peptides in complex biological matrixes. The analysis of hard protein corona on SAMNs led to the determination of a biomarker for cow mastitis in milk by MALDI-TOF mass spectrometry.

Synergistic foaming and surface properties of a weakly interacting mixture of soy glycinin and biosurfactant stevioside.

PubMed

Wan, Zhi-Li; Wang, Li-Ying; Wang, Jin-Mei; Yuan, Yang; Yang, Xiao-Quan

2014-07-16

The adsorption of the mixtures of soy glycinin (11S) with a biosurfactant stevioside (STE) at the air-water interface was studied to understand its relation with foaming properties. A combination of several techniques such as dynamic surface tension, dilatational rheology, fluorescence spectroscopy, and isothermal titration calorimetry (ITC) was used. In the presence of intermediate STE concentrations (0.25-0.5%), the weak binding of STE with 11S in bulk occurred by hydrophobic interactions, which could induce conformational changes of 11S, as evidenced by fluorescence and ITC. Accordingly, the strong synergy in reducing surface tension and the plateau in surface elasticity for mixed 11S-STE layers formed from the weakly interacting mixtures were clearly observed. This effect could be explained by the complexation with STE, which might facilitate the partial dissociation and further unfolding of 11S upon adsorption, thus enhancing the protein-protein and protein-STE interfacial interactions. These surface properties were positively reflected in foams produced by the weakly interacting system, which exhibited good foaming capacity and considerable stability probably due to better response to external stresses. However, at high STE concentrations (1-2%), as a consequence of the interface dominated by STE due to the preferential adsorption of STE molecules, the surface elasticity of layers dramatically decreased, and the resultant foams became less stable.

Single-Molecule Light-Sheet Imaging of Suspended T Cells.

PubMed

Ponjavic, Aleks; McColl, James; Carr, Alexander R; Santos, Ana Mafalda; Kulenkampff, Klara; Lippert, Anna; Davis, Simon J; Klenerman, David; Lee, Steven F

2018-05-08

Adaptive immune responses are initiated by triggering of the T cell receptor. Single-molecule imaging based on total internal reflection fluorescence microscopy at coverslip/basal cell interfaces is commonly used to study this process. These experiments have suggested, unexpectedly, that the diffusional behavior and organization of signaling proteins and receptors may be constrained before activation. However, it is unclear to what extent the molecular behavior and cell state is affected by the imaging conditions, i.e., by the presence of a supporting surface. In this study, we implemented single-molecule light-sheet microscopy, which enables single receptors to be directly visualized at any plane in a cell to study protein dynamics and organization in live, resting T cells. The light sheet enabled the acquisition of high-quality single-molecule fluorescence images that were comparable to those of total internal reflection fluorescence microscopy. By comparing the apical and basal surfaces of surface-contacting T cells using single-molecule light-sheet microscopy, we found that most coated-glass surfaces and supported lipid bilayers profoundly affected the diffusion of membrane proteins (T cell receptor and CD45) and that all the surfaces induced calcium influx to various degrees. Our results suggest that, when studying resting T cells, surfaces are best avoided, which we achieve here by suspending cells in agarose. Copyright © 2018. Published by Elsevier Inc.

Activation of classical protein kinase C reduces the expression of human cationic amino acid transporter 3 (hCAT-3) in the plasma membrane

PubMed Central

Rotmann, Alexander; Vékony, Nicole; Gassner, Davina; Niegisch, Günter; Strand, Dennis; Martiné, Ursula; Closs, Ellen I.

2005-01-01

We have previously shown that activation of PKC (protein kinase C) results in internalization of hCAT-1 [human CAT-1 (cationic amino acid transporter 1)] and a decrease in arginine transport [Rotmann, Strand, Martiné and Closs (2004) J. Biol. Chem. 279, 54185–54192]. However, others found increased transport rates for arginine in response to PKC activation, suggesting a differential effect of PKC on different CAT isoforms. Therefore we investigated the effect of PKC on hCAT-3, an isoform expressed in thymus, brain, ovary, uterus and mammary gland. In Xenopus laevis oocytes and human U373MG glioblastoma cells, hCAT-3-mediated L-arginine transport was significantly reduced upon treatment with compounds that activate classical PKC. In contrast, inactive phorbol esters and an activator of novel PKC isoforms had no effect. PKC inhibitors (including the PKCα-preferring Ro 31-8280) reduced the inhibitory effect of the PKC-activating compounds. Microscopic analyses revealed a PMA-induced reduction in the cell-surface expression of fusion proteins between hCAT-3 and enhanced green fluorescent protein expressed in X. laevis oocytes and glioblastoma cells. Western-blot analysis of biotinylated surface proteins demonstrated a PMA-induced decrease in hCAT-3 in the plasma membrane, but not in total protein lysates. Pretreatment with a PKC inhibitor also reduced this PMA effect. It is concluded that similar to hCAT-1, hCAT-3 activity is decreased by PKC via reduction of transporter molecules in the plasma membrane. Classical PKC isoforms seem to be responsible for this effect. PMID:16332251

Molecular classification of liver cirrhosis in a rat model by proteomics and bioinformatics.

PubMed

Xu, Xiu-Qin; Leow, Chon K; Lu, Xin; Zhang, Xuegong; Liu, Jun S; Wong, Wing-Hung; Asperger, Arndt; Deininger, Sören; Eastwood Leung, Hon-Chiu

2004-10-01

Liver cirrhosis is a worldwide health problem. Reliable, noninvasive methods for early detection of liver cirrhosis are not available. Using a three-step approach, we classified sera from rats with liver cirrhosis following different treatment insults. The approach consisted of: (i) protein profiling using surface-enhanced laser desorption/ionization (SELDI) technology; (ii) selection of a statistically significant serum biomarker set using machine learning algorithms; and (iii) identification of selected serum biomarkers by peptide sequencing. We generated serum protein profiles from three groups of rats: (i) normal (n=8), (ii) thioacetamide-induced liver cirrhosis (n=22), and (iii) bile duct ligation-induced liver fibrosis (n=5) using a weak cation exchanger surface. Profiling data were further analyzed by a recursive support vector machine algorithm to select a panel of statistically significant biomarkers for class prediction. Sensitivity and specificity of classification using the selected protein marker set were higher than 92%. A consistently down-regulated 3495 Da protein in cirrhosis samples was one of the selected significant biomarkers. This 3495 Da protein was purified on-chip and trypsin digested. Further structural characterization of this biomarkers candidate was done by using cross-platform matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) peptide mass fingerprinting (PMF) and matrix-assisted laser desorption/ionization time of flight/time of flight (MALDI-TOF/TOF) tandem mass spectrometry (MS/MS). Combined data from PMF and MS/MS spectra of two tryptic peptides suggested that this 3495 Da protein shared homology to a histidine-rich glycoprotein. These results demonstrated a novel approach to discovery of new biomarkers for early detection of liver cirrhosis and classification of liver diseases.

Insulin-induced translocation of IR to the nucleus in insulin responsive cells requires a nuclear translocation sequence.

PubMed

Kesten, Dov; Horovitz-Fried, Miriam; Brutman-Barazani, Tamar; Sampson, Sanford R

2018-04-01

Insulin binding to its cell surface receptor (IR) activates a cascade of events leading to its biological effects. The Insulin-IR complex is rapidly internalized and then is either recycled back to the plasma membrane or sent to lysosomes for degradation. Although most of the receptor is recycled or degraded, a small amount may escape this pathway and migrate to the nucleus of the cell where it might be important in promulgation of receptor signals. In this study we explored the mechanism by which insulin induces IR translocation to the cell nucleus. Experiments were performed cultured L6 myoblasts, AML liver cells and 3T3-L1 adipocytes. Insulin treatment induced a rapid increase in nuclear IR protein levels within 2 to 5 min. Treatment with WGA, an inhibitor of nuclear import, reduced insulin-induced increases nuclear IR protein; IR was, however, translocated to a perinuclear location. Bioinformatics tools predicted a potential nuclear localization sequence (NLS) on IR. Immunofluorescence staining showed that a point mutation on the predicted NLS blocked insulin-induced IR nuclear translocation. In addition, blockade of nuclear IR activation in isolated nuclei by an IR blocking antibody abrogated insulin-induced increases in IR tyrosine phosphorylation and nuclear PKCδ levels. Furthermore, over expression of mutated IR reduced insulin-induced glucose uptake and PKB phosphorylation. When added to isolated nuclei, insulin induced IR phosphorylation but had no effect on nuclear IR protein levels. These results raise questions regarding the possible role of nuclear IR in IR signaling and insulin resistance. Copyright © 2018 Elsevier B.V. All rights reserved.

Surface Induced Dissociation Coupled with High Resolution Mass Spectrometry Unveils Heterogeneity of a 211 kDa Multicopper Oxidase Protein Complex

NASA Astrophysics Data System (ADS)

Zhou, Mowei; Yan, Jing; Romano, Christine A.; Tebo, Bradley M.; Wysocki, Vicki H.; Paša-Tolić, Ljiljana

2018-01-01

Manganese oxidation is an important biogeochemical process that is largely regulated by bacteria through enzymatic reactions. However, the detailed mechanism is poorly understood due to challenges in isolating and characterizing these unknown enzymes. A manganese oxidase, Mnx, from Bacillus sp. PL-12 has been successfully overexpressed in active form as a protein complex with a molecular mass of 211 kDa. We have recently used surface induced dissociation (SID) and ion mobility-mass spectrometry (IM-MS) to release and detect folded subcomplexes for determining subunit connectivity and quaternary structure. The data from the native mass spectrometry experiments led to a plausible structural model of this multicopper oxidase, which has been difficult to study by conventional structural biology methods. It was also revealed that each Mnx subunit binds a variable number of copper ions. Becasue of the heterogeneity of the protein and limited mass resolution, ambiguities in assigning some of the observed peaks remained as a barrier to fully understanding the role of metals and potential unknown ligands in Mnx. In this study, we performed SID in a modified Fourier transform-ion cyclotron resonance (FTICR) mass spectrometer. The high mass accuracy and resolution offered by FTICR unveiled unexpected artificial modifications on the protein that had been previously thought to be iron bound species based on lower resolution spectra. Additionally, isotopically resolved spectra of the released subcomplexes revealed the metal binding stoichiometry at different structural levels. This method holds great potential for in-depth characterization of metalloproteins and protein-ligand complexes. [Figure not available: see fulltext.

Modification of solubility and heat-induced gelation of amaranth 11S globulin by protein engineering.

PubMed

Carrazco-Peña, Laura; Osuna-Castro, Juan A; De León-Rodríguez, Antonio; Maruyama, Nobuyuki; Toro-Vazquez, Jorge F; Morales-Rueda, Juan A; Barba de la Rosa, Ana P

2013-04-10

The primary structure of amaranth 11S globulin (Ah11S) was engineered with the aim to improve its functional properties. Four continuous methionines were inserted in variable region V, obtaining the Ah11Sr+4M construction. Changes on protein structure and surface characteristics were analyzed in silico. Solubility and heat-induced gelation of recombinant amaranth 11S proglobulin (Ah11Sr and Ah11Sr+4M) were compared with the native protein (Ah11Sn) purified from amaranth seed flour. The Ah11Sr+4 M showed the highest surface hydrophobicity, but as consequence the solubility was reduced. At low ionic strength (μ = 0.2) and acidic pH (<4.1), the recombinant proteins Ah11Sr and Ah11Sr+4 M had the highest and lowest solubility values, respectively. All globulins samples formed gels at 90 °C and low ionic strength, but Ah11Sn produced the weakest and Ah11Sr the strongest gels. Differential scanning calorimetry analysis under gel forming conditions revealed only exothermic transitions for all amaranth 11S globulins analyzed. In conclusion, the 3D structure analysis has revealed interesting molecular features that could explain the thermal resistance and gel forming ability of amaranth 11S globulins. The incorporation of four continuous methionines in amaranth increased the hydrophobicity, and self-supporting gels formed had intermediate hardness between Ah11Sn and Ah11Sr. These functional properties could be used in the food industry for the development of new products based on amaranth proteins.

Novel photonic technique creates micrometer resolution protein arrays and provides a new approach to coupling of genes, peptide hormones and drugs to nanoparticle carriers

NASA Astrophysics Data System (ADS)

Duroux, M.; Duroux, L.; Neves-Petersen, M. T.; Skovsen, E.; Petersen, S. B.

2007-07-01

We demonstrate that ultraviolet light can be used to make sterically oriented covalent immobilization of a large variety of protein molecules onto either thiolated quartz, gold or silicon. The reaction mechanism behind the reported new technology involves light-induced breakage of disulphide bridges in proteins upon UV illumination of nearby aromatic amino acids, resulting in the formation of free, reactive thiol groups that will form covalent bonds with thiol reactive surfaces. In general, the protein molecules retain their function. The size of the immobilization spot is limited to the focal point of illumination being as small as a few micrometers. This new technology allows for dense packing of different bio-molecules on a surface, allowing the creation of multi-potent functionalised new materials, such as nano-biosensors. We have developed the necessary technology for preparing large protein arrays of enzymes and fragments of monoclonal antibodies. Dedicated image processing software has been developed for making quality assessment of the protein arrays. This novel technology is ideal to couple drugs and other bio-molecules to nanoparticles which can be used as carriers into cells for therapeutic purposes.

Immunising with the transmembrane envelope proteins of different retroviruses including HIV-1

PubMed Central

Denner, Joachim

2013-01-01

The induction of neutralizing antibodies is a promising way to prevent retrovirus infections. Neutralizing antibodies are mainly directed against the envelope proteins, which consist of two molecules, the surface envelope (SU) protein and the transmembrane envelope (TM) protein. Antibodies broadly neutralizing the human immunodeficiencvy virus-1 (HIV-1) and binding to the TM protein gp41 of the virus have been isolated from infected individuals. Their epitopes are located in the membrane proximal external region (MPER). Since there are difficulties to induce such neutralizing antibodies as basis for an effective AIDS vaccine, we performed a comparative analysis immunising with the TM proteins of different viruses from the family Retroviridae. Both subfamilies, the Orthoretrovirinae and the Spumaretrovirinae were included. In this study, the TM proteins of three gammaretroviruses including (1) the porcine endogenous retrovirus (PERV), (2) the Koala retrovirus (KoRV), (3) the feline leukemia virus (FeLV), of two lentiviruses, HIV-1, HIV-2, and of two spumaviruses, the feline foamy virus (FFV) and the primate foamy virus (PFV) were used for immunisation. Whereas in all immunisation studies binding antibodies were induced, neutralizing antibodies were only found in the case of the gammaretroviruses. The induced antibodies were directed against the MPER and the fusion peptide proximal region (FPPR) of their TM proteins; however only the antibodies against the MPER were neutralizing. Most importantly, the epitopes in the MPER were localized in the same position as the epitopes of the antibodies broadly neutralizing HIV-1 in the TM protein gp41 of HIV-1, indicating that the MPER is an effective target for the neutralization of retroviruses. PMID:23249763

The stress response of bacterium Cupriavidus metallidurans CH34 into simulated microgravity

NASA Astrophysics Data System (ADS)

van Houdt, Rob; de Boever, Patrick; Coninx, Ilse; Janssen, Ann; Benotmane, Rafi; Leys, Natalie; Mergeay, Max

The stress response of bacterium Cupriavidus metallidurans CH34 into simulated microgravity R. Van Houdt, P. De Boever, I. Coninx, A. Janssen, M.A. Benotmane, N. Leys, and M. Mergeay Expertise group for Molecular and Cellular Biology, Institute for Environment, Health and Safety, Belgian Nuclear Research Centre (SCK•CEN), Boeretang 200, B-2400 Mol, Belgium. We have studied the response of Cupriavidus (formerly Ralstonia) metallidurans CH34 to simulated microgravity by culturing in a Rotating Wall Vessel (RWV) bioreactor. This bioreactor technology generates a unique Low-Shear Modeled Microgravity (LSMMG) environment and is exploited as analogue for in vivo medical and space environments. Cupriavidus and Ralstonia species are relevant model bacteria since they are often isolated from the floor, air and surfaces of spacecraft assembly rooms and not only contaminate the clean rooms but have also been found prior-to-flight on surfaces of space robots such as the Mars Odyssey Orbiter and even in-flight in ISS cooling water and Shuttle drinking water. In addition, C. metallidurans CH34 is also being used in fundamental space flight experiments aimed to gain a better insight in the bacterial adaptation to space. The first objective was to elucidate the stress response of C. metallidurans CH34 grown in LSMMG compared to a normal gravity control. Transcriptomic analysis revealed that a significant part of the heat shock response was induced in LSMMG. Transcription of d naK, encoding the major heat-shock protein and a prokaryotic homologue of the eukaryotic Hsp70 protein, was induced 6.4 fold in LSMMG. DnaK is assisted by partner chaperones DnaJ and GrpE for which transcription respectively were induced 2.0 and 2.6 fold. Transcription of other chaperones known to belong to the heat shock response was also induced in LSMMG: hslV and hsl U, encoding the HslVU protease, were induced respectively 5.5 and 3.4 fold; htpG, encoding a Hsp90 family chaperone, was induced 4.6 fold and clpB was induced 4.7 fold. Transcription of the Lon protease was induced 2.5 fold. It appears that C. metallidurans CH34 experiences growth in Low-Shear Modelled Microgravity as a stressful condition eliciting the need to express the heat-shock proteins which assist protein folding, assembly, transport, repair and degradation. Challenging cells grown in simulated gravity (LSMMG) to a heat-shock for 30 min at 50° C resulted indeed in a smaller reduction (1.7 log) in cultivable cells compared to the reduction observed for cells grown in normal earth gravity (Low-Shear Gravity LSG) (4.0 log). Next to genes involved in the heat shock response, 5 of the 11 copies of uspA, encoding a widely conserved protein belonging to a superfamily whose physiological function is unknown but which is induced in response to a variety of stresses, were induced from 2.7 to 8.7 fold. In addition, LSMMG resulted in the upregulation of various genes encoding site-specific tyrosine recombinases, site-specific serine recombinase and transposases possibly indicating that Low-Shear Modeled Microgravity could elicit an adaptive response by genetic rearrangements. Finally, the parA and parB genes from pMOL30, one of the two plasmids carried by CH34 and specialized in heavy metals resistance, were strongly induced in LSMMG respectively 19.6 and 7.0 fold. The overproduction of similar proteins was also detected in C. metallidurans cells, cultured in during space flight.

High-Throughput Genetic Identification of Functionally Important Regions of the Yeast DEAD-Box Protein Mss116p

DOE Office of Scientific and Technical Information (OSTI.GOV)

Mohr, Georg; Del Campo, Mark; Turner, Kathryn G.

The Saccharomyces cerevisiae DEAD-box protein Mss116p is a general RNA chaperone that functions in splicing mitochondrial group I and group II introns. Recent X-ray crystal structures of Mss116p in complex with ATP analogs and single-stranded RNA show that the helicase core induces a bend in the bound RNA, as in other DEAD-box proteins, while a C-terminal extension (CTE) induces a second bend, resulting in RNA crimping. Here, we illuminate these structures by using high-throughput genetic selections, unigenic evolution, and analyses of in vivo splicing activity to comprehensively identify functionally important regions and permissible amino acid substitutions throughout Mss116p. The functionallymore » important regions include those containing conserved sequence motifs involved in ATP and RNA binding or interdomain interactions, as well as previously unidentified regions, including surface loops that may function in protein-protein interactions. The genetic selections recapitulate major features of the conserved helicase motifs seen in other DEAD-box proteins but also show surprising variations, including multiple novel variants of motif III (SAT). Patterns of amino acid substitutions indicate that the RNA bend induced by the helicase core depends on ionic and hydrogen-bonding interactions with the bound RNA; identify a subset of critically interacting residues; and indicate that the bend induced by the CTE results primarily from a steric block. Finally, we identified two conserved regions - one the previously noted post II region in the helicase core and the other in the CTE - that may help displace or sequester the opposite RNA strand during RNA unwinding.« less

Severe Burn-Induced Intestinal Epithelial Barrier Dysfunction Is Associated With Endoplasmic Reticulum Stress and Autophagy in Mice

PubMed Central

Huang, Yalan; Feng, Yanhai; Wang, Yu; Wang, Pei; Wang, Fengjun; Ren, Hui

2018-01-01

The disruption of intestinal barrier plays a vital role in the pathophysiological changes after severe burn injury, however, the underlying mechanisms are poorly understood. Severe burn causes the disruption of intestinal tight junction (TJ) barrier. Previous studies have shown that endoplasmic reticulum (ER) stress and autophagy are closely associated with the impairment of intestinal mucosa. Thus, we hypothesize that ER stress and autophagy are likely involved in burn injury-induced intestinal epithelial barrier dysfunction. Mice received a 30% total body surface area (TBSA) full-thickness burn, and were sacrificed at 0, 1, 2, 6, 12 and 24 h postburn. The results showed that intestinal permeability was increased significantly after burn injury, accompanied by the damage of mucosa and the alteration of TJ proteins. Severe burn induced ER stress, as indicated by increased intraluminal chaperone binding protein (BIP), CCAAT/enhancer-binding protein homologous protein (CHOP) and inositol-requiring enzyme 1(IRE1)/X-box binding protein 1 splicing (XBP1). Autophagy was activated after burn injury, as evidenced by the increase of autophagy related protein 5 (ATG5), Beclin 1 and LC3II/LC3I ratio and the decrease of p62. Besides, the number of autophagosomes was also increased after burn injury. The levels of p-PI3K(Ser191), p-PI3K(Ser262), p-AKT(Ser473), and p-mTOR were decreased postburn, suggesting that autophagy-related PI3K/AKT/mTOR pathway is involved in the intestinal epithelial barrier dysfunction following severe burn. In summary, severe burn injury induces the ER stress and autophagy in intestinal epithelia, leading to the disruption of intestinal barrier. PMID:29740349

The role of mAKAPβ in the process of cardiomyocyte hypertrophy induced by angiotensin II

PubMed Central

GUO, HUIXIN; LIU, BAOXIN; HOU, LEI; THE, ERLINDA; LI, GANG; WANG, DONGZHI; JIE, QIQIANG; CHE, WENLIANG; WEI, YIDONG

2015-01-01

Angiotensin II (AngII) is the central product of the renin-angiotensin system (RAS) and this octapeptide contributes to the pathophysiology of cardiac hypertrophy and remodeling. mAKAPβ is an A-kinase anchoring protein (AKAP) that has the function of binding to the regulatory subunit of protein kinase A (PKA) and confining the holoenzyme to discrete locations within the cell. In this study, we aimed to investigate the role of mAKAPβ in AngII-induced cardiomyocyte hypertrophy and the possible mechanisms involved. Cultured cardiomyocytes from neonatal rats were treated with AngII. Subsequently, the morphology of the cardiomyocytes was observed and the expression of mAKAPβ and cardiomyocyte hypertrophic markers was measured. mAKAPβ-shRNA was constructed for RNA interference; the expression of mAKAPβ and hypertrophic markers, the cell surface area and the [3H]Leucine incorporation rate in the AngII-treated rat cardiomyocytes were detected following RNA interference. Simultaneously, changes in the expression levels of phosphorylated extracellular signal-regulated kinase (p-ERK)2 in the cardiomyocytes were assessed. The cell size of the AngII-treated cardiaomyocytes was significantly larger than that of the untreated cardiomyocytes. The expression of hypertrophic markers and p-ERK2, the cell surface area and the [3H]Leucine incorporation rate were all significantly increased in the AngII-treated cells. However, the expression of mAKAPβ remained unaltered in this process. RNA interference simultaneously inhibited the protein expression of mAKAPβ and p-ERK2, and the hypertrophy of the cardiomyocytes induced by AngII was attenuated. These results demonstrate that AngII induces hypertrophy in cardiomyocytes, and mAKAPβ is possibly involved in this process. The effects of mAKAPβ on AngII-induced cardiomyocyte hypertrophy may be associated with p-ERK2 expression. PMID:25739102

Lipid rafts are required for signal transduction by angiotensin II receptor type 1 in neonatal glomerular mesangial cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Adebiyi, Adebowale, E-mail: aadebiyi@uthsc.edu; Soni, Hitesh; John, Theresa A.

Angiotensin II (ANG-II) receptors (AGTRs) contribute to renal physiology and pathophysiology, but the underlying mechanisms that regulate AGTR function in glomerular mesangium are poorly understood. Here, we show that AGTR1 is the functional AGTR subtype expressed in neonatal pig glomerular mesangial cells (GMCs). Cyclodextrin (CDX)-mediated cholesterol depletion attenuated cell surface AGTR1 protein expression and ANG-II-induced intracellular Ca{sup 2+} ([Ca{sup 2+}]{sub i}) elevation in the cells. The COOH-terminus of porcine AGTR1 contains a caveolin (CAV)-binding motif. However, neonatal GMCs express CAV-1, but not CAV-2 and CAV-3. Colocalization and in situ proximity ligation assay detected an association between endogenous AGTR1 and CAV-1more » in the cells. A synthetic peptide corresponding to the CAV-1 scaffolding domain (CSD) sequence also reduced ANG-II-induced [Ca{sup 2+}]{sub i} elevation in the cells. Real-time imaging of cell growth revealed that ANG-II stimulates neonatal GMC proliferation. ANG-II-induced GMC growth was attenuated by EMD 66684, an AGTR1 antagonist; BAPTA, a [Ca{sup 2+}]{sub i} chelator; KN-93, a Ca{sup 2+}/calmodulin-dependent protein kinase II inhibitor; CDX; and a CSD peptide, but not PD 123319, a selective AGTR2 antagonist. Collectively, our data demonstrate [Ca{sup 2+}]{sub i}-dependent proliferative effect of ANG-II and highlight a critical role for lipid raft microdomains in AGTR1-mediated signal transduction in neonatal GMCs. - Highlights: • AGTR1 is the functional AGTR subtype expressed in neonatal mesangial cells. • Endogenous AGTR1 associates with CAV-1 in neonatal mesangial cells. • Lipid raft disruption attenuates cell surface AGTR1 protein expression. • Lipid raft disruption reduces ANG-II-induced [Ca{sup 2+}]{sub i} elevation in neonatal mesangial cells. • Lipid raft disruption inhibits ANG-II-induced neonatal mesangial cell growth.« less

Evaluation of Trichodysplasia Spinulosa-Associated Polyomavirus Capsid Protein as a New Carrier for Construction of Chimeric Virus-Like Particles Harboring Foreign Epitopes

PubMed Central

Gedvilaite, Alma; Kucinskaite-Kodze, Indre; Lasickiene, Rita; Timinskas, Albertas; Vaitiekaite, Ausra; Ziogiene, Danguole; Zvirbliene, Aurelija

2015-01-01

Recombinant virus-like particles (VLPs) represent a promising tool for protein engineering. Recently, trichodysplasia spinulosa-associated polyomavirus (TSPyV) viral protein 1 (VP1) was efficiently produced in yeast expression system and shown to self-assemble to VLPs. In the current study, TSPyV VP1 protein was exploited as a carrier for construction of chimeric VLPs harboring selected B and T cell-specific epitopes and evaluated in comparison to hamster polyomavirus VP1 protein. Chimeric VLPs with inserted either hepatitis B virus preS1 epitope DPAFR or a universal T cell-specific epitope AKFVAAWTLKAAA were produced in yeast Saccharomyces cerevisiae. Target epitopes were incorporated either at the HI or BC loop of the VP1 protein. The insertion sites were selected based on molecular models of TSPyV VP1 protein. The surface exposure of the insert positions was confirmed using a collection of monoclonal antibodies raised against the intact TSPyV VP1 protein. All generated chimeric proteins were capable to self-assemble to VLPs, which induced a strong immune response in mice. The chimeric VLPs also activated dendritic cells and T cells as demonstrated by analysis of cell surface markers and cytokine production profiles in spleen cell cultures. In conclusion, TSPyV VP1 protein represents a new potential carrier for construction of chimeric VLPs harboring target epitopes. PMID:26230706

Structural centrosome aberrations sensitize polarized epithelia to basal cell extrusion.

PubMed

Ganier, Olivier; Schnerch, Dominik; Nigg, Erich A

2018-06-01

Centrosome aberrations disrupt tissue architecture and may confer invasive properties to cancer cells. Here we show that structural centrosome aberrations, induced by overexpression of either Ninein-like protein (NLP) or CEP131/AZI1, sensitize polarized mammalian epithelia to basal cell extrusion. While unperturbed epithelia typically dispose of damaged cells through apical dissemination into luminal cavities, certain oncogenic mutations cause a switch in directionality towards basal cell extrusion, raising the potential for metastatic cell dissemination. Here we report that NLP-induced centrosome aberrations trigger the preferential extrusion of damaged cells towards the basal surface of epithelial monolayers. This switch in directionality from apical to basal dissemination coincides with a profound reorganization of the microtubule cytoskeleton, which in turn prevents the contractile ring repositioning that is required to support extrusion towards the apical surface. While the basal extrusion of cells harbouring NLP-induced centrosome aberrations requires exogenously induced cell damage, structural centrosome aberrations induced by excess CEP131 trigger the spontaneous dissemination of dying cells towards the basal surface from MDCK cysts. Thus, similar to oncogenic mutations, structural centrosome aberrations can favour basal extrusion of damaged cells from polarized epithelia. Assuming that additional mutations may promote cell survival, this process could sensitize epithelia to disseminate potentially metastatic cells. © 2018 The Authors.

Crystal Nucleation Using Surface-Energy-Modified Glass Substrates.

PubMed

Nordquist, Kyle A; Schaab, Kevin M; Sha, Jierui; Bond, Andrew H

2017-08-02

Systematic surface energy modifications to glass substrates can induce nucleation and improve crystallization outcomes for small molecule active pharmaceutical ingredients (APIs) and proteins. A comparatively broad probe for function is presented in which various APIs, proteins, organic solvents, aqueous media, surface energy motifs, crystallization methods, form factors, and flat and convex surface energy modifications were examined. Replicate studies ( n ≥ 6) have demonstrated an average reduction in crystallization onset times of 52(4)% (alternatively 52 ± 4%) for acetylsalicylic acid from 91% isopropyl alcohol using two very different techniques: bulk cooling to 0 °C using flat surface energy modifications or microdomain cooling to 4 °C from the interior of a glass capillary having convex surface energy modifications that were immersed in the solution. For thaumatin and bovine pancreatic trypsin, a 32(2)% reduction in crystallization onset times was demonstrated in vapor diffusion experiments ( n ≥ 15). Nucleation site arrays have been engineered onto form factors frequently used in crystallization screening, including microscope slides, vials, and 96- and 384-well high-throughput screening plates. Nucleation using surface energy modifications on the vessels that contain the solutes to be crystallized adds a layer of useful variables to crystallization studies without requiring significant changes to workflows or instrumentation.

Quantitative Comparison of Protein Adsorption and Conformational Changes on Dielectric-Coated Nanoplasmonic Sensing Arrays.

PubMed

Ferhan, Abdul Rahim; Jackman, Joshua A; Sut, Tun Naw; Cho, Nam-Joon

2018-04-22

Nanoplasmonic sensors are a popular, surface-sensitive measurement tool to investigate biomacromolecular interactions at solid-liquid interfaces, opening the door to a wide range of applications. In addition to high surface sensitivity, nanoplasmonic sensors have versatile surface chemistry options as plasmonic metal nanoparticles can be coated with thin dielectric layers. Within this scope, nanoplasmonic sensors have demonstrated promise for tracking protein adsorption and substrate-induced conformational changes on oxide film-coated arrays, although existing studies have been limited to single substrates. Herein, we investigated human serum albumin (HSA) adsorption onto silica- and titania-coated arrays of plasmonic gold nanodisks by localized surface plasmon resonance (LSPR) measurements and established an analytical framework to compare responses across multiple substrates with different sensitivities. While similar responses were recorded on the two substrates for HSA adsorption under physiologically-relevant ionic strength conditions, distinct substrate-specific behavior was observed at lower ionic strength conditions. With decreasing ionic strength, larger measurement responses occurred for HSA adsorption onto silica surfaces, whereas HSA adsorption onto titania surfaces occurred independently of ionic strength condition. Complementary quartz crystal microbalance-dissipation (QCM-D) measurements were also performed, and the trend in adsorption behavior was similar. Of note, the magnitudes of the ionic strength-dependent LSPR and QCM-D measurement responses varied, and are discussed with respect to the measurement principle and surface sensitivity of each technique. Taken together, our findings demonstrate how the high surface sensitivity of nanoplasmonic sensors can be applied to quantitatively characterize protein adsorption across multiple surfaces, and outline broadly-applicable measurement strategies for biointerfacial science applications.

Interaction of tau protein with model lipid membranes induces tau structural compaction and membrane disruption

PubMed Central

Jones, Emmalee M.; Dubey, Manish; Camp, Phillip J.; Vernon, Briana C.; Biernat, Jacek; Mandelkow, Eckhard; Majewski, Jaroslaw; Chi, Eva Y.

2012-01-01

The misfolding and aggregation of the intrinsically disordered, microtubule-associated tau protein into neurofibrillary tangles is implicated in the pathogenesis of Alzheimer's disease. However, the mechanisms of tau aggregation and toxicity remain unknown. Recent work has shown that lipid membrane can induce tau aggregation and that membrane permeabilization may serve as a pathway by which protein aggregates exert toxicity, suggesting that the plasma membrane may play dual roles in tau pathology. This prompted our investigation to assess tau's propensity to interact with membranes and to elucidate the mutually disruptive structural perturbations the interactions induce in both tau and the membrane. We show that although highly charged and soluble, the full-length tau (hTau40) is also highly surface active, selectively inserts into anionic DMPG lipid monolayers and induces membrane morphological changes. To resolve molecular-scale structural details of hTau40 associated with lipid membranes, X-ray and neutron scattering techniques are utilized. X-ray reflectivity indicates hTau40's presence underneath a DMPG monolayer and penetration into the lipid headgroups and tailgroups, whereas grazing incidence X-ray diffraction shows that hTau40 insertion disrupts lipid packing. Moreover, both air/water and DMPG lipid membrane interfaces induce the disordered hTau40 to partially adopt a more compact conformation with density similar to that of a folded protein. Neutron reflectivity shows that tau completely disrupts supported DMPG bilayers while leaving the neutral DPPC bilayer intact. Our results show that hTau40's strong interaction with anionic lipids induces tau structural compaction and membrane disruption, suggesting possible membrane-based mechanisms of tau aggregation and toxicity in neurodegenerative diseases. PMID:22401494

Neutral endopeptidase inhibits neuropeptide-mediated transactivation of the insulin-like growth factor receptor-Akt cell survival pathway.

PubMed

Sumitomo, M; Milowsky, M I; Shen, R; Navarro, D; Dai, J; Asano, T; Hayakawa, M; Nanus, D M

2001-04-15

G-protein coupled receptor (GPCR) agonists such as neuropeptides activate the insulin-like growth factor-1 receptor (IGF-IR) or the serine-threonine protein kinase Akt, suggesting that neuropeptides-GPCR signaling can cross-communicate with IGF-IR-Akt signaling pathways. Neutral endopeptidase 24.11 (NEP) is a cell-surface peptidase that cleaves and inactivates the neuropeptides endothelin-1 (ET-1) and bombesin, which are implicated in progression to androgen-independent prostate cancer (PC). We investigated the mechanisms of NEP regulation of neuropeptide-mediated cell survival in PC cells, including whether neuropeptide substrates of NEP induce phosphorylations of IGF-IR and Akt in PC cells. Western analyses revealed ET-1 and bombesin treatment induced phosphorylation of IGF-IRbeta and Akt independent of IGF-I in TSU-Pr1, DU145, and PC-3 PC cells, which lack NEP expression, but not in NEP-expressing LNCaP cells. Recombinant NEP and induced NEP expression in TSU-Pr1 cells using a tetracycline-repressive expression system inhibited ET-1-mediated phosphorylation of IGF-IRbeta and Akt, and blocked the protective effects of ET-1 against apoptosis induced by serum starvation. Incubation of TSU-Pr1 cells with specific kinase inhibitors together with ET-1 or bombesin showed that IGF-IR activation is required for neuropeptide-induced Akt phosphorylation, and that neuropeptide-induced Akt activation is predominantly mediated by Src and phosphatidylinositol 3-kinase but not by mitogen-activated protein kinase or protein kinase C. These data show that the neuropeptides ET-1 and bombesin stimulate ligand-independent activation of the IGF-IR, which results in Akt activation, and that this cross-communication between GPCR and IGF-IR signaling is inhibited by NEP.

Endomorphin-2: A Biased Agonist at the μ-Opioid Receptor

PubMed Central

Rivero, Guadalupe; Llorente, Javier; McPherson, Jamie; Cooke, Alex; Mundell, Stuart J.; McArdle, Craig A.; Rosethorne, Elizabeth M.; Charlton, Steven J.; Krasel, Cornelius; Bailey, Christopher P.; Henderson, Graeme

2012-01-01

Previously we correlated the efficacy for G protein activation with that for arrestin recruitment for a number of agonists at the μ-opioid receptor (MOPr) stably expressed in HEK293 cells. We suggested that the endomorphins (endomorphin-1 and -2) might be biased toward arrestin recruitment. In the present study, we investigated this phenomenon in more detail for endomorphin-2, using endogenous MOPr in rat brain as well as MOPr stably expressed in HEK293 cells. For MOPr in neurons in brainstem locus ceruleus slices, the peptide agonists [d-Ala2,N-Me-Phe4,Gly5-ol]-enkephalin (DAMGO) and endomorphin-2 activated inwardly rectifying K+ current in a concentration-dependent manner. Analysis of these responses with the operational model of pharmacological agonism confirmed that endomorphin-2 had a much lower operational efficacy for G protein-mediated responses than did DAMGO at native MOPr in mature neurons. However, endomorphin-2 induced faster desensitization of the K+ current than did DAMGO. In addition, in HEK293 cells stably expressing MOPr, the ability of endomorphin-2 to induce phosphorylation of Ser375 in the COOH terminus of the receptor, to induce association of arrestin with the receptor, and to induce cell surface loss of receptors was much more efficient than would be predicted from its efficacy for G protein-mediated signaling. Together, these results indicate that endomorphin-2 is an arrestin-biased agonist at MOPr and the reason for this is likely to be the ability of endomorphin-2 to induce greater phosphorylation of MOPr than would be expected from its ability to activate MOPr and to induce activation of G proteins. PMID:22553358

Endomorphin-2: a biased agonist at the μ-opioid receptor.

PubMed

Rivero, Guadalupe; Llorente, Javier; McPherson, Jamie; Cooke, Alex; Mundell, Stuart J; McArdle, Craig A; Rosethorne, Elizabeth M; Charlton, Steven J; Krasel, Cornelius; Bailey, Christopher P; Henderson, Graeme; Kelly, Eamonn

2012-08-01

Previously we correlated the efficacy for G protein activation with that for arrestin recruitment for a number of agonists at the μ-opioid receptor (MOPr) stably expressed in HEK293 cells. We suggested that the endomorphins (endomorphin-1 and -2) might be biased toward arrestin recruitment. In the present study, we investigated this phenomenon in more detail for endomorphin-2, using endogenous MOPr in rat brain as well as MOPr stably expressed in HEK293 cells. For MOPr in neurons in brainstem locus ceruleus slices, the peptide agonists [d-Ala(2),N-Me-Phe(4),Gly(5)-ol]-enkephalin (DAMGO) and endomorphin-2 activated inwardly rectifying K(+) current in a concentration-dependent manner. Analysis of these responses with the operational model of pharmacological agonism confirmed that endomorphin-2 had a much lower operational efficacy for G protein-mediated responses than did DAMGO at native MOPr in mature neurons. However, endomorphin-2 induced faster desensitization of the K(+) current than did DAMGO. In addition, in HEK293 cells stably expressing MOPr, the ability of endomorphin-2 to induce phosphorylation of Ser375 in the COOH terminus of the receptor, to induce association of arrestin with the receptor, and to induce cell surface loss of receptors was much more efficient than would be predicted from its efficacy for G protein-mediated signaling. Together, these results indicate that endomorphin-2 is an arrestin-biased agonist at MOPr and the reason for this is likely to be the ability of endomorphin-2 to induce greater phosphorylation of MOPr than would be expected from its ability to activate MOPr and to induce activation of G proteins.

Structural elucidation of the interaction between neurodegenerative disease-related tau protein with model lipid membranes

NASA Astrophysics Data System (ADS)

Jones, Emmalee M.

A protein's sequence of amino acids determines how it folds. That folded structure is linked to protein function, and misfolding to dysfunction. Protein misfolding and aggregation into beta-sheet rich fibrillar aggregates is connected with over 20 neurodegenerative diseases, including Alzheimer's disease (AD). AD is characterized in part by misfolding, aggregation and deposition of the microtubule associated tau protein into neurofibrillary tangles (NFTs). However, two questions remain: What is tau's fibrillization mechanism, and what is tau's cytotoxicity mechanism? Tau is prone to heterogeneous interactions, including with lipid membranes. Lipids have been found in NFTs, anionic lipid vesicles induced aggregation of the microtubule binding domain of tau, and other protein aggregates induced ion permeability in cells. This evidence prompted our investigation of tau's interaction with model lipid membranes to elucidate the structural perturbations those interactions induced in tau protein and in the membrane. We show that although tau is highly charged and soluble, it is highly surface active and preferentially interacts with anionic membranes. To resolve molecular-scale structural details of tau and model membranes, we utilized X-ray and neutron scattering techniques. X-ray reflectivity indicated tau aggregated at air/water and anionic lipid membrane interfaces and penetrated into membranes. More significantly, membrane interfaces induced tau protein to partially adopt a more compact conformation with density similar to folded protein and ordered structure characteristic of beta-sheet formation. This suggests possible membrane-based mechanisms of tau aggregation. Membrane morphological changes were seen using fluorescence microscopy, and X-ray scattering techniques showed tau completely disrupts anionic membranes, suggesting an aggregate-based cytotoxicity mechanism. Further investigation of protein constructs and a "hyperphosphorylation" disease mimic helped clarify the role of the microtubule binding domain in anionic lipid affinity and demonstrated even "hyperphosphorylation" did not prevent interaction with anionic membranes. Additional studies investigated more complex membrane models to increase physiological relevance. These insights revealed structural changes in tau protein and lipid membranes after interaction. We observed tau's affinity for interfaces, and aggregation and compaction once tau partitions to interfaces. We observed the beginnings of beta-sheet formation in tau at anionic lipid membranes. We also examined disruption to the membrane on a molecular scale.

Physiological response of Pichia pastoris GS115 to methanol-induced high level production of the Hepatitis B surface antigen: catabolic adaptation, stress responses, and autophagic processes

PubMed Central

2012-01-01

Background Pichia pastoris is an established eukaryotic host for the production of recombinant proteins. Most often, protein production is under the control of the strong methanol-inducible aox1 promoter. However, detailed information about the physiological alterations in P. pastoris accompanying the shift from growth on glycerol to methanol-induced protein production under industrial relevant conditions is missing. Here, we provide an analysis of the physiological response of P. pastoris GS115 to methanol-induced high-level production of the Hepatitis B virus surface antigen (HBsAg). High product titers and the retention of the protein in the endoplasmic reticulum (ER) are supposedly of major impact on the host physiology. For a more detailed understanding of the cellular response to methanol-induced HBsAg production, the time-dependent changes in the yeast proteome and ultrastructural cell morphology were analyzed during the production process. Results The shift from growth on glycerol to growth and HBsAg production on methanol was accompanied by a drastic change in the yeast proteome. In particular, enzymes from the methanol dissimilation pathway started to dominate the proteome while enzymes from the methanol assimilation pathway, e.g. the transketolase DAS1, increased only moderately. The majority of methanol was metabolized via the energy generating dissimilatory pathway leading to a corresponding increase in mitochondrial size and numbers. The methanol-metabolism related generation of reactive oxygen species induced a pronounced oxidative stress response (e.g. strong increase of the peroxiredoxin PMP20). Moreover, the accumulation of HBsAg in the ER resulted in the induction of the unfolded protein response (e.g. strong increase of the ER-resident disulfide isomerase, PDI) and the ER associated degradation (ERAD) pathway (e.g. increase of two cytosolic chaperones and members of the AAA ATPase superfamily) indicating that potential degradation of HBsAg could proceed via the ERAD pathway and through the proteasome. However, the amount of HBsAg did not show any significant decline during the cultivation revealing its general protection from proteolytic degradation. During the methanol fed-batch phase, induction of vacuolar proteases (e.g. strong increase of APR1) and constitutive autophagic processes were observed. Vacuolar enclosures were mainly found around peroxisomes and not close to HBsAg deposits and, thus, were most likely provoked by peroxisomal components damaged by reactive oxygen species generated by methanol oxidation. Conclusions In the methanol fed-batch phase P. pastoris is exposed to dual stress; stress resulting from methanol degradation and stress resulting from the production of the recombinant protein leading to the induction of oxidative stress and unfolded protein response pathways, respectively. Finally, the modest increase of methanol assimilatory enzymes compared to the strong increase of methanol dissimilatory enzymes suggests here a potential to increase methanol incorporation into biomass/product through metabolic enhancement of the methanol assimilatory pathway. PMID:22873405