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.

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.

Sequestration of GPI-anchored proteins in caveolae triggered by cross-linking.

PubMed

Mayor, S; Rothberg, K G; Maxfield, F R

1994-06-24

Glycosyl-phosphatidylinositol (GPI)-anchored proteins have been reported to reside in clusters collected over small membrane invaginations called caveolae. The detection of different GPI-anchored proteins with fluorescently labeled monoclonal antibodies showed that these proteins are not constitutively concentrated in caveolae; they enter these structures independently after cross-linking with polyclonal secondary antibodies. Analysis of the cell surface distribution of the GPI-anchored folate receptor by electron microscopy confirms these observations. Thus, multimerization of GPI-anchored proteins regulates their sequestration in caveolae, but in the absence of agents that promote clustering they are diffusely distributed over the plasma membrane.

Surface plasmon resonance biosensing of the monomer and the linked dimer of the variants of protein G under mass transport limitation.

PubMed

Imamura, Hiroshi; Honda, Shinya

2016-12-01

This article presented the data related to the research article entitled "Calibration-free concentration analysis for an analyte prone to self-association" (H. Imamura, S. Honda, 2017) [1]. The data included surface plasmon resonance (SPR) responses of the variants of protein G with different masses under mass transport limitation. The friction factors of the proteins analyzed by an ultracentrifugation were recorded. Calculation of the SPR response of the proteins was also described.

Studying the protein organization of the postsynaptic density by a novel solid phase- and chemical cross-linking-based technology.

PubMed

Liu, Szu-Heng; Cheng, Huei-Hsuan; Huang, San-Yuan; Yiu, Pei-Chun; Chang, Yen-Chung

2006-06-01

Agarose beads carrying a cleavable, fluorescent, and photoreactive cross-linking reagent on the surface were synthesized and used to selectively pull out the proteins lining the surface of supramolecules. A quantitative comparison of the abundances of various proteins in the sample pulled out by the beads from supramolecules with their original abundances could provide information on the spatial arrangement of these proteins in the supramolecule. The usefulness of these synthetic beads was successfully verified by trials using a synthetic protein complex consisting of three layers of different proteins on glass coverslips. By using these beads, we determined the interior or superficial locations of five major and 19 minor constituent proteins in the postsynaptic density (PSD), a large protein complex and the landmark structure of asymmetric synapses in the mammalian central nervous system. The results indicate that alpha,beta-tubulins, dynein heavy chain, microtubule-associated protein 2, spectrin, neurofilament H and M subunits, an hsp70 protein, alpha-internexin, dynamin, and PSD-95 protein reside in the interior of the PSD. Dynein intermediate chain, alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate receptors, kainate receptors, N-cadherin, beta-catenin, N-ethylmaleimide-sensitive factor, an hsc70 protein, and actin reside on the surface of the PSD. The results further suggest that the N-methyl-d-aspartate receptors and the alpha-subunits of calcium/calmodulin-dependent protein kinase II are likely to reside on the surface of the PSD although with unique local protein organizations. Based on our results and the known interactions between various PSD proteins from data mining, a model for the molecular organization of the PSD is proposed.

A General Method for Targeted Quantitative Cross-Linking Mass Spectrometry.

PubMed

Chavez, Juan D; Eng, Jimmy K; Schweppe, Devin K; Cilia, Michelle; Rivera, Keith; Zhong, Xuefei; Wu, Xia; Allen, Terrence; Khurgel, Moshe; Kumar, Akhilesh; Lampropoulos, Athanasios; Larsson, Mårten; Maity, Shuvadeep; Morozov, Yaroslav; Pathmasiri, Wimal; Perez-Neut, Mathew; Pineyro-Ruiz, Coriness; Polina, Elizabeth; Post, Stephanie; Rider, Mark; Tokmina-Roszyk, Dorota; Tyson, Katherine; Vieira Parrine Sant'Ana, Debora; Bruce, James E

2016-01-01

Chemical cross-linking mass spectrometry (XL-MS) provides protein structural information by identifying covalently linked proximal amino acid residues on protein surfaces. The information gained by this technique is complementary to other structural biology methods such as x-ray crystallography, NMR and cryo-electron microscopy[1]. The extension of traditional quantitative proteomics methods with chemical cross-linking can provide information on the structural dynamics of protein structures and protein complexes. The identification and quantitation of cross-linked peptides remains challenging for the general community, requiring specialized expertise ultimately limiting more widespread adoption of the technique. We describe a general method for targeted quantitative mass spectrometric analysis of cross-linked peptide pairs. We report the adaptation of the widely used, open source software package Skyline, for the analysis of quantitative XL-MS data as a means for data analysis and sharing of methods. We demonstrate the utility and robustness of the method with a cross-laboratory study and present data that is supported by and validates previously published data on quantified cross-linked peptide pairs. This advance provides an easy to use resource so that any lab with access to a LC-MS system capable of performing targeted quantitative analysis can quickly and accurately measure dynamic changes in protein structure and protein interactions.

Intracellular transport and processing of the Marburg virus surface protein in vertebrate and insect cells.

PubMed

Becker, S; Klenk, H D; Mühlberger, E

1996-11-01

The surface protein (GP) of Marburg virus (MBG) is synthesized as a 90-kDa precursor protein which is cotranslationally modified by the addition of high-mannose sugars (140 kDa). This step is followed by the conversion of the N-linked sugars to endoglycosidase H (endo H)-resistant species and the addition of O-linked oliosaccharides leading to a mature protein of 170-200 kDa approximately 30 min after pulse labelling. The mature form of GP is efficiently transported to the plasma membrane. GP synthesized using the T7 polymerase-driven vaccinia virus expression system was transported with essentially the same kinetics as the authentic GP. However, the protein that is shown to appear 30 min after pulse labeling at the plasma membrane was slighly smaller (160 kDa) than GP incorporated into the virions (170 kDa). Using a recombinant baculovirus, GP was expressed at high levels in insect cells. Three different species could be identified: a 90-kDa unglycosylated GP localized in the cytoplasm and two 140-kDa glycosylated proteins. Characterization of the glycosylated GPs revealed that processing of the oligosaccharides of GP was less efficient in insect cells than in mammalian cells. The majority of GP remained endo H sensitive containing high-mannose type N-linked glycans, whereas only a small fraction became endo H resistant carrying processed N-glycans and O-glycans. Tunicamycin treatment of the GP-expressing cells demonstrated that N-glycosylation is essential for the transport of the MBG surface protein.

ABI domain containing proteins contribute to surface protein display and cell division in Staphylococcus aureus

PubMed Central

Frankel, Matthew B.; Wojcik, Brandon; DeDent, Andrea C.; Missiakas, Dominique M.; Schneewind, Olaf

2012-01-01

Summary The human pathogen Staphyloccocus aureus requires cell wall anchored surface proteins to cause disease. During cell division, surface proteins with YSIRK signal peptides are secreted into the cross wall, a layer of newly synthesized peptidoglycan between separating daughter cells. The molecular determinants for the trafficking of surface proteins are, however, still unknown. We screened mutants with non-redundant transposon insertions by fluorescence-activated cell sorting for reduced deposition of protein A (SpA) into the staphylococcal envelope. Three mutants, each of which harbored transposon insertions in genes for transmembrane proteins, displayed greatly reduced envelope abundance of SpA and surface proteins with YSIRK signal peptides. Characterization of the corresponding mutations identified three transmembrane proteins with abortive infectivity (ABI) domains, elements first described in lactococci for their role in phage exclusion. Mutations in genes for ABI domain proteins, designated spdA, spdB and spdC (surface protein display), diminish the expression of surface proteins with YSIRK signal peptides, but not of precursor proteins with conventional signal peptides. spdA, spdB and spdC mutants display an increase in the thickness of cross walls and in the relative abundance of staphylococci with cross walls, suggesting that spd mutations may represent a possible link between staphylococcal cell division and protein secretion. PMID:20923422

ABI domain-containing proteins contribute to surface protein display and cell division in Staphylococcus aureus.

PubMed

Frankel, Matthew B; Wojcik, Brandon M; DeDent, Andrea C; Missiakas, Dominique M; Schneewind, Olaf

2010-10-01

The human pathogen Staphylococcus aureus requires cell wall anchored surface proteins to cause disease. During cell division, surface proteins with YSIRK signal peptides are secreted into the cross-wall, a layer of newly synthesized peptidoglycan between separating daughter cells. The molecular determinants for the trafficking of surface proteins are, however, still unknown. We screened mutants with non-redundant transposon insertions by fluorescence-activated cell sorting for reduced deposition of protein A (SpA) into the staphylococcal envelope. Three mutants, each of which harboured transposon insertions in genes for transmembrane proteins, displayed greatly reduced envelope abundance of SpA and surface proteins with YSIRK signal peptides. Characterization of the corresponding mutations identified three transmembrane proteins with abortive infectivity (ABI) domains, elements first described in lactococci for their role in phage exclusion. Mutations in genes for ABI domain proteins, designated spdA, spdB and spdC (surface protein display), diminish the expression of surface proteins with YSIRK signal peptides, but not of precursor proteins with conventional signal peptides. spdA, spdB and spdC mutants display an increase in the thickness of cross-walls and in the relative abundance of staphylococci with cross-walls, suggesting that spd mutations may represent a possible link between staphylococcal cell division and protein secretion. © 2010 Blackwell Publishing Ltd.

Characterization of a novel wheat endosperm protein belonging to the prolamin superfamily

USDA-ARS?s Scientific Manuscript database

Starch granule surface-associated proteins were separated by HPLC and identified by direct protein sequencing. Among the proteins identified was one that consisted of two polypeptide chains of 11 kDa and 19 kDa linked by disulfide bonds. Sequencing of tryptic peptides from each of the polypeptide ch...

Surface Sites for Engineering Allosteric Control in Proteins

PubMed Central

Lee, Jeeyeon; Natarajan, Madhusudan; Nashine, Vishal C.; Socolich, Michael; Vo, Tina; Russ, William P.; Benkovic, Stephen J.; Ranganathan, Rama

2010-01-01

Statistical analyses of protein families reveal networks of coevolving amino acids that functionally link distantly positioned functional surfaces. Such linkages suggest a concept for engineering allosteric control into proteins: The intramolecular networks of two proteins could be joined across their surface sites such that the activity of one protein might control the activity of the other. We tested this idea by creating PAS-DHFR, a designed chimeric protein that connects a light-sensing signaling domain from a plant member of the Per/Arnt/Sim (PAS) family of proteins with Escherichia coli dihydrofolate reductase (DHFR). With no optimization, PAS-DHFR exhibited light-dependent catalytic activity that depended on the site of connection and on known signaling mechanisms in both proteins. PAS-DHFR serves as a proof of concept for engineering regulatory activities into proteins through interface design at conserved allosteric sites. PMID:18927392

Effect of surface modification on protein retention and cell proliferation under strain.

PubMed

Dunkers, J P; Lee, H-J; Matos, M A; Pakstis, L M; Taboas, J M; Hudson, S D; Cicerone, M T

2011-07-01

When culturing cells on flexible surfaces, it is important to consider extracellular matrix treatments that will remain on the surface under mechanical strain. Here we investigate differences in laminin deposited on oxidized polydimethylsiloxane (PDMS) with plasma treatment (plasma-only) vs. plasma and aminopropyltrimethoxysilane treatment (silane-linked). We use specular X-ray reflectivity (SXR), transmission electron microscopy (TEM), and immunofluorescence to probe the quantity and uniformity of laminin. The surface coverage of laminin is approximately 45% for the plasma-only and 50% for the silane-linked treatment as determined by SXR. TEM and immunofluorescence reveal additional islands of laminin aggregates on the plasma-only PDMS compared with the relatively smooth and uniform silane-linked laminin surface. We also examine laminin retention under strain and vascular smooth muscle cell viability and proliferation under static and strain conditions. Equibiaxial stretching of the PDMS surfaces shows greatly improved retention of the silane-linked laminin over plasma-only. There are significantly more cells on the silane-linked surface after 4 days of equibiaxial strain. Published by Elsevier Ltd.

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

Surface proteins and the formation of biofilms by Staphylococcus aureus.

PubMed

Kim, Sung Joon; Chang, James; Rimal, Binayak; Yang, Hao; Schaefer, Jacob

2018-03-01

Staphylococcus aureus biofilms pose a serious clinical threat as reservoirs for persistent infections. Despite this clinical significance, the composition and mechanism of formation of S. aureus biofilms are unknown. To address these problems, we used solid-state NMR to examine S. aureus (SA113), a strong biofilm-forming strain. We labeled whole cells and cell walls of planktonic cells, young biofilms formed for 12-24h after stationary phase, and more mature biofilms formed for up to 60h after stationary phase. All samples were labeled either by (i) [ 15 N]glycine and l-[1- 13 C]threonine, or in separate experiments, by (ii) l-[2- 13 C, 15 N]leucine. We then measured 13 C- 15 N direct bonds by C{N} rotational-echo double resonance (REDOR). The increase in peptidoglycan stems that have bridges connected to a surface protein was determined directly by a cell-wall double difference (biofilm REDOR difference minus planktonic REDOR difference). This procedure eliminates errors arising from differences in 15 N isotopic enrichments and from the routing of 13 C label from threonine degradation to glycine. For both planktonic cells and the mature biofilm, 20% of pentaglycyl bridges are not cross-linked and are potential surface-protein attachment sites. None of these sites has a surface protein attached in the planktonic cells, but one-fourth have a surface protein attached in the mature biofilm. Moreover, the leucine-label shows that the concentration of β-strands in leucine-rich regions doubles in the mature biofilm. Thus, a primary event in establishing a S. aureus biofilm is extensive decoration of the cell surface with surface proteins that are linked covalently to the cell wall and promote cell-cell adhesion. Copyright © 2017 Elsevier B.V. All rights reserved.

Surface Localization of Zein Storage Proteins in Starch Granules from Maize Endosperm1

PubMed Central

Mu-Forster, Chen; Wasserman, Bruce P.

1998-01-01

Starch granules from maize (Zea mays) contain a characteristic group of polypeptides that are tightly associated with the starch matrix (C. Mu-Forster, R. Huang, J.R. Powers, R.W. Harriman, M. Knight, G.W. Singletary, P.L. Keeling, B.P. Wasserman [1996] Plant Physiol 111: 821–829). Zeins comprise about 50% of the granule-associated proteins, and in this study their spatial distribution within the starch granule was determined. Proteolysis of starch granules at subgelatinization temperatures using the thermophilic protease thermolysin led to selective removal of the zeins, whereas granule-associated proteins of 32 kD or above, including the waxy protein, starch synthase I, and starch-branching enzyme IIb, remained refractory to proteolysis. Granule-associated proteins from maize are therefore composed of two distinct classes, the surface-localized zeins of 10 to 27 kD and the granule-intrinsic proteins of 32 kD or higher. The origin of surface-localized δ-zein was probed by comparing δ-zein levels of starch granules obtained from homogenized whole endosperm with granules isolated from amyloplasts. Starch granules from amyloplasts contained markedly lower levels of δ-zein relative to granules prepared from whole endosperm, thus indicating that δ-zein adheres to granule surfaces after disruption of the amyloplast envelope. Cross-linking experiments show that the zeins are deposited on the granule surface as aggregates. In contrast, the granule-intrinsic proteins are prone to covalent modification, but do not form intermolecular cross-links. We conclude that individual granule intrinsic proteins exist as monomers and are not deposited in the form of multimeric clusters within the starch matrix. PMID:9536075

Disease-linked mutations in factor H reveal pivotal role of cofactor activity in self-surface-selective regulation of complement activation.

PubMed

Kerr, Heather; Wong, Edwin; Makou, Elisavet; Yang, Yi; Marchbank, Kevin; Kavanagh, David; Richards, Anna; Herbert, Andrew P; Barlow, Paul N

2017-08-11

Spontaneous activation enables the complement system to respond very rapidly to diverse threats. This activation is efficiently suppressed by complement factor H (CFH) on self-surfaces but not on foreign surfaces. The surface selectivity of CFH, a soluble protein containing 20 complement-control protein modules (CCPs 1-20), may be compromised by disease-linked mutations. However, which of the several functions of CFH drives this self-surface selectivity remains unknown. To address this, we expressed human CFH mutants in Pichia pastoris We found that recombinant I62-CFH (protective against age-related macular degeneration) and V62-CFH functioned equivalently, matching or outperforming plasma-derived CFH, whereas R53H-CFH, linked to atypical hemolytic uremic syndrome (aHUS), was defective in C3bBb decay-accelerating activity (DAA) and factor I cofactor activity (CA). The aHUS-linked CCP 19 mutant D1119G-CFH had virtually no CA on (self-like) sheep erythrocytes ( E S ) but retained DAA. The aHUS-linked CCP 20 mutant S1191L/V1197A-CFH (LA-CFH) had dramatically reduced CA on E S but was less compromised in DAA. D1119G-CFH and LA-CFH both performed poorly at preventing complement-mediated hemolysis of E S PspCN, a CFH-binding Streptococcus pneumoniae protein domain, binds CFH tightly and increases accessibility of CCPs 19 and 20. PspCN did not improve the DAA of any CFH variant on E S Conversely, PspCN boosted the CA, on E S , of I62-CFH, R53H-CFH, and LA-CFH and also enhanced hemolysis protection by I62-CFH and LA-CFH. We conclude that CCPs 19 and 20 are critical for efficient CA on self-surfaces but less important for DAA. Exposing CCPs 19 and 20 with PspCN and thus enhancing CA on self-surfaces may reverse deficiencies of some CFH variants. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Ligand-induced association of surface immunoglobulin with the detergent insoluble cytoskeleton may involve an 89K protein

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

Gupta, S.K.; Woda, B.

1986-03-01

Membrane immunoglobulin of B-lymphocytes is thought to play an important role in antigen recognition and cellular activation. Binding of cross-linking ligands to surface immunoglobulin (SIg) on intact cells converts it to a detergent insoluble state, and this conversion is associated with the transmission of a mitogenic signal. Insolubilized membrane proteins may be solubilized by incubating the detergent insoluble cytoskeletons in buffers which convert F-actin to G-actin ((Buffer 1), 0.34M sucrose, 0.5mM ATP, 0.5mM Dithiothrietol and lmM EDTA). Immunoprecipitation of SIg from the detergent soluble fraction of /sup 35/S-methionine labeled non ligand treated rat B-cells results in the co-isolation of anmore » 89K protein and a 44K protein, presumably actin. The 89K protein is not associated with the fraction of endogenous detergent insoluble SIg. On treatment of rat B cells with cross-linking ligand (anti-Ig) the 89K protein becomes detergent insoluble along with most of the SIg and co-isolates with SIg on immunoprecipitation of the detergent insoluble, buffer l solubilized fraction. The migration of the SIg-associated 89K protein from the detergent soluble fraction to the detergent insoluble fraction after ligand treatment, suggests that this protein might be involved in linking SIg to the underlying cytoskeleton and could be involved in the transmission of a mitogenic signal.« less

Computational investigation of kinetics of cross-linking reactions in proteins: importance in structure prediction.

PubMed

Bandyopadhyay, Pradipta; Kuntz, Irwin D

2009-01-01

The determination of protein structure using distance constraints is a new and promising field of study. One implementation involves attaching residues of a protein using a cross-linking agent, followed by protease digestion, analysis of the resulting peptides by mass spectroscopy, and finally sequence threading to detect the protein folds. In the present work, we carry out computational modeling of the kinetics of cross-linking reactions in proteins using the master equation approach. The rate constants of the cross-linking reactions are estimated using the pKas and the solvent-accessible surface areas of the residues involved. This model is tested with fibroblast growth factor (FGF) and cytochrome C. It is consistent with the initial experimental rate data for individual lysine residues for cytochrome C. Our model captures all observed cross-links for FGF and almost 90% of the observed cross-links for cytochrome C, although it also predicts cross-links that were not observed experimentally (false positives). However, the analysis of the false positive results is complicated by the fact that experimental detection of cross-links can be difficult and may depend on specific experimental conditions such as pH, ionic strength. Receiver operator characteristic plots showed that our model does a good job in predicting the observed cross-links. Molecular dynamics simulations showed that for cytochrome C, in general, the two lysines come closer for the observed cross-links as compared to the false positive ones. For FGF, no such clear pattern exists. The kinetic model and MD simulation can be used to study proposed cross-linking protocols.

Evidence for tyrosine-linked glycosaminoglycan in a bacterial surface protein.

PubMed

Peters, J; Rudolf, S; Oschkinat, H; Mengele, R; Sumper, M; Kellermann, J; Lottspeich, F; Baumeister, W

1992-04-01

The S-layer protein of Acetogenium kivui was subjected to proteolysis with different proteases and several high molecular mass glycosaminoglycan peptides containing glucose, galactosamine and an unidentified sugar-related component were separated by molecular sieve chromatography and reversed-phase HPLC and subjected to N-terminal sequence analysis. By methylation analysis glucose was found to be uniformly 1,6-linked, whereas galactosamine was exclusively 1,4-linked. Hydrazinolysis and subsequent amino-acid analysis as well as two-dimensional NMR spectroscopy were used to demonstrate that in these peptides carbohydrate was covalently linked to tyrosine. As all of the four Tyr-glycosylation sites were found to be preceded by valine, a new recognition sequence for glycosylation is suggested.

Compositions and methods for cancer treatment using targeted carbon nanotubes

DOEpatents

Harrison, Jr., Roger G; Resasco, Daniel E; Neves, Luis Filipe Ferreira

2013-08-27

The present invention is a method for detecting and destroying cancer tumors. The method is based on the concept of associating a linking protein or linking peptide such as, but not limited to, annexin V or other annexins to carbon nanotubes such as single-walled carbon nanotubes (SWNTs) to form a protein-CNT complex. Said linking protein or peptide can selectively bind to cancerous cells, especially tumor vasculature endothelial cells, rather than to healthy ones by binding to cancer-specific external receptors such as anionic phospholipids including phosphatidylserine expressed on the outer surfaces of cancer cells only. Irradiation of bound CNTs with one or more specific electromagnetic wavelengths is then used to detect and destroy those cells to which the CNTs are bound via the linking protein or peptide thereby destroying the tumor or cancer cells and preferably an immunostimulant is provided to the patient to enhance the immune response against antigens released from the tumor or cancer cells.

A protein secretion system linked to bacteroidete gliding motility and pathogenesis

PubMed Central

Sato, Keiko; Naito, Mariko; Yukitake, Hideharu; Hirakawa, Hideki; Shoji, Mikio; McBride, Mark J.; Rhodes, Ryan G.; Nakayama, Koji

2009-01-01

Porphyromonas gingivalis secretes strong proteases called gingipains that are implicated in periodontal pathogenesis. Protein secretion systems common to other Gram-negative bacteria are lacking in P. gingivalis, but several proteins, including PorT, have been linked to gingipain secretion. Comparative genome analysis and genetic experiments revealed 11 additional proteins involved in gingipain secretion. Six of these (PorK, PorL, PorM, PorN, PorW, and Sov) were similar in sequence to Flavobacterium johnsoniae gliding motility proteins, and two others (PorX and PorY) were putative two-component system regulatory proteins. Real-time RT-PCR analysis revealed that porK, porL, porM, porN, porP, porT, and sov were down-regulated in P. gingivalis porX and porY mutants. Disruption of the F. johnsoniae porT ortholog resulted in defects in motility, chitinase secretion, and translocation of a gliding motility protein, SprB adhesin, to the cell surface, providing a link between a unique protein translocation system and a motility apparatus in members of the Bacteroidetes phylum. PMID:19966289

Trifunctional cross-linker for mapping protein-protein interaction networks and comparing protein conformational states

PubMed Central

Tan, Dan; Li, Qiang; Zhang, Mei-Jun; Liu, Chao; Ma, Chengying; Zhang, Pan; Ding, Yue-He; Fan, Sheng-Bo; Tao, Li; Yang, Bing; Li, Xiangke; Ma, Shoucai; Liu, Junjie; Feng, Boya; Liu, Xiaohui; Wang, Hong-Wei; He, Si-Min; Gao, Ning; Ye, Keqiong; Dong, Meng-Qiu; Lei, Xiaoguang

2016-01-01

To improve chemical cross-linking of proteins coupled with mass spectrometry (CXMS), we developed a lysine-targeted enrichable cross-linker containing a biotin tag for affinity purification, a chemical cleavage site to separate cross-linked peptides away from biotin after enrichment, and a spacer arm that can be labeled with stable isotopes for quantitation. By locating the flexible proteins on the surface of 70S ribosome, we show that this trifunctional cross-linker is effective at attaining structural information not easily attainable by crystallography and electron microscopy. From a crude Rrp46 immunoprecipitate, it helped identify two direct binding partners of Rrp46 and 15 protein-protein interactions (PPIs) among the co-immunoprecipitated exosome subunits. Applying it to E. coli and C. elegans lysates, we identified 3130 and 893 inter-linked lysine pairs, representing 677 and 121 PPIs. Using a quantitative CXMS workflow we demonstrate that it can reveal changes in the reactivity of lysine residues due to protein-nucleic acid interaction. DOI: http://dx.doi.org/10.7554/eLife.12509.001 PMID:26952210

Phosphorylation of actin-binding protein (ABP-280; filamin) by tyrosine kinase p56lck modulates actin filament cross-linking.

PubMed

Pal Sharma, C; Goldmann, Wolfgang H

2004-01-01

Actin-binding protein (ABP-280; filamin) is a phosphoprotein present in the periphery of the cytoplasm where it can cross-link actin filaments, associate with lipid membranes, and bind to membrane surface receptors. Given its function and localization in the cell, we decided to investigate the possibility of whether it serves as substrate for p56lck, a lymphocyte-specific member of the src family of protein tyrosine kinases associated with cell surface glycoproteins. The interaction of p56lck with membrane glycoproteins is important for cell development and functional activation. Here, we show that purified p56lck interacts and catalyzes in vitro kinase reactions. Tyrosine phosphorylation by p56lck is restricted to a single peptide of labeled ABP-280 shown by protease digest. The addition of phorbol ester to cells results in the inhibition of phosphorylation of ABP-280 by p56lck. These results show a decrease in phosphorylation suggesting conformationally induced regulation. Dynamic light scattering confirmed increased actin filament cross-linking due to phosphorylation of ABP-280 by p56lck.

Non-interacting surface solvation and dynamics in protein-protein interactions.

PubMed

Visscher, Koen M; Kastritis, Panagiotis L; Bonvin, Alexandre M J J

2015-03-01

Protein-protein interactions control a plethora of cellular processes, including cell proliferation, differentiation, apoptosis, and signal transduction. Understanding how and why proteins interact will inevitably lead to novel structure-based drug design methods, as well as design of de novo binders with preferred interaction properties. At a structural and molecular level, interface and rim regions are not enough to fully account for the energetics of protein-protein binding, even for simple lock-and-key rigid binders. As we have recently shown, properties of the global surface might also play a role in protein-protein interactions. Here, we report on molecular dynamics simulations performed to understand solvent effects on protein-protein surfaces. We compare properties of the interface, rim, and non-interacting surface regions for five different complexes and their free components. Interface and rim residues become, as expected, less mobile upon complexation. However, non-interacting surface appears more flexible in the complex. Fluctuations of polar residues are always lower compared with charged ones, independent of the protein state. Further, stable water molecules are often observed around polar residues, in contrast to charged ones. Our analysis reveals that (a) upon complexation, the non-interacting surface can have a direct entropic compensation for the lower interface and rim entropy and (b) the mobility of the first hydration layer, which is linked to the stability of the protein-protein complex, is influenced by the local chemical properties of the surface. These findings corroborate previous hypotheses on the role of the hydration layer in shielding protein-protein complexes from unintended protein-protein interactions. © 2014 Wiley Periodicals, Inc.

Barnacle cement: a polymerization model based on evolutionary concepts

PubMed Central

Dickinson, Gary H.; Vega, Irving E.; Wahl, Kathryn J.; Orihuela, Beatriz; Beyley, Veronica; Rodriguez, Eva N.; Everett, Richard K.; Bonaventura, Joseph; Rittschof, Daniel

2009-01-01

Summary Enzymes and biochemical mechanisms essential to survival are under extreme selective pressure and are highly conserved through evolutionary time. We applied this evolutionary concept to barnacle cement polymerization, a process critical to barnacle fitness that involves aggregation and cross-linking of proteins. The biochemical mechanisms of cement polymerization remain largely unknown. We hypothesized that this process is biochemically similar to blood clotting, a critical physiological response that is also based on aggregation and cross-linking of proteins. Like key elements of vertebrate and invertebrate blood clotting, barnacle cement polymerization was shown to involve proteolytic activation of enzymes and structural precursors, transglutaminase cross-linking and assembly of fibrous proteins. Proteolytic activation of structural proteins maximizes the potential for bonding interactions with other proteins and with the surface. Transglutaminase cross-linking reinforces cement integrity. Remarkably, epitopes and sequences homologous to bovine trypsin and human transglutaminase were identified in barnacle cement with tandem mass spectrometry and/or western blotting. Akin to blood clotting, the peptides generated during proteolytic activation functioned as signal molecules, linking a molecular level event (protein aggregation) to a behavioral response (barnacle larval settlement). Our results draw attention to a highly conserved protein polymerization mechanism and shed light on a long-standing biochemical puzzle. We suggest that barnacle cement polymerization is a specialized form of wound healing. The polymerization mechanism common between barnacle cement and blood may be a theme for many marine animal glues. PMID:19837892

Surface derivatization strategy for combinatorial analysis of cell response to mixtures of protein domains.

PubMed

Chiang, Chunyi; Karuri, Stella W; Kshatriya, Pradnya P; Schwartz, Jeffrey; Schwarzbauer, Jean E; Karuri, Nancy W

2012-01-10

We report a robust strategy for conjugating mixtures of two or more protein domains to nonfouling polyurethane surfaces. In our strategy, the carbamate groups of polyurethane are reacted with zirconium alkoxide from the vapor phase to give a surface-bound oxide that serves as a chemical layer that can be used to bond organics to the polymer substrate. A hydroxyalkylphosphonate monolayer was synthesized on this layer, which was then used to covalently bind primary amine groups in protein domains using chloroformate-derived cross-linking. The effectiveness of this synthesis strategy was gauged by using an ELISA to measure competitive, covalent bonding of cell-binding (III(9-10)) and fibronectin-binding (III(1-2)) domains of the cell adhesion protein fibronectin. Cell adhesion, spreading, and fibronectin matrix assembly were examined on surfaces conjugated with single domains, a 1:1 surface mixture of III(1-2) and III(9-10), and a recombinant protein "duplex" containing both domains in one fusion protein. The mixture performed as well as or better than the other surfaces in these assays. Our surface activation strategy is amenable to a wide range of polymer substrates and free amino group-containing protein fragments. As such, this technique may be used to create biologically specific materials through the immobilization of specific protein groups or mixtures thereof on a substrate surface.

N-Glycosylation of Campylobacter jejuni Surface Proteins Promotes Bacterial Fitness

PubMed Central

Nothaft, Harald; Zheng, Jing

2013-01-01

Campylobacter jejuni is the etiologic agent of human bacterial gastroenteritis worldwide. In contrast, despite heavy colonization, C. jejuni maintains a commensal mode of existence in chickens. The consumption of contaminated chicken products is thought to be the principal mode of C. jejuni transmission to the human population. C. jejuni harbors a system for N-linked protein glycosylation that has been well characterized and modifies more than 60 periplasmic and membrane-bound proteins. However, the precise role of this modification in the biology of C. jejuni remains unexplored. We hypothesized that the N-glycans protect C. jejuni surface proteins from the action of gut proteases. The C. jejuni pglB mutant, deficient in the expression of the oligosaccharyltransferase, exhibited reduced growth in medium supplemented with chicken cecal contents (CCC) compared with that of wild-type (WT) cells. Inactivation of the cecal proteases by heat treatment or with protease inhibitors completely restored bacterial viability and partially rescued bacterial growth. Physiological concentrations of trypsin, but not chymotrypsin, also reduced C. jejuni pglB mutant CFU. Live or dead staining indicated that CCC preferentially influenced C. jejuni growth as opposed to bacterial viability. We identified multiple chicken cecal proteases by mass fingerprinting. The use of protease inhibitors that target specific classes indicated that both metalloproteases and serine proteases were involved in the attenuated growth of the oligosaccharyltransferase mutant. In conclusion, protein N-linked glycosylation of surface proteins may enhance C. jejuni fitness by protecting bacterial proteins from cleavage due to gut proteases. PMID:23460522

Novel Strategy to Create Hypoallergenic Peanut Protein-Polyphenol Edible Matrices for Oral Immunotherapy

USDA-ARS?s Scientific Manuscript database

Peanut allergy is an IgE-mediated hypersensitivity. Upon peanut consumption by an allergic individual, epitopes on peanut proteins bind and cross-link peanut-specific IgE on mast cell and basophil surfaces triggering the cells to release inflammatory mediators responsible for allergic reactions. P...

Plantaricin A, a cationic peptide produced by Lactobacillus plantarum, permeabilizes eukaryotic cell membranes by a mechanism dependent on negative surface charge linked to glycosylated membrane proteins.

PubMed

Sand, Sverre L; Nissen-Meyer, Jon; Sand, Olav; Haug, Trude M

2013-02-01

Lactobacillus plantarum C11 releases plantaricin A (PlnA), a cationic peptide pheromone that has a membrane-permeabilizing, antimicrobial effect. We have previously shown that PlnA may also permeabilize eukaryotic cells, with a potency that differs between cell types. It is generally assumed that cationic antimicrobial peptides exert their effects through electrostatic attraction to negatively charged phospholipids in the membrane. The aim of the present study was to investigate if removal of the negative charge linked to glycosylated proteins at the cell surface reduces the permeabilizing potency of PlnA. The effects of PlnA were tested on clonal rat anterior pituitary cells (GH(4) cells) using patch clamp and microfluorometric techniques. In physiological extracellular solution, GH(4) cells are highly sensitive to PlnA, but the sensitivity was dramatically reduced in solutions that partly neutralize the negative surface charge of the cells, in agreement with the notion that electrostatic interactions are probably important for the PlnA effects. Trypsination of cells prior to PlnA exposure also rendered the cells less sensitive to the peptide, suggesting that negative charges linked to membrane proteins are involved in the permeabilizing action. Finally, pre-exposure of cells to a mixture of enzymes that split carbohydrate residues from the backbone of glycosylated proteins also impeded the PlnA-induced membrane permeabilization. We conclude that electrostatic attraction between PlnA and glycosylated membrane proteins is probably an essential first step before PlnA can interact with membrane phospholipids. Deviating glycosylation patterns may contribute to the variation in PlnA sensitivity of different cell types, including cancerous cells and their normal counterparts. Copyright © 2012 Elsevier B.V. All rights reserved.

Protein secretion and surface display in Gram-positive bacteria

PubMed Central

Schneewind, Olaf; Missiakas, Dominique M.

2012-01-01

The cell wall peptidoglycan of Gram-positive bacteria functions as a surface organelle for the transport and assembly of proteins that interact with the environment, in particular, the tissues of an infected host. Signal peptide-bearing precursor proteins are secreted across the plasma membrane of Gram-positive bacteria. Some precursors carry C-terminal sorting signals with unique sequence motifs that are cleaved by sortase enzymes and linked to the cell wall peptidoglycan of vegetative forms or spores. The sorting signals of pilin precursors are cleaved by pilus-specific sortases, which generate covalent bonds between proteins leading to the assembly of fimbrial structures. Other precursors harbour surface (S)-layer homology domains (SLH), which fold into a three-pronged spindle structure and bind secondary cell wall polysaccharides, thereby associating with the surface of specific Gram-positive microbes. Type VII secretion is a non-canonical secretion pathway for WXG100 family proteins in mycobacteria. Gram-positive bacteria also secrete WXG100 proteins and carry unique genes that either contribute to discrete steps in secretion or represent distinctive substrates for protein transport reactions. PMID:22411983

Identification of novel direct protein-protein interactions by irradiating living cells with femtosecond UV laser pulses.

PubMed

Itri, Francesco; Monti, Daria Maria; Chino, Marco; Vinciguerra, Roberto; Altucci, Carlo; Lombardi, Angela; Piccoli, Renata; Birolo, Leila; Arciello, Angela

2017-10-07

The identification of protein-protein interaction networks in living cells is becoming increasingly fundamental to elucidate main biological processes and to understand disease molecular bases on a system-wide level. We recently described a method (LUCK, Laser UV Cross-linKing) to cross-link interacting protein surfaces in living cells by UV laser irradiation. By using this innovative methodology, that does not require any protein modification or cell engineering, here we demonstrate that, upon UV laser irradiation of HeLa cells, a direct interaction between GAPDH and alpha-enolase was "frozen" by a cross-linking event. We validated the occurrence of this direct interaction by co-immunoprecipitation and Immuno-FRET analyses. This represents a proof of principle of the LUCK capability to reveal direct protein interactions in their physiological environment. Copyright © 2017 Elsevier Inc. All rights reserved.

S-Layer Protein-Based Biosensors.

PubMed

Schuster, Bernhard

2018-04-11

The present paper highlights the application of bacterial surface (S-) layer proteins as versatile components for the fabrication of biosensors. One technologically relevant feature of S-layer proteins is their ability to self-assemble on many surfaces and interfaces to form a crystalline two-dimensional (2D) protein lattice. The S-layer lattice on the surface of a biosensor becomes part of the interface architecture linking the bioreceptor to the transducer interface, which may cause signal amplification. The S-layer lattice as ultrathin, highly porous structure with functional groups in a well-defined special distribution and orientation and an overall anti-fouling characteristics can significantly raise the limit in terms of variety and the ease of bioreceptor immobilization, compactness of bioreceptor molecule arrangement, sensitivity, specificity, and detection limit for many types of biosensors. The present paper discusses and summarizes examples for the successful implementation of S-layer lattices on biosensor surfaces in order to give a comprehensive overview on the application potential of these bioinspired S-layer protein-based biosensors.

Clustering of adhesion receptors following exposure of insect blood cells to foreign surfaces.

PubMed

Nardi, James B; Zhuang, Shufei; Pilas, Barbara; Bee, Charles Mark; Kanost, Michael R

2005-05-01

Cell-mediated immune responses of insects involve interactions of two main classes of blood cells (hemocytes) known as granular cells and plasmatocytes. In response to a foreign surface, these hemocytes suddenly transform from circulating, non-adherent cells to cells that interact and adhere to each other and the foreign surface. This report presents evidence that during this adhesive transformation the extracellular matrix (ECM) proteins lacunin and a ligand for peanut agglutinin (PNA) lectin are released by granular cells and bind to surfaces of both granular cells and plasmatocytes. ECM protein co-localizes on cell surfaces with the adhesive receptors integrin and neuroglian, a member of the immunoglobulin superfamily. The ECM protein(s) secreted by granular cells are hypothesized to interact with adhesion receptors such as neuroglian and integrin by cross linking and clustering them on hemocyte surfaces. This clustering of receptors is known to enhance the adhesiveness (avidity) of interacting mammalian immune cells. The formation of ring-shaped clusters of these adhesion receptors on surfaces of insect immune cells represents an evolutionary antecedent of the mammalian immunological synapse.

Improved diagnostic performance of a commercial anaplasma antibody competitive enzyme-linked immunosorbent assay using recombinant major surface protein 5–glutathione S-transferase fusion protein as antigen

USDA-ARS?s Scientific Manuscript database

This study tested the hypothesis that removal of maltose binding protein from recombinant antigen used for plate coating would improve the specificity of Anaplasma antibody competitive ELISA. Three hundred and eight sera with significant MBP antibody binding (=30%I) in Anaplasma negative herds was 1...

Novel electrospun nanofibers of modified gelatin-tyrosine in cartilage tissue engineering.

PubMed

Agheb, Maria; Dinari, Mohammad; Rafienia, Mohammad; Salehi, Hossein

2017-02-01

In natural cartilage tissues, chondrocytes are linked to extracellular matrix (ECM) through cell-surface binding proteins. Surface modification of gelatin can provide a new generation of biopolymers and fibrous scaffolds with chemical, mechanical, and biological properties. In this study tyrosine protein and 1,2,3-triazole ring were utilized to functionalize gelatin without Cu catalyst. Their molecular structure was characterized by Fourier transform infrared spectroscopy (FTIR) and nuclear magnetic resonance spectroscopy ( 1 HNMR). Chemical cross-linkers such as glutaraldehyde (GA) and 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC)/N-hydroxysulfosuccinimide (NHS) were used to electrospin the modified gelatin. The modification of gelatin and cross-linking effects were confirmed by scanning electron microscopy (SEM), contact angle measurement, and mechanical tests. MTT assay using chondrocyte cells showed cell viability of electrospun modified gelatin scaffolds. In vitro cell culture studies showed that electrospun engineered protein scaffolds would support the attachment and growth of cells. The results also showed that cross-linked nanofibers with EDC/NHS could be considered excellent matrices in cell adhesion and proliferation before electrospinning process and their potential substrate in tissue engineering applications, especially in the field of cartilage engineering. Copyright © 2016. Published by Elsevier B.V.

Myo1c regulates lipid raft recycling to control cell spreading, migration and Salmonella invasion

PubMed Central

Brandstaetter, Hemma; Kendrick-Jones, John; Buss, Folma

2012-01-01

A balance between endocytosis and membrane recycling regulates the composition and dynamics of the plasma membrane. Internalization and recycling of cholesterol- and sphingolipid-enriched lipid rafts is an actin-dependent process that is mediated by a specialized Arf6-dependent recycling pathway. Here, we identify myosin1c (Myo1c) as the first motor protein that drives the formation of recycling tubules emanating from the perinuclear recycling compartment. We demonstrate that the single-headed Myo1c is a lipid-raft-associated motor protein that is specifically involved in recycling of lipid-raft-associated glycosylphosphatidylinositol (GPI)-linked cargo proteins and their delivery to the cell surface. Whereas Myo1c overexpression increases the levels of these raft proteins at the cell surface, in cells depleted of Myo1c function through RNA interference or overexpression of a dominant-negative mutant, these tubular transport carriers of the recycling pathway are lost and GPI-linked raft markers are trapped in the perinuclear recycling compartment. Intriguingly, Myo1c only selectively promotes delivery of lipid raft membranes back to the cell surface and is not required for recycling of cargo, such as the transferrin receptor, which is mediated by parallel pathways. The profound defect in lipid raft trafficking in Myo1c-knockdown cells has a dramatic impact on cell spreading, cell migration and cholesterol-dependent Salmonella invasion; processes that require lipid raft transport to the cell surface to deliver signaling components and the extra membrane essential for cell surface expansion and remodeling. Thus, Myo1c plays a crucial role in the recycling of lipid raft membrane and proteins that regulate plasma membrane plasticity, cell motility and pathogen entry. PMID:22328521

Myo1c regulates lipid raft recycling to control cell spreading, migration and Salmonella invasion.

PubMed

Brandstaetter, Hemma; Kendrick-Jones, John; Buss, Folma

2012-04-15

A balance between endocytosis and membrane recycling regulates the composition and dynamics of the plasma membrane. Internalization and recycling of cholesterol- and sphingolipid-enriched lipid rafts is an actin-dependent process that is mediated by a specialized Arf6-dependent recycling pathway. Here, we identify myosin1c (Myo1c) as the first motor protein that drives the formation of recycling tubules emanating from the perinuclear recycling compartment. We demonstrate that the single-headed Myo1c is a lipid-raft-associated motor protein that is specifically involved in recycling of lipid-raft-associated glycosylphosphatidylinositol (GPI)-linked cargo proteins and their delivery to the cell surface. Whereas Myo1c overexpression increases the levels of these raft proteins at the cell surface, in cells depleted of Myo1c function through RNA interference or overexpression of a dominant-negative mutant, these tubular transport carriers of the recycling pathway are lost and GPI-linked raft markers are trapped in the perinuclear recycling compartment. Intriguingly, Myo1c only selectively promotes delivery of lipid raft membranes back to the cell surface and is not required for recycling of cargo, such as the transferrin receptor, which is mediated by parallel pathways. The profound defect in lipid raft trafficking in Myo1c-knockdown cells has a dramatic impact on cell spreading, cell migration and cholesterol-dependent Salmonella invasion; processes that require lipid raft transport to the cell surface to deliver signaling components and the extra membrane essential for cell surface expansion and remodeling. Thus, Myo1c plays a crucial role in the recycling of lipid raft membrane and proteins that regulate plasma membrane plasticity, cell motility and pathogen entry.

Calculating the mean time to capture for tethered ligands and its effect on the chemical equilibrium of bound ligand pairs

NASA Astrophysics Data System (ADS)

Shen, Lu; Decker, Caitlin; Maynard, Heather; Levine, Alex

Cells interact with a number of extracellular proteins including growth factors, which are essential for e.g., wound healing and development. Some of these growth factors must form dimers on the cell surface to initiate their signaling pathway. This suggests one can more efficiently induce signaling via polymer-linked proteins. Motivated by experiments on a family of fibroblast growth factors linked by polymers of varying molecular weight we investigate theoretically the effect of the length of the linking polymer on the binding kinetics of the dimers to a receptor-covered surface. We show, through a first-passage time calculation, how the number of bound dimers in chemical equilibrium depends on the linker molecular weight. We discuss more broadly the implications for a variety of signaling molecules. This work was supported by the NSF-DMR-1309188. HDM thanks the NIH NIBIB (R01EB013674) for support of the cell assay data.

Ferric reductase genes involved in high-affinity iron uptake are differentially regulated in yeast and hyphae of Candida albicans.

PubMed

Jeeves, Rose E; Mason, Robert P; Woodacre, Alexandra; Cashmore, Annette M

2011-09-01

The pathogenic yeast Candida albicans possesses a reductive iron uptake system which is active in iron-restricted conditions. The sequestration of iron by this mechanism initially requires the reduction of free iron to the soluble ferrous form, which is catalysed by ferric reductase proteins. Reduced iron is then taken up into the cell by a complex of a multicopper oxidase protein and an iron transport protein. Multicopper oxidase proteins require copper to function and so reductive iron and copper uptake are inextricably linked. It has previously been established that Fre10 is the major cell surface ferric reductase in C. albicans and that transcription of FRE10 is regulated in response to iron levels. We demonstrate here that Fre10 is also a cupric reductase and that Fre7 also makes a significant contribution to cell surface ferric and cupric reductase activity. It is also shown, for the first time, that transcription of FRE10 and FRE7 is lower in hyphae compared to yeast and that this leads to a corresponding decrease in cell surface ferric, but not cupric, reductase activity. This demonstrates that the regulation of two virulence determinants, the reductive iron uptake system and the morphological form of C. albicans, are linked. Copyright © 2011 John Wiley & Sons, Ltd.

Application of bioconjugation chemistry on biosensor fabrication for detection of TAR-DNA binding protein 43.

PubMed

Dai, Yifan; Wang, Chunlai; Chiu, Liang-Yuan; Abbasi, Kevin; Tolbert, Blanton S; Sauvé, Geneviève; Yen, Yun; Liu, Chung-Chiun

2018-06-01

A simple-prepare, single-use and cost-effective, in vitro biosensor for the detection of TAR DNA-binding protein 43 (TDP-43), a biomarker of neuro-degenerative disorders, was designed, manufactured and tested. This study reports the first biosensor application for the detection of TDP-43 using a novel biosensor fabrication methodology. Bioconjugation mechanism was applied by conjugating anti-TDP 43 with N-succinimidyl S-acetylthioacetate (SATA) producing a thiol-linked anti-TDP 43, which was used to directly link with gold electrode surface, minimizing the preparation steps for biosensor fabrication and simplifying the biosensor surface. The effectiveness of this bioconjugation mechanism was evaluated and confirmed by FqRRM12 protein, using nuclear magnetic resonance (NMR). The surface coverage of the electrode was analyzed by Time-of-Flight-Secondary Ion Mass Spectrometry (TOF-SIMS). Differential pulse voltammetry (DPV) was acted as the detection transduction mechanism with the use of [Fe(CN) 6 ] 3-/4- redox probe. Human TDP-43 peptide of 0.0005 µg/mL to 2 µg/mL in undiluted human serum was analyzed using this TDP-43 biosensor. Interference study of the TDP-43 biosensor using β-amyloid 42 protein and T-tau protein confirmed the specificity of this TDP-43 biosensor. This bioconjugation chemistry based approach for biosensor fabrication circumvents tedious gold surface modification and functionalization while enabling specific detection of TDP-43 in less than 1 h with a low fabrication cost of a single biosensor less than $3. Copyright © 2018 Elsevier B.V. All rights reserved.

Protein-Nanoparticle Interactions: Improving Immobilized Lytic Enzyme Activity and Surface Energy Effects

NASA Astrophysics Data System (ADS)

Downs, Emily Elizabeth

Protein-nanostructure conjugates, particularly particles, are a subject of significant interest due to changes in their fundamental behavior compared to bulk surfaces. As the size scale of nano-structured materials and proteins are on the same order of magnitude, nanomaterial properties can heavily influence how proteins adsorb and conform to the surface. Previous work has demonstrated the ability of nanoscale surfaces to modulate protein activity, conformation, and retention by modifying the particle surface curvature, morphology, and surface charge. This work has improved our understanding of the protein material interactions, but a complete understanding is still lacking. The goal of this thesis is to investigate two missing areas of understanding using two distinct systems. The first system utilizes a particle with controlled surface energy to observe the impact of surface energy on protein-particle interactions, while the second system uses a modified Listeria-specific protein to determine how protein structure and flexibility affects protein adsorption and activity on particles. Spherical, amorphous, and uniformly doped Zn-silica particles with tailored surface energies were synthesized to understand the impact of surface energy on protein adsorption behavior. Particle surface energy increased with a decrease in particle size and greater dopant concentrations. Protein adsorption and structural loss increased with both particle size and particle surface energy. Higher surface energies promoted protein-particle association and increased protein unfolding. Particle curvature and protein steric hindrance effects limited adsorption and structural loss on smaller particles. Protein surface charge heterogeneity was also found to be linked to both protein adsorption and unfolding behavior on larger particles. Greater surface charge heterogeneity led to higher adsorption concentrations and multilayer formation. These multilayers transitioned from protein-particle interactions to protein-protein interactions and were thicker with greater surface energy, which resulted in the recovery of secondary structure in the outermost layer. To help understand the impact of protein structure on nano-bio conjugate interactions, a listeria specific protein was used. This system was chosen as it has applications in the food industry in preventing bacterial contamination. The insertion of an amino acid linker between the enzymatic and binding domain of the protein improved the flexibility between domains, leading to increased adsorption, and improved activity in both cell-wall and plating assays. Additionally, linker modified protein incorporated into the silica-polymer nanocomposite showed significant activity in a real-world example of contaminated lettuce. This thesis study has isolated the impact of surface energy and protein flexibility on protein adsorption and structure. Particle surface energy affects adsorbed protein concentration and conformation. Coupled with protein surface charge, surface energy was also found to dictate multilayer thickness. The conformational flexibility of the protein was shown to help in controlling not only protein adsorption concentration but also in retaining protein activity after immobilization. Also, a controllable synthesis method for particles with adjustable surface energy, an ideal platform for studying protein-particle interactions, has been established.

Hotspots for allosteric regulation on protein surfaces

PubMed Central

Reynolds, Kimberly A.; McLaughlin, Richard N.; Ranganathan, Rama

2012-01-01

Recent work indicates a general architecture for proteins in which sparse networks of physically contiguous and co-evolving amino acids underlie basic aspects of structure and function. These networks, termed sectors, are spatially organized such that active sites are linked to many surface sites distributed throughout the structure. Using the metabolic enzyme dihydrofolate reductase as a model system, we show that (1) the sector is strongly correlated to a network of residues undergoing millisecond conformational fluctuations associated with enzyme catalysis and (2) sector-connected surface sites are statistically preferred locations for the emergence of allosteric control in vivo. Thus, sectors represent an evolutionarily conserved “wiring” mechanism that can enable perturbations at specific surface positions to rapidly initiate conformational control over protein function. These findings suggest that sectors enable the evolution of intermolecular communication and regulation. PMID:22196731

Probing the surface of a sweet protein: NMR study of MNEI with a paramagnetic probe

PubMed Central

Niccolai, Neri; Spadaccini, Roberta; Scarselli, Maria; Bernini, Andrea; Crescenzi, Orlando; Spiga, Ottavia; Ciutti, Arianna; Di Maro, Daniela; Bracci, Luisa; Dalvit, Claudio; Temussi, Piero A.

2001-01-01

The design of safe sweeteners is very important for people who are affected by diabetes, hyperlipemia, and caries and other diseases that are linked to the consumption of sugars. Sweet proteins, which are found in several tropical plants, are many times sweeter than sucrose on a molar basis. A good understanding of their structure–function relationship can complement traditional SAR studies on small molecular weight sweeteners and thus help in the design of safe sweeteners. However, there is virtually no sequence homology and very little structural similarity among known sweet proteins. Studies on mutants of monellin, the best characterized of sweet proteins, proved not decisive in the localization of the main interaction points of monellin with its receptor. Accordingly, we resorted to an unbiased approach to restrict the search of likely areas of interaction on the surface of a typical sweet protein. It has been recently shown that an accurate survey of the surface of proteins by appropriate paramagnetic probes may locate interaction points on protein surface. Here we report the survey of the surface of MNEI, a single chain monellin, by means of a paramagnetic probe, and a direct assessment of bound water based on an application of ePHOGSY, an NMR experiment that is ideally suited to detect interactions of small ligands to a protein. Detailed surface mapping reveals the presence, on the surface of MNEI, of interaction points that include residues previously predicted by ELISA tests and by mutagenesis. PMID:11468346

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

Fibrillar Structure and Charge Determine the Interaction of Polyglutamine Protein Aggregates with the Cell Surface*

PubMed Central

Trevino, R. Sean; Lauckner, Jane E.; Sourigues, Yannick; Pearce, Margaret M.; Bousset, Luc; Melki, Ronald; Kopito, Ron R.

2012-01-01

The pathogenesis of most neurodegenerative diseases, including transmissible diseases like prion encephalopathy, inherited disorders like Huntington disease, and sporadic diseases like Alzheimer and Parkinson diseases, is intimately linked to the formation of fibrillar protein aggregates. It is becoming increasingly appreciated that prion-like intercellular transmission of protein aggregates can contribute to the stereotypical spread of disease pathology within the brain, but the mechanisms underlying the binding and uptake of protein aggregates by mammalian cells are largely uninvestigated. We have investigated the properties of polyglutamine (polyQ) aggregates that endow them with the ability to bind to mammalian cells in culture and the properties of the cell surface that facilitate such uptake. Binding and internalization of polyQ aggregates are common features of mammalian cells and depend upon both trypsin-sensitive and trypsin-resistant saturable sites on the cell surface, suggesting the involvement of cell surface proteins in this process. polyQ aggregate binding depends upon the presence of a fibrillar amyloid-like structure and does not depend upon electrostatic interaction of fibrils with the cell surface. Sequences in the huntingtin protein that flank the amyloid-forming polyQ tract also influence the extent to which aggregates are able to bind to cell surfaces. PMID:22753412

Reversibly immobilized biological materials in monolayer films on electrodes

DOEpatents

Weaver, P.F.; Frank, A.J.

1993-05-04

Methods and techniques are described for reversibly binding charged biological particles in a fluid medium to an electrode surface. The methods are useful in a variety of applications. The biological materials may include microbes, proteins, and viruses. The electrode surface may consist of reversibly electroactive materials such as polyvinylferrocene, silicon-linked ferrocene or quinone.

Reversibly immobilized biological materials in monolayer films on electrodes

DOEpatents

Weaver, Paul F.; Frank, Arthur J.

1993-01-01

Methods and techniques are described for reversibly binding charged biological particles in a fluid medium to an electrode surface. The methods are useful in a variety of applications. The biological materials may include microbes, proteins, and viruses. The electrode surface may consist of reversibly electroactive materials such as polyvinylferrocene, silicon-linked ferrocene or quinone.

Protein immobilization on the surface of polydimethylsiloxane and polymethyl methacrylate microfluidic devices.

PubMed

Khnouf, Ruba; Karasneh, Dina; Albiss, Borhan Aldeen

2016-02-01

PDMS and PMMA are two of the most used polymers in the fabrication of lab-on-chip or microfluidic devices. In order to use these polymers in biological applications, it is sometimes essential to be able to bind biomolecules such as proteins and DNA to the surface of these materials. In this work, we have evaluated a number of processes that have been developed to bind protein to PDMS surfaces which include passive adsorption, passive adsorption with glutaraldehyde cross-linking, (3-aminopropyl) triethoxysilane functionalization followed by glutaraldehyde or 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride cross-linkers. It has been shown that the latter technique--using 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride--results in more than twice the bonding of protein to the surface of PDMS microchannels than proteins binding passively. We have also evaluated a few techniques that have been tested for the functionalization of PMMA microchannels where we have found that the use of polyethyleneimine (PEI) has led to the strongest protein-PMMA microchannel bond. We finally demonstrated the effect of PDMS curing methodology on protein adsorption to its surface, and showed that increased curing time is the factor that reduces passive adsorption the most. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Gelation Mechanisms and Characterization of Electrochemically Generated Protein Films at Metal Interfaces

NASA Astrophysics Data System (ADS)

Martin, Elizabeth J.

Although the electrochemical behavior of metals used in orthopedic implants has been studied extensively, the material interactions with proteins during corrosion processes remains poorly understood. Some studies suggest that metal-protein interactions accelerate corrosion, while others suggest that proteins protect the material from degradation. Corrosion of implant materials is a major concern due to the metal ion release that can sometimes cause adverse local tissue reactions and ultimately, failure of the implant. The initial purpose of this research was therefore to study the corrosion behavior of CoCrMo, an alloy commonly used in hip replacements, with a quartz crystal microbalance (QCM) in physiologically relevant media. The QCM enables in situ characterization of surface changes accompanying corrosion and is sensitive to viscoelastic effects at its surface. Results of QCM studies in proteinaceous media showed film deposition on the alloy surface under electrochemical conditions that otherwise produced mass loss if proteins were not present in the electrolyte. Additional studies on pure Co, Cr, and Mo demonstrated that the protein films also form on Mo surfaces after a release of molybdate ions, suggesting that these ions are essential for film formation. The electrochemically generated protein films are reminiscent of carbonaceous films that form on implant surfaces in vivo, therefore a second goal of the research was to delineate mechanisms that cause the films to form. In the second stage of this research, electrochemical QCM tests were conducted on models of the CoCrMo system consisting of Cr electrodes in proteinaceous or polymeric media containing dissolved molybdate ions. Studies indicated that films can be generated through electrochemical processes so long as both amine functional groups and molybdate ions are present in the electrolyte solution. These results suggest that the films form due to an ionic cross-linking reaction between the positively charged amine groups in the proteins and the negatively charged molybdate ions. Results also indicated that film generation is controlled by the potential at the electrode surface. Numerical analysis on the model systems suggest that a drop in the local pH at the corroding electrode surface may influence film generation, but a critical concentration of molybdate-amine cross-links must be exceeded for gels to form. A final goal of this research was to develop a technique to characterize the viscoelastic properties of polymer films in liquid media using the QCM as a high-frequency rheometer. The work showed that by measuring frequency and dissipation shifts at multiple harmonics of the QCM resonant frequency, the viscoelastic phase angle, density-modulus product, and areal mass of a film submersed in liquid can be quantified in situ. The method was successfully applied to characterize the electrochemically generated protein films. Results implied that the films are composed of a weakly cross-linked network with properties similar to concentrated albumin solutions containing 40 wt% protein. The analysis technique can be extended to characterize any polymer film in a liquid environment, with applications including adsorption, self-assembly, or cell-substrate interactions.

Structural and Functional Characterization of an Ancient Bacterial Transglutaminase Sheds Light on the Minimal Requirements for Protein Cross-Linking.

PubMed

Fernandes, Catarina G; Plácido, Diana; Lousa, Diana; Brito, José A; Isidro, Anabela; Soares, Cláudio M; Pohl, Jan; Carrondo, Maria A; Archer, Margarida; Henriques, Adriano O

2015-09-22

Transglutaminases are best known for their ability to catalyze protein cross-linking reactions that impart chemical and physical resilience to cellular structures. Here, we report the crystal structure and characterization of Tgl, a transglutaminase from the bacterium Bacillus subtilis. Tgl is produced during sporulation and cross-links the surface of the highly resilient spore. Tgl-like proteins are found only in spore-forming bacteria of the Bacillus and Clostridia classes, indicating an ancient origin. Tgl is a single-domain protein, produced in active form, and the smallest transglutaminase characterized to date. We show that Tgl is structurally similar to bacterial cell wall endopeptidases and has an NlpC/P60 catalytic core, thought to represent the ancestral unit of the cysteine protease fold. We show that Tgl functions through a unique partially redundant catalytic dyad formed by Cys116 and Glu187 or Glu115. Strikingly, the catalytic Cys is insulated within a hydrophobic tunnel that traverses the molecule from side to side. The lack of similarity of Tgl to other transglutaminases together with its small size suggests that an NlpC/P60 catalytic core and insulation of the active site during catalysis may be essential requirements for protein cross-linking.

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.

Cyclic Nucleotide-gated Channel α-3 (CNGA3) Interacts with Stereocilia Tip-Link Cadherin 23 + Exon 68 or Alternatively with Myosin VIIa, Two Proteins Required for Hair Cell Mechanotransduction*

PubMed Central

Selvakumar, Dakshnamurthy; Drescher, Marian J.; Drescher, Dennis G.

2013-01-01

Previously, we obtained evidence for a photoreceptor/olfactory type of CNGA3 transcript in a purified teleost vestibular hair cell preparation with immunolocalization of CNGA3 protein to stereocilia of teleost vestibular and mammalian cochlear hair cells. The carboxyl terminus of highly Ca2+-permeable CNGA3 expressed in the mammalian organ of Corti and saccular hair cells was found to interact with an intracellular domain of microfibril interface-located protein 1 (EMILIN 1), a member of the elastin superfamily, also immunolocalizd to hair cell stereocilia (Selvakumar, D., Drescher, M. J., Dowdall, J. R., Khan, K. M., Hatfield, J. S., Ramakrishnan, N. A., and Drescher, D. G. (2012) Biochem. J. 443, 463–476). Here, we provide evidence for organ of Corti proteins, of Ca2+-dependent binding of the amino terminus of CNGA3 specifically to the carboxyl terminus of stereocilia tip-link protein CDH23 +68 (cadherin 23 with expressed exon 68) by yeast two-hybrid mating and co-transformation protocols, pulldown assays, and surface plasmon resonance analysis. Myosin VIIa, required for adaptation of hair cell mechanotransduction (MET) channel(s), competed with CDH23 +68, with direct Ca2+-dependent binding to the amino terminus of CNGA3. Based upon the premise that hair cell stereocilia tip-link proteins are closely coupled with MET, these results are consistent with the possibility that CNGA3 participates in hair-cell MET. Together with the demonstration of protein-protein interaction between HCN1 and tip-link protein protocadherin 15 CD3 (Ramakrishnan, N. A., Drescher, M. J., Barretto, R. L., Beisel, K. W., Hatfield, J. S., and Drescher, D. G. (2009) J. Biol. Chem. 284, 3227–3238; Ramakrishnan, N. A., Drescher, M. J., Khan, K. M., Hatfield, J. S., and Drescher, D. G. (2012) J. Biol. Chem. 287, 37628–37646), a protein-protein interaction for CNGA3 and a second tip-link protein, CDH23 +68, further suggests possible association of two different channels with a single stereocilia tip link. PMID:23329832

Cyclic nucleotide-gated channel α-3 (CNGA3) interacts with stereocilia tip-link cadherin 23 + exon 68 or alternatively with myosin VIIa, two proteins required for hair cell mechanotransduction.

PubMed

Selvakumar, Dakshnamurthy; Drescher, Marian J; Drescher, Dennis G

2013-03-08

Previously, we obtained evidence for a photoreceptor/olfactory type of CNGA3 transcript in a purified teleost vestibular hair cell preparation with immunolocalization of CNGA3 protein to stereocilia of teleost vestibular and mammalian cochlear hair cells. The carboxyl terminus of highly Ca(2+)-permeable CNGA3 expressed in the mammalian organ of Corti and saccular hair cells was found to interact with an intracellular domain of microfibril interface-located protein 1 (EMILIN 1), a member of the elastin superfamily, also immunolocalizd to hair cell stereocilia (Selvakumar, D., Drescher, M. J., Dowdall, J. R., Khan, K. M., Hatfield, J. S., Ramakrishnan, N. A., and Drescher, D. G. (2012) Biochem. J. 443, 463-476). Here, we provide evidence for organ of Corti proteins, of Ca(2+)-dependent binding of the amino terminus of CNGA3 specifically to the carboxyl terminus of stereocilia tip-link protein CDH23 +68 (cadherin 23 with expressed exon 68) by yeast two-hybrid mating and co-transformation protocols, pulldown assays, and surface plasmon resonance analysis. Myosin VIIa, required for adaptation of hair cell mechanotransduction (MET) channel(s), competed with CDH23 +68, with direct Ca(2+)-dependent binding to the amino terminus of CNGA3. Based upon the premise that hair cell stereocilia tip-link proteins are closely coupled with MET, these results are consistent with the possibility that CNGA3 participates in hair-cell MET. Together with the demonstration of protein-protein interaction between HCN1 and tip-link protein protocadherin 15 CD3 (Ramakrishnan, N. A., Drescher, M. J., Barretto, R. L., Beisel, K. W., Hatfield, J. S., and Drescher, D. G. (2009) J. Biol. Chem. 284, 3227-3238; Ramakrishnan, N. A., Drescher, M. J., Khan, K. M., Hatfield, J. S., and Drescher, D. G. (2012) J. Biol. Chem. 287, 37628-37646), a protein-protein interaction for CNGA3 and a second tip-link protein, CDH23 +68, further suggests possible association of two different channels with a single stereocilia tip link.

Mechanics of Lamellipodia

NASA Astrophysics Data System (ADS)

Quint, D. A.; Schwarz, J. M.

2008-03-01

The actin cytoskeleton is a morphologically-complex assembly of cross-linked F-actin filaments. The cytoskeleton provides rigidity for the cell within appropriate time scales so that it can change its shape to, for example, crawl along surfaces. In addition to cross-linking proteins, many other proteins are involved in the assembly of the actin cytoskeleton such as branching proteins, capping proteins, and severing proteins. Presumably these proteins work cooperatively toward the dynamic formation of rigidity. We will initially focus on the role of branching proteins. The F-actin filaments in lamellipodia---protrusions of the mobile edge of a crawling cell---have some overall orientation due to the branching. Branched filaments emerge at a 70 degree angle from the mother filament's growing end.^1 This overall orientation is modelled as an anisotropy in an effective medium theory determining the cytoskeleton's elasticity in the static regime. The potential for a splay rigid phase, in addition to a rigid phase, is also investigated. ^1T. M. Svitkina and G. G. Borisy, J. Cell Biol. 145, 1009 (1999).

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.

Insolubility and redistribution of GPI-anchored proteins at the cell surface after detergent treatment.

PubMed Central

Mayor, S; Maxfield, F R

1995-01-01

A diverse set of cell surface eukaryotic proteins including receptors, enzymes, and adhesion molecules have a glycosylphosphoinositol-lipid (GPI) modification at the carboxy-terminal end that serves as their sole means of membrane anchoring. These GPI-anchored proteins are poorly solubilized in nonionic detergent such as Triton X-100. In addition these detergent-insoluble complexes from plasma membranes are significantly enriched in several cytoplasmic proteins including nonreceptor-type tyrosine kinases and caveolin/VIP-21, a component of the striated coat of caveolae. These observations have suggested that the detergent-insoluble complexes represent purified caveolar membrane preparations. However, we have recently shown by immunofluorescence and electron microscopy that GPI-anchored proteins are diffusely distributed at the cell surface but may be enriched in caveolae only after cross-linking. Although caveolae occupy only a small fraction of the cell surface (< 4%), almost all of the GPI-anchored protein at the cell surface becomes incorporated into detergent-insoluble low-density complexes. In this paper we show that upon detergent treatment the GPI-anchored proteins are redistributed into a significantly more clustered distribution in the remaining membranous structures. These results show that GPI-anchored proteins are intrinsically detergent-insoluble in the milieu of the plasma membrane, and their co-purification with caveolin is not reflective of their native distribution. These results also indicate that the association of caveolae, GPI-anchored proteins, and signalling proteins must be critically re-examined. Images PMID:7579703

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

PubMed

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

2018-01-23

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

Biotin-transfer from a trifunctional crosslinker for identification of cell surface receptors of soluble protein ligands

PubMed Central

Tremblay, Tammy-Lynn; Hill, Jennifer J.

2017-01-01

Here we describe a novel crosslinker and its application as a biotin-transfer reagent to identify cell surface receptors of soluble protein ligands on live cells. This crosslinker contains three functional groups: an aldehyde-reactive aminooxy group, a sulfhydryl, and a biotin (ASB). It is readily synthesized via a 3-step addition reaction using standard solid-phase peptide synthesis methods and commercially available intermediates, allowing access to laboratories without specialized synthetic chemistry capabilities. For the biotin-transfer method, ASB is linked to a protein ligand through the sulfhydryl group in a two-step process that allows the introduction of a disulfide bond between the ligand and the crosslinker. Incubation of the labelled ligand with oxidized live cells leads to the formation of crosslinks with aldehyde-containing glycans on the cell surface receptor. Subsequent reduction of the disulfide bond results in biotin transfer from the ligand to the cell surface receptor. Protein biotinylation that is mediated by ligand binding to its receptor is differentiated from background biotinylation events by comparison with a similarly labelled control protein using comparative proteomic mass spectrometry to quantify streptavidin-bound proteins. Using this method, we successfully identified the cell surface receptors of a peptide hormone, a monoclonal antibody, and a single-domain antibody-Fc fusion construct. PMID:28422167

Construction of a Functional S-Layer Fusion Protein Comprising an Immunoglobulin G-Binding Domain for Development of Specific Adsorbents for Extracorporeal Blood Purification

PubMed Central

Völlenkle, Christine; Weigert, Stefan; Ilk, Nicola; Egelseer, Eva; Weber, Viktoria; Loth, Fritz; Falkenhagen, Dieter; Sleytr, Uwe B.; Sára, Margit

2004-01-01

The chimeric gene encoding a C-terminally-truncated form of the S-layer protein SbpA from Bacillus sphaericus CCM 2177 and two copies of the Fc-binding Z-domain was constructed, cloned, and heterologously expressed in Escherichia coli HMS174(DE3). The Z-domain is a synthetic analogue of the B-domain of protein A, capable of binding the Fc part of immunoglobulin G (IgG). The S-layer fusion protein rSbpA31-1068/ZZ retained the specific properties of the S-layer protein moiety to self-assemble in suspension and to recrystallize on supports precoated with secondary cell wall polymer (SCWP), which is the natural anchoring molecule for the S-layer protein in the bacterial cell wall. Due to the construction principle of the S-layer fusion protein, the ZZ-domains remained exposed on the outermost surface of the protein lattice. The binding capacity of the native or cross-linked monolayer for human IgG was determined by surface plasmon resonance measurements. For batch adsorption experiments, 3-μm-diameter, biocompatible cellulose-based, SCWP-coated microbeads were used for recrystallization of the S-layer fusion protein. In the case of the native monolayer, the binding capacity for human IgG was 5.1 ng/mm2, whereas after cross-linking with dimethyl pimelimidate, 4.4 ng of IgG/mm2 was bound. This corresponded to 78 and 65% of the theoretical saturation capacity of a planar surface for IgGs aligned in the upright position, respectively. Compared to commercial particles used as immunoadsorbents to remove autoantibodies from sera of patients suffering from an autoimmune disease, the IgG binding capacity of the S-layer fusion protein-coated microbeads was at least 20 times higher. For that reason, this novel type of microbeads should find application in the microsphere-based detoxification system. PMID:15006773

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.

Processing and surface modification of novel natural-origin architectures aimed for biomedical applications

NASA Astrophysics Data System (ADS)

Silva, Simone dos Santos

In the last decades, tissue engineering has emerged as a potential therapeutical tool aimed at developing substitutes that are able to restore proper function of the damaged organs/tissues. Nature-inspired routes involving natural origin polymer-based systems represent an attractive alternative to produce novel materials by mimicking the tissue environment required for tissue regeneration. Moreover, further modifications of these systems allow the adjustment of their properties in accordance with the requirements for successful biomedical applications. The main goal of the present thesis is to develop and modify natural origin polymer-based systems using simple methodologies such as sol-gel, surface modification by means of plasma treatment and blending of chitosan with proteins (soy protein isolate and silk fibroin). A sol-gel method was used to improve the bulk properties of chitosan by the incorporation of an inorganic component at the sub-nanometric level. Chitosan/siloxane hybrid materials were synthesised, where essentially urea bridges covalently bond the chitosan to the polysiloxane network. These bifunctional materials exhibit interesting photoluminescence features and a bioactive behaviour. In most situations in the biomedical field, the surface of a biomaterial is in direct contact with living tissues. Therefore, the surface characteristics play a fundamental role on the implant biocompatibility. In this thesis, nitrogen and argon plasma treatment was applied on chitosan membranes in order to improve their surface properties. The applied modifications promoted differences on surface chemistry, wettability and roughness, which reflected in a significant improvement of fibroblast adhesion and proliferation onto chitosan membranes. Besides the surface modification, blending of chitosan with proteins such as soy protein isolate and silk fibroin was also used to modify the bulk properties of chitosan. In situ cross-linking with glutaraldehyde solutions was used to enhance the interaction between the components of the blend. Hence, membranes with different morphologies, water absorption and degradability were obtained. The biological assays suggested that the cross-linking with lower glutaraldehyde concentration promotes better cell adhesion on the membranes. The morphological characterization showed that both surface roughness surface and surface energy were dependent on soy protein content. Structural investigations by FTIR and NMR indicated that the blends are not completely miscible due to a weak polysaccharide-protein interaction. In another related work, novel hydrogels were produced combining Bombyx mori silk fibroin and chitosan. In this case, these systems were cross-linked with genipin. These hydrogels were freeze dried to obtain cross-linked chitosan/silk sponges. Rheological and mechanical properties, structural aspects and morphological features of the porous structures were evaluated. The results revealed stable and ordered structures, similar porosities, and swelling capability that depended on the pH. The cytotoxicity assay indicated that cellular viability was about 100% in all sponges and for all time points studied (1, 3, 7 and 14 days), demonstrating the extremely low cytotoxicity levels of the materials. Cell studies using chondrocytes-like cells seeded onto sponges, including cell viability (MTS assay), proliferation (DNA test), morphology (SEM analysis) and matrix production (GAGs quantification), showed a significant high adhesion, proliferation and matrix production with the time of culture. The findings in this work suggested that the properties of the sponges can be manipulated by either change chitosan/silk fibroin ratio or through genipin cross-linking. Parallel to this study, the possibility of obtaining modified silk nanometric nets using electrospinning processing from regenerated silk fibroin/formic acid with addition of genipin was explored. Modified silk nanofibers with diameters ranging from 140 nm to 590 nm were developed. The changes on the secondary structure of nanofibers, induced by the reaction of silk fibroin with genipin, promoted a higher integrity of these modified nanofibers in water. In summary, the findings from these works demonstrated the potential and versatility of the proposed strategies in obtaining different structures (e.g. membranes, hydrogels) using mixtures of chitosan with proteins or with inorganic agents for improving the performance of natural origin polymer-based materials to be used in biomedical applications.

Photoreactive, core-shell cross-linked/hollow microspheres prepared by delayed addition of cross-linker in dispersion polymerization for antifouling and immobilization of protein.

PubMed

Wang, Shengliu; Yue, Kai; Liu, Lianying; Yang, Wantai

2013-01-01

When dispersion polymerization of styrene (St) had run for 3h, after particle rapidly growing stage, 4,4'-dimethacryloyloxybenzophenone (DMABP) cross-linker was added to reaction system and photoreactive, core(PSt)-shell(Poly(St-co-DMABP)) particles with rich benzophenone (BP) groups on surface were prepared. Polymerization of DMABP could occurred mainly on the preformed core of PSt because its diffusion could be impeded by (1) compactness of particles formed at the moment of cross-linker addition (more than 80% of monomer had been consumed, particles were no longer fully swollen by monomer), (2) reduced polarity of continuous phase, and (3) immediate occurrence of cross-linking. Subsequently, photoreactive, cross-linked hollow particles were yielded by removal of uncross-linked core in THF. SEM and TEM observation demonstrated the formation of core-shell structure and improvement of shell thickness when DMABP content increased. UV-vis spectra analysis on polymer dissolved in THF indicated that there is no polymer of DMABP in core. FTIR spectra analysis and XPS measurement further revealed that BP component on particle surface was enriched when amount of DMABP increased. Finally, an anti-fouling polymer (poly (ethylene glycol), PEG) and protein of mouse IgG was immobilized on particle surface under UV irradiation, as confirmed by FTIR spectra analysis, SEM observation and TMB color reaction. Crown Copyright © 2012. Published by Elsevier Inc. All rights reserved.

Investigating the Link between Molecular Subtypes of Glioblastoma, Epithelial-Mesenchymal Transition, and CD133 Cell Surface Protein

PubMed Central

Zarkoob, Hadi; Taube, Joseph H.; Singh, Sheila K.; Mani, Sendurai A.; Kohandel, Mohammad

2013-01-01

In this manuscript, we use genetic data to provide a three-faceted analysis on the links between molecular subclasses of glioblastoma, epithelial-to-mesenchymal transition (EMT) and CD133 cell surface protein. The contribution of this paper is three-fold: First, we use a newly identified signature for epithelial-to-mesenchymal transition in human mammary epithelial cells, and demonstrate that genes in this signature have significant overlap with genes differentially expressed in all known GBM subtypes. However, the overlap between genes up regulated in the mesenchymal subtype of GBM and in the EMT signature was more significant than other GBM subtypes. Second, we provide evidence that there is a negative correlation between the genetic signature of EMT and that of CD133 cell surface protein, a putative marker for neural stem cells. Third, we study the correlation between GBM molecular subtypes and the genetic signature of CD133 cell surface protein. We demonstrate that the mesenchymal and neural subtypes of GBM have the strongest correlations with the CD133 genetic signature. While the mesenchymal subtype of GBM displays similarity with the signatures of both EMT and CD133, it also exhibits some differences with each of these signatures that are partly due to the fact that the signatures of EMT and CD133 are inversely related to each other. Taken together these data shed light on the role of the mesenchymal transition and neural stem cells, and their mutual interaction, in molecular subtypes of glioblastoma multiforme. PMID:23734191

Adsorption and bioactivity studies of albumin onto hydroxyapatite surface.

PubMed

Mavropoulos, Elena; Costa, Andréa M; Costa, Lilian T; Achete, Carlos A; Mello, Alexandre; Granjeiro, José M; Rossi, Alexandre M

2011-03-01

Bovine serum albumin (BSA) may have an inhibitory or promoter effect on hydroxyapatite (HA) nucleation when apatite is precipitated in a medium containing the protein. In this study we evaluated the influence of BSA on the precipitation of calcium phosphate phases (CP) from simulated body fluid (SBF) when the protein was previously bounded to HA surface. The kinetics of BSA immobilization onto hydroxyapatite surface was performed in different buffers and protein concentrations in order to adjust experimental conditions in which BSA was tightly linked to HA surface for long periods in SBF solution. It was shown that for BSA concentration higher than 0.1mg/mL the adsorption to HA surface followed Langmuir-Freundlich mechanisms, which confirmed the existence of cooperative protein-protein interactions on HA surface. Fourier Transformed Infrared Attenuated Total Reflectance Microscopy (FTIRM-ATR) evidenced changes in BSA conformational state in favor of less-ordered structure. Analyses from high resolution grazing incident X-ray diffraction using synchrotron radiation (GIXRD), Scanning Electron Microscopy (SEM) and Atomic Force Microscopy (AFM) showed that a poorly crystalline calcium phosphate was precipitated on the surface of HA discs coated with BSA, after the immersion in SBF for 4 days. The new bioactive layer had morphological characteristics similar to the one formed on the HA surface without protein. It was identified as a carbonated apatite with preferential crystal growth along apatite 002 direction. The GIXRD results also revealed that BSA layer bound to the surface inhibited the HA dissolution leading to a reduction on the formation of new calcium phosphate phase. 2010 Elsevier B.V. All rights reserved.

Heat transfer and vascular cambium necrosis in the boles of trees during surface fires

Treesearch

M. B. Dickinson

2002-01-01

Heat-transfer and cell-survival models are used to link surface fire behavior with vascular cambium necrosis from heating by flames. Vascular cambium cell survival was predicted with a numerical model based on the kinetics of protein denaturation and parameterized with data from the literature. Cell survival was predicted for vascular cambium temperature regimes...

Identification of a Linked Set of Genes in Serpulina hyodysenteriae (B204) Predicted To Encode Closely Related 39-Kilodalton Extracytoplasmic Proteins

PubMed Central

Gabe, Jeffrey D.; Dragon, Elizabeth; Chang, Ray-Jen; McCaman, Michael T.

1998-01-01

A tandem pair of nearly identical genes from Serpulina hyodysenteriae (B204) were cloned and sequenced. The full open reading frame of one gene and the partial open reading frame of the neighboring gene appear to encode secreted proteins which are homologous to, yet distinct from, the 39-kDa extracytoplasmic protein purified from the membrane fraction of S. hyodysenteriae. We have designated these newly identified genes vspA and vspB (for variable surface protein). PMID:9440540

Surfaceome and Proteosurfaceome in Parietal Monoderm Bacteria: Focus on Protein Cell-Surface Display

PubMed Central

Desvaux, Mickaël; Candela, Thomas; Serror, Pascale

2018-01-01

The cell envelope of parietal monoderm bacteria (archetypal Gram-positive bacteria) is formed of a cytoplasmic membrane (CM) and a cell wall (CW). While the CM is composed of phospholipids, the CW is composed at least of peptidoglycan (PG) covalently linked to other biopolymers, such as teichoic acids, polysaccharides, and/or polyglutamate. Considering the CW is a porous structure with low selective permeability contrary to the CM, the bacterial cell surface hugs the molecular figure of the CW components as a well of the external side of the CM. While the surfaceome corresponds to the totality of the molecules found at the bacterial cell surface, the proteinaceous complement of the surfaceome is the proteosurfaceome. Once translocated across the CM, secreted proteins can either be released in the extracellular milieu or exposed at the cell surface by associating to the CM or the CW. Following the gene ontology (GO) for cellular components, cell-surface proteins at the CM can either be integral (GO: 0031226), i.e., the integral membrane proteins, or anchored to the membrane (GO: 0046658), i.e., the lipoproteins. At the CW (GO: 0009275), cell-surface proteins can be covalently bound, i.e., the LPXTG-proteins, or bound through weak interactions to the PG or wall polysaccharides, i.e., the cell wall binding proteins. Besides monopolypeptides, some proteins can associate to each other to form supramolecular protein structures of high molecular weight, namely the S-layer, pili, flagella, and cellulosomes. After reviewing the cell envelope components and the different molecular mechanisms involved in protein attachment to the cell envelope, perspectives in investigating the proteosurfaceome in parietal monoderm bacteria are further discussed. PMID:29491848

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

PubMed

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

2016-03-29

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

Role of Mitochondria in Prostate Cancer

DTIC Science & Technology

2005-12-01

outer surface of the inner mitochondrial membrane. This enzyme acts in concert with the cytoplasmic NAD-linked glycerophosphate dehydrogenase (cGPDH...dependent ROS production (Fig. 4). This observation may be attributable to the absence of a CoQ -binding protein in mGPDH. This CoQ -binding protein...evidently has a natural protection of ubisemiquinone formed during CoQ reduction by succinate dehydrogenase [28]. Evaluation of cell proliferation

Binding-, intracellular transport-, and biosynthesis-defective mutants of vasopressin type 2 receptor in patients with X-linked nephrogenic diabetes insipidus.

PubMed Central

Tsukaguchi, H; Matsubara, H; Taketani, S; Mori, Y; Seido, T; Inada, M

1995-01-01

Nephrogenic diabetes insipidus (NDI) is most often an X-linked disorder in which urine is not concentrated due to renal resistance to arginine vasopressin. We recently identified four vasopressin type 2 receptor gene mutations in unrelated X-linked NDI families, including R143P, delta V278, R202C, and 804insG. All these mutations reduced ligand binding activity to < 10% of the normal without affecting mRNA accumulation. To elucidate whether the receptors are expressed on the cell surface, we analyzed biosynthesis and localization of tagged or untagged receptors stably expressed in Chinese hamster ovary (CHO) cells, using two antibodies directed against distinct termini. Whole-cell and surface labeling studies revealed that the R202C clone had both surface-localized (50-55 kD) and intracellular proteins (40 and 75 kD), similar to the wild-type AVPR2 clone, whereas the R143P and delta V278 clones lacked the surface receptors, despite relatively increased intracellular components. The 804insG mutant cell produced no proteins despite an adequate mRNA level. Immunofluorescence staining confirmed that the R202C mutant reaches the cell surface, whereas the R143P and delta V278 mutants are retained within the cytoplasmic compartment. Thus, R202C, R143P/delta V278, and 804insG result in three distinct phenotypes, that is, a simple binding impairment at the cell surface, blocked intracellular transport, and ineffective biosynthesis or/and accelerated degradation of the receptor, respectively, and therefore are responsible for NDI. This phenotypic classification will help understanding of molecular pathophysiology of this disorder. Images PMID:7560098

Mild Oxidation Promotes and Advanced Oxidation Impairs Remodeling of Human High-Density Lipoprotein in vitro

PubMed Central

Gao, Xuan; Jayaraman, Shobini; Gursky, Olga

2008-01-01

SUMMARY High-density lipoproteins (HDL) prevent atherosclerosis by removing cholesterol from macrophages and by exerting anti-oxidant and anti-inflammatory effects. Oxidation is thought to impair HDL functions, yet certain oxidative modifications may be advantageous; thus, mild oxidation reportedly enhances cell cholesterol uptake by HDL whereas extensive oxidation impairs it. To elucidate the underlying energetic and structural basis, we analyzed the effects of copper and hypochlorite (that preferentially oxidize lipids and proteins, respectively) on thermal stability of plasma spherical HDL. Circular dichroism, light scattering, calorimetry, gel electrophoresis and electron microscopy showed that mild oxidation destabilizes HDL and accelerates protein dissociation and lipoprotein fusion, while extensive oxidation inhibits these reactions; this inhibition correlates with massive protein cross-linking and lipolysis. We propose that mild oxidation lowers kinetic barriers for HDL remodeling due to diminished apolipoprotein affinity for lipids resulting from oxidation of methionine and aromatic residues in apolipoproteins A-I and A-II followed by protein cross-linking into dimers and/or trimers. In contrast, advanced oxidation inhibits protein dissociation and HDL fusion due to lipid re-distribution from core to surface upon lipolysis and to massive protein cross-linking. Our results help reconcile the apparent controversy in the studies of oxidized HDL and suggest that mild oxidation may benefit HDL functions. PMID:18190928

Catechol chemistry inspired approach to construct self-cross-linked polymer nanolayers as versatile biointerfaces.

PubMed

Liu, Xinyue; Deng, Jie; Ma, Lang; Cheng, Chong; Nie, Chuanxiong; He, Chao; Zhao, Changsheng

2014-12-16

In this study, we proposed a catechol chemistry inspired approach to construct surface self-cross-linked polymer nanolayers for the design of versatile biointerfaces. Several representative biofunctional polymers, P(SS-co-AA), P(SBMA-co-AA), P(EGMA-co-AA), P(VP-co-AA), and P(MTAC-co-AA), were first synthesized via reversible addition-fragmentation chain transfer (RAFT) polymerization, and then the catecholic molecules (dopamine, DA) were conjugated to the acrylic acid (AA) units by the facile carbodiimide chemistry. Then, the catechol (Cat) group conjugated biofunctional polymers, named PSS-Cat, PSBMA-Cat, PEGMA-Cat, PVP-Cat, and PMTAC-Cat, were applied for the construction of self-cross-linked nanolayers on polymeric substrates via the pH induced catechol cross-linking and immobilization. The XPS spectra, surface morphology, and wettability gave robust evidence that the catechol conjugated polymers were successfully coated, and the coated substrates possessed increased surface roughness and hydrophilicity. Furthermore, the systematic in vitro investigation of protein adsorption, platelet adhesion, activated partial thromboplastin time (APTT), thrombin time (TT), cell viability, and antibacterial ability confirmed that the coated nanolayers conferred the substrates with versatile biological performances. The PSS-Cat coated substrate had low blood component activation and excellent anticoagulant activity; while the PEGMA-Cat and PSBMA-Cat showed ideal resistance to protein fouling and inhibition of platelet activation. The PSS-Cat and PVP-Cat coated substrates exhibited promoted endothelial cell proliferation and viability. The PMTAC-Cat coated substrate showed an outstanding activity on bacterial inhibition. In conclusion, the catechol chemistry inspired approach allows the self-cross-linked nanolayers to be easily immobilized on polymeric substrates with the stable conformation and multiple biofunctionalities. It is expected that this low-cost and facile bioinspired coating system will present great potential in creating novel and versatile biointerfaces.

A Simultaneously Antimicrobial, Protein-Repellent, and Cell-Compatible Polyzwitterion Network.

PubMed

Kurowska, Monika; Eickenscheidt, Alice; Guevara-Solarte, Diana-Lorena; Widyaya, Vania Tanda; Marx, Franziska; Al-Ahmad, Ali; Lienkamp, Karen

2017-04-10

A simultaneously antimicrobial, protein-repellent, and cell-compatible surface-attached polymer network is reported, which reduces the growth of bacterial biofilms on surfaces through its multifunctionality. The coating was made from a poly(oxonorbornene)-based zwitterion (PZI), which was surface-attached and cross-linked in one step by simultaneous UV-activated CH insertion and thiol-ene reaction. The process was applicable to both laboratory surfaces like silicon, glass, and gold and real-life surfaces like polyurethane foam wound dressings. The chemical structure and physical properties of the PZI surface and the two reference surfaces SMAMP ("synthetic mimic of an antimicrobial peptide"), an antimicrobial but protein-adhesive polymer coating, and PSB (poly(sulfobetaine)), a protein-repellent but not antimicrobial polyzwitterion coating were characterized by Fourier transform infrared spectroscopy, ellipsometry, contact angle measurements, photoelectron spectroscopy, swellability measurements (using surface plasmon resonance spectroscopy, SPR), zeta potential measurements, and atomic force microscopy. The time-dependent antimicrobial activity assay (time-kill assay) confirmed the high antimicrobial activity of the PZI; SPR was used to demonstrate that it was also highly protein-repellent. Biofilm formation studies showed that the material effectively reduced the growth of Escherichia coli and Staphylococcus aureus biofilms. Additionally, it was shown that the PZI was highly compatible with immortalized human mucosal gingiva keratinocytes and human red blood cells using the Alamar Blue assay, the live-dead stain, and the hemolysis assay. PZI thus may be an attractive coating for biomedical applications, particularly for the fight against bacterial biofilms on medical devices and in other applications.

Library Screen Identifies Enterococcus faecalis CcpA, the Catabolite Control Protein A, as an Effector of Ace, a Collagen Adhesion Protein Linked to Virulence

PubMed Central

Gao, Peng; Pinkston, Kenneth L.; Bourgogne, Agathe; Cruz, Melissa R.; Garsin, Danielle A.; Murray, Barbara E.

2013-01-01

The Enterococcus faecalis cell wall-anchored protein Ace is an important virulence factor involved in cell adhesion and infection. Expression of Ace on the cell surface is affected by many factors, including stage of growth, culture temperature, and environmental components, such as serum, urine, and collagen. However, the mechanisms that regulate or modulate Ace display are not well understood. With interest in identifying genes associated with Ace expression, we utilized a whole-cell enzyme-linked immunosorbent assay (ELISA)-based screening method to identify mutants from a transposon insertion mutant library which exhibited distinct Ace surface expression profiles. We identified a ccpA insertion mutant which showed significantly decreased levels of Ace surface expression at early growth phase versus those of wild-type OG1RF. Confirmation of the observation was achieved through flow cytometry and complementation analysis. Compared to the wild type, the E. faecalis ccpA mutant had an impaired ability to adhere to collagen when grown to early exponential phase, consistent with the lack of Ace expression in the early growth phase. As a key component of carbon catabolite regulation, CcpA has been previously reported to play a critical role in regulating expression of proteins involved in E. faecalis carbohydrate uptake and utilization. Our discovery is the first to associate CcpA with the production of a major E. faecalis virulence factor, providing new insights into the regulation of E. faecalis pathogenesis. PMID:23974022

A Rhizavidin Monomer with Nearly Multimeric Avidin-Like Binding Stability Against Biotin Conjugates.

PubMed

Lee, Jeong Min; Kim, Jung A; Yen, Tzu-Chi; Lee, In Hwan; Ahn, Byungjun; Lee, Younghoon; Hsieh, Chia-Lung; Kim, Ho Min; Jung, Yongwon

2016-03-01

Developing a monomeric form of an avidin-like protein with highly stable biotin binding properties has been a major challenge in biotin-avidin linking technology. Here we report a monomeric avidin-like protein-enhanced monoavidin-with off-rates almost comparable to those of multimeric avidin proteins against various biotin conjugates. Enhanced monoavidin (eMA) was developed from naturally dimeric rhizavidin by optimally maintaining protein rigidity during monomerization and additionally shielding the bound biotin by diverse engineering of the surface residues. eMA allowed the monovalent and nonperturbing labeling of head-group-biotinylated lipids in bilayer membranes. In addition, we fabricated an unprecedented 24-meric avidin probe by fusing eMA to a multimeric cage protein. The 24-meric avidin and eMA were utilized to demonstrate how artificial clustering of cell-surface proteins greatly enhances the internalization rates of assembled proteins on live cells. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

An internal thioester in a pathogen surface protein mediates covalent host binding

PubMed Central

Walden, Miriam; Edwards, John M; Dziewulska, Aleksandra M; Bergmann, Rene; Saalbach, Gerhard; Kan, Su-Yin; Miller, Ona K; Weckener, Miriam; Jackson, Rosemary J; Shirran, Sally L; Botting, Catherine H; Florence, Gordon J; Rohde, Manfred; Banfield, Mark J; Schwarz-Linek, Ulrich

2015-01-01

To cause disease and persist in a host, pathogenic and commensal microbes must adhere to tissues. Colonization and infection depend on specific molecular interactions at the host-microbe interface that involve microbial surface proteins, or adhesins. To date, adhesins are only known to bind to host receptors non-covalently. Here we show that the streptococcal surface protein SfbI mediates covalent interaction with the host protein fibrinogen using an unusual internal thioester bond as a ‘chemical harpoon’. This cross-linking reaction allows bacterial attachment to fibrin and SfbI binding to human cells in a model of inflammation. Thioester-containing domains are unexpectedly prevalent in Gram-positive bacteria, including many clinically relevant pathogens. Our findings support bacterial-encoded covalent binding as a new molecular principle in host-microbe interactions. This represents an as yet unexploited target to treat bacterial infection and may also offer novel opportunities for engineering beneficial interactions. DOI: http://dx.doi.org/10.7554/eLife.06638.001 PMID:26032562

Cross-linking reveals laminin coiled-coil architecture

PubMed Central

Armony, Gad; Jacob, Etai; Moran, Toot; Levin, Yishai; Mehlman, Tevie; Levy, Yaakov; Fass, Deborah

2016-01-01

Laminin, an ∼800-kDa heterotrimeric protein, is a major functional component of the extracellular matrix, contributing to tissue development and maintenance. The unique architecture of laminin is not currently amenable to determination at high resolution, as its flexible and narrow segments complicate both crystallization and single-particle reconstruction by electron microscopy. Therefore, we used cross-linking and MS, evaluated using computational methods, to address key questions regarding laminin quaternary structure. This approach was particularly well suited to the ∼750-Å coiled coil that mediates trimer assembly, and our results support revision of the subunit order typically presented in laminin schematics. Furthermore, information on the subunit register in the coiled coil and cross-links to downstream domains provide insights into the self-assembly required for interaction with other extracellular matrix and cell surface proteins. PMID:27815530

Improvement of LysM-Mediated Surface Display of Designed Ankyrin Repeat Proteins (DARPins) in Recombinant and Nonrecombinant Strains of Lactococcus lactis and Lactobacillus Species

PubMed Central

Zadravec, Petra; Štrukelj, Borut

2015-01-01

Safety and probiotic properties make lactic acid bacteria (LAB) attractive hosts for surface display of heterologous proteins. Protein display on nonrecombinant microorganisms is preferred for therapeutic and food applications due to regulatory requirements. We displayed two designed ankyrin repeat proteins (DARPins), each possessing affinity for the Fc region of human IgG, on the surface of Lactococcus lactis by fusing them to the Usp45 secretion signal and to the peptidoglycan-binding C terminus of AcmA, containing lysine motif (LysM) repeats. Growth medium containing a secreted fusion protein was used to test its heterologous binding to 10 strains of species of the genus Lactobacillus, using flow cytometry, whole-cell enzyme-linked immunosorbent assay (ELISA), and fluorescence microscopy. The fusion proteins bound to the surfaces of all lactobacilli; however, binding to the majority of bacteria was only 2- to 5-fold stronger than that of the control. Lactobacillus salivarius ATCC 11741 demonstrated exceptionally strong binding (32- to 55-fold higher than that of the control) and may therefore be an attractive host for nonrecombinant surface display. Genomic comparison of the species indicated the exopolysaccharides of Lb. salivarius as a possible reason for the difference. Additionally, a 15-fold concentration-dependent increase in nonrecombinant surface display on L. lactis was demonstrated by growing bacteria with sublethal concentrations of the antibiotics chloramphenicol and erythromycin. Nonrecombinant surface display on LAB, based on LysM repeats, was optimized by selecting Lactobacillus salivarius ATCC 11741 as the optimal host and by introducing antibiotics as additives for increasing surface display on L. lactis. Additionally, effective display of DARPins on the surfaces of nonrecombinant LAB has opened up several new therapeutic possibilities. PMID:25576617

Quantitative evaluation of Candia antarctica lipase B displayed on the cell surface of a Pichia pastoris based on an FS anchor system.

PubMed

Liang, Xing-xiang; Wang, Bei-bei; Sun, Yu-fei; Lin, Ying; Han, Shuang-yan; Zheng, Sui-ping; Cui, Tang-bing

2013-03-01

A new approach is described to quantify the number of enzyme molecules, such as Candia antarctica lipase B, that are displayed on the cell surface of Pichia pastoris. Enhanced green fluorescent protein (EGFP) and Candida antarctica lipase B (CALB) were fused and displayed on the surface of P. pastoris by linking to the anchor flocculation functional domain of FLO1p from Saccharomyces cerevisiae. Confocal laser scanning microscopy, flow cytometry, and fluorescence spectrophotometry were used to monitor the fluorescence intensity of fused EGFP. Combined with the corresponding protein concentration detected in the medium, a standard curve describing the relationship between the fusion protein concentration and fluorescence intensity were obtained and could be used to number CALB displayed on the cell surface. The results showed that approx. 10(4) molecules of CALB molecules were immobilized on the single P. pastoris cell wall based on FS anchor system.

Self-assembly of protein-based biomaterials initiated by titania nanotubes.

PubMed

Forstater, Jacob H; Kleinhammes, Alfred; Wu, Yue

2013-12-03

Protein-based biomaterials are a promising strategy for creating robust highly selective biocatalysts. The assembled biomaterials must sufficiently retain the near-native structure of proteins and provide molecular access to catalytically active sites. These requirements often exclude the use of conventional assembly techniques, which rely on covalent cross-linking of proteins or entrapment within a scaffold. Here we demonstrate that titania nanotubes can initiate and template the self-assembly of enzymes, such as ribonuclease A, while maintaining their catalytic activity. Initially, the enzymes form multilayer thick ellipsoidal aggregates centered on the nanotube surface; subsequently, these nanosized entities assemble into a micrometer-sized enzyme material that has enhanced enzymatic activity and contains as little as 0.1 wt % TiO2 nanotubes. This phenomenon is uniquely associated with the active anatase (001)-like surface of titania nanotubes and does not occur on other anatase nanomaterials, which contain significantly fewer undercoordinated Ti surface sites. These findings present a nanotechnology-enabled mechanism of biomaterial growth and open a new route for creating stable protein-based biomaterials and biocatalysts without the need for chemical modification.

The Fate of Nascent APP in Hippocampal Neurons: A Live Cell Imaging Study.

PubMed

DelBove, Claire E; Deng, Xian-Zhen; Zhang, Qi

2018-06-21

Amyloid precursor protein (APP) is closely associated with Alzheimer's disease (AD) because its proteolytic products form amyloid plaques and its mutations are linked to familial AD patients. As a membrane protein, APP is involved in neuronal development and plasticity. However, it remains unclear how nascent APP is distributed and transported to designated membrane compartments to execute its diverse functions. Here, we employed a dual-tagged APP fusion protein in combination with a synaptic vesicle marker to study the surface trafficking and cleavage of APP in hippocampal neurons immediately after its synthesis. Using long-term time-lapse imaging, we found that a considerable amount of nascent APP was directly transported to the somatodendritic surface, from which it propagates to distal neurites. Some APP in the plasma membrane was endocytosed and some was cleaved by α-secretase. Hence, we conclude that surface transportation of APP is a major step preceding its proteolytic processing and neuritic distribution.

A force-based protein biochip

NASA Astrophysics Data System (ADS)

Blank, K.; Mai, T.; Gilbert, I.; Schiffmann, S.; Rankl, J.; Zivin, R.; Tackney, C.; Nicolaus, T.; Spinnler, K.; Oesterhelt, F.; Benoit, M.; Clausen-Schaumann, H.; Gaub, H. E.

2003-09-01

A parallel assay for the quantification of single-molecule binding forces was developed based on differential unbinding force measurements where ligand-receptor interactions are compared with the unzipping forces of DNA hybrids. Using the DNA zippers as molecular force sensors, the efficient discrimination between specific and nonspecific interactions was demonstrated for small molecules binding to specific receptors, as well as for protein-protein interactions on protein arrays. Finally, an antibody sandwich assay with different capture antibodies on one chip surface and with the detection antibodies linked to a congruent surface via the DNA zippers was used to capture and quantify a recombinant hepatitis C antigen from solution. In this case, the DNA zippers enable not only discrimination between specific and nonspecific binding, but also allow for the local application of detection antibodies, thereby eliminating false-positive results caused by cross-reactive antibodies and nonspecific binding.

Ultra-high vacuum surface analysis study of rhodopsin incorporation into supported lipid bilayers.

PubMed

Michel, Roger; Subramaniam, Varuni; McArthur, Sally L; Bondurant, Bruce; D'Ambruoso, Gemma D; Hall, Henry K; Brown, Michael F; Ross, Eric E; Saavedra, S Scott; Castner, David G

2008-05-06

Planar supported lipid bilayers that are stable under ambient atmospheric and ultra-high-vacuum conditions were prepared by cross-linking polymerization of bis-sorbylphosphatidylcholine (bis-SorbPC). X-ray photoelectron spectroscopy (XPS) and time-of-flight secondary ion mass spectrometry (ToF-SIMS) were employed to investigate bilayers that were cross-linked using either redox-initiated radical polymerization or ultraviolet photopolymerization. The redox method yields a more structurally intact bilayer; however, the UV method is more compatible with incorporation of transmembrane proteins. UV polymerization was therefore used to prepare cross-linked bilayers with incorporated bovine rhodopsin, a light-activated, G-protein-coupled receptor (GPCR). A previous study (Subramaniam, V.; Alves, I. D.; Salgado, G. F. J.; Lau, P. W.; Wysocki, R. J.; Salamon, Z.; Tollin, G.; Hruby, V. J.; Brown, M. F.; Saavedra, S. S. J. Am. Chem. Soc. 2005, 127, 5320-5321) showed that rhodopsin retains photoactivity after incorporation into UV-polymerized bis-SorbPC, but did not address how the protein is associated with the bilayer. In this study, we show that rhodopsin is retained in supported bilayers of poly(bis-SorbPC) under ultra-high-vacuum conditions, on the basis of the increase in the XPS nitrogen concentration and the presence of characteristic amino acid peaks in the ToF-SIMS data. Angle-resolved XPS data show that the protein is inserted into the bilayer, rather than adsorbed on the bilayer surface. This is the first study to demonstrate the use of ultra-high-vacuum techniques for structural studies of supported proteolipid bilayers.

Tricomponent Immunopotentiating System as a Novel Molecular Design Strategy for Malaria Vaccine Development ▿

PubMed Central

Miyata, Takeshi; Harakuni, Tetsuya; Tsuboi, Takafumi; Sattabongkot, Jetsumon; Ikehara, Ayumu; Tachibana, Mayumi; Torii, Motomi; Matsuzaki, Goro; Arakawa, Takeshi

2011-01-01

The creation of subunit vaccines to prevent malaria infection has been hampered by the intrinsically weak immunogenicity of the recombinant antigens. We have developed a novel strategy to increase immune responses by creating genetic fusion proteins to target specific antigen-presenting cells (APCs). The fusion complex was composed of three physically linked molecular entities: (i) a vaccine antigen, (ii) a multimeric α-helical coiled-coil core, and (iii) an APC-targeting ligand linked to the core via a flexible linker. The vaccine efficacy of the tricomponent complex was evaluated using an ookinete surface protein of Plasmodium vivax, Pvs25, and merozoite surface protein-1 of Plasmodium yoelii. Immunization of mice with the tricomponent complex induced a robust antibody response and conferred substantial levels of P. vivax transmission blockade as evaluated by a membrane feed assay, as well as protection from lethal P. yoelii infection. The observed effect was strongly dependent on the presence of all three components physically integrated as a fusion complex. This system, designated the tricomponent immunopotentiating system (TIPS), onto which any recombinant protein antigens or nonproteinaceous substances could be loaded, may be a promising strategy for devising subunit vaccines or adjuvants against various infectious diseases, including malaria. PMID:21807905

Novel function of the endoplasmic reticulum degradation-enhancing α-mannosidase-like proteins in the human hepatitis B virus life cycle, mediated by the middle envelope protein.

PubMed

Lazar, Catalin; Uta, Mihaela; Petrescu, Stefana Maria; Branza-Nichita, Norica

2017-02-01

Cells replicating the human hepatitis B virus (HBV) express high levels of degradation-enhancing α-mannosidase-like proteins (EDEMs), a family of proteins involved in the endoplasmic reticulum associated degradation, one of the pathways activated during the unfolded protein response. Owing to their α-1,2 mannosidase activity, the EDEM1-3 proteins are able to process the N-linked glycans of misfolded or incompletely folded proteins, providing the recognition signal for their subsequent degradation. The HBV small (S), medium (M), and large (L) surface proteins bear an N-linked glycosylation site in the common S domain that is partially occupied in all proteins. The M protein contains an additional site in its preS2 domain, which is always functional. Here, we report that these oligosaccharides are processed by EDEMs, more efficiently by EDEM3, which induces degradation of L and S proteins, accompanied by a reduction of subviral particles production. In striking contrast, M not only is spared from degradation but its trafficking is also accelerated leading to an improved secretion. This unusual behavior of the M protein requires strictly the mannose trimming of the preS2 N-linked glycan. Furthermore, we show that HBV secretion is significantly inhibited under strong endoplasmic reticulum stress conditions when M expression is prevented by mutagenesis of the viral genome. These observations unfold unique properties of the M protein in the HBV life cycle during unfolded protein response and point to alternative mechanisms employed by EDEMs to alleviate this stress in case of necessity by promoting glycoprotein trafficking rather than degradation. © 2016 John Wiley & Sons Ltd.

Drastic differences in glycosylation of related S-layer glycoproteins from moderate and extreme halophiles.

PubMed

Mengele, R; Sumper, M

1992-04-25

The outer surface of the moderate halophilic archaebacterium Haloferax volcanii (formerly named Halobacterium volcanii) is covered with a hexagonally packed surface (S) layer glycoprotein. The polypeptide (794 amino acid residues) contains 7 N-glycosylation sites. Four of these sites were isolated as glycopeptides and the structure of one of the corresponding saccharides was determined. Oligosaccharides consisting of beta-1,4-linked glucose residues are attached to the protein via the linkage unit asparaginyl-glucose. In the related glycoprotein from the extreme halophile Halobacterium halobium, the glucose residues are replaced by sulfated glucuronic acid residues, causing a drastic increase in surface charge density. This is discussed in terms of a recent model explaining the stability of halophilic proteins.

Role of N-linked oligosaccharides in processing and intracellular transport of E2 glycoprotein of rubella virus.

PubMed Central

Qiu, Z; Hobman, T C; McDonald, H L; Seto, N O; Gillam, S

1992-01-01

The role of N-linked glycosylation in processing and intracellular transport of rubella virus glycoprotein E2 has been studied by expressing glycosylation mutants of E2 in COS cells. A panel of E2 glycosylation mutants were generated by oligonucleotide-directed mutagenesis. Each of the three potential N-linked glycosylation sites was eliminated separately as well as in combination with the other two sites. Expression of the E2 mutant proteins in COS cells indicated that in rubella virus M33 strain, all three sites are used for the addition of N-linked oligosaccharides. Removal of any of the glycosylation sites resulted in slower glycan processing, lower stability, and aberrant disulfide bonding of the mutant proteins, with the severity of defect depending on the number of deleted carbohydrate sites. The mutant proteins were transported to the endoplasmic reticulum and Golgi complex but were not detected on the cell surface. However, the secretion of the anchor-free form of E2 into the medium was not completely blocked by the removal of any one of its glycosylation sites. This effect was dependent on the position of the deleted glycosylation site. Images PMID:1583721

Dendrimer-Linked Antifreeze Proteins Have Superior Activity and Thermal Recovery.

PubMed

Stevens, Corey A; Drori, Ran; Zalis, Shiran; Braslavsky, Ido; Davies, Peter L

2015-09-16

By binding to ice, antifreeze proteins (AFPs) depress the freezing point of a solution and inhibit ice recrystallization if freezing does occur. Previous work showed that the activity of an AFP was incrementally increased by fusing it to another protein. Even larger increases in activity were achieved by doubling the number of ice-binding sites by dimerization. Here, we have combined the two strategies by linking multiple outward-facing AFPs to a dendrimer to significantly increase both the size of the molecule and the number of ice-binding sites. Using a heterobifunctional cross-linker, we attached between 6 and 11 type III AFPs to a second-generation polyamidoamine (G2-PAMAM) dendrimer with 16 reactive termini. This heterogeneous sample of dendrimer-linked type III constructs showed a greater than 4-fold increase in freezing point depression over that of monomeric type III AFP. This multimerized AFP was particularly effective at ice recrystallization inhibition activity, likely because it can simultaneously bind multiple ice surfaces. Additionally, attachment to the dendrimer has afforded the AFP superior recovery from heat denaturation. Linking AFPs together via polymers can generate novel reagents for controlling ice growth and recrystallization.

A novel cross-linked enzyme aggregates (CLEAs) of papain and neutrase-production, partial characterization and application.

PubMed

Chen, Zhongqin; Wang, Yanwei; Liu, Wei; Wang, Jingya; Chen, Haixia

2017-02-01

The neutrase (EC 3.4.24.4) and papain (EC 3.4.22.2) were together immobilized ascross-linked enzyme aggregates (N-P-CLEAs) and their properties were characterized. The influence of the precipitant, cross-linking ratio of glutaraldehyde and cross-linking time were investigated. Ethanol was selected as the more efficient precipitant compared with ammonium sulfate. The proper cross-linking ratio of enzyme and glutaraldehyde was 1:5 (v/v) and the optimized cross-linking time was 4h. N-P-CLEAs showed obvious improvement in thermal stability and pH stability than the free enzyme (P<0.05) and could hold relatively high activity retention in nonpolar and hydrophilic solvents and without activity loss at 4°C for more than six months. The cross-linking reaction had been appeared in N-P-CLEAs and more orderly microscopic surface morphology of N-P-CLEAs was observed. The molecular weight and thermal denaturation temperature of N-P-CLEAs were increased while the isoelectric point was decreased compared with those of the free enzymes. Application of N-P-CLEAs in bean proteins and zein showed a higher degree of hydrolysis, such as the hydrolysis degree of mung bean protein hydrolyzed by N-P-CLEAs was 12%, increased by approximately 4.5% compared to that of free enzyme. The results demonstrated that the N-P-CLEAs was suitable for application in food protein hydrolysis. Copyright © 2016 Elsevier B.V. All rights reserved.

Isolation of recombinant antibodies directed against surface proteins of Clostridium difficile.

PubMed

Shirvan, Ali Nazari; Aitken, Robert

2016-01-01

Clostridium difficile has emerged as an increasingly important nosocomial pathogen and the prime causative agent of antibiotic-associated diarrhoea and pseudomembranous colitis in humans. In addition to toxins A and B, immunological studies using antisera from patients infected with C. difficile have shown that a number of other bacterial factors contribute to the pathogenesis, including surface proteins, which are responsible for adhesion, motility and other interactions with the human host. In this study, various clostridial targets, including FliC, FliD and cell wall protein 66, were expressed and purified. Phage antibody display yielded a large panel of specific recombinant antibodies, which were expressed, purified and characterised. Reactions of the recombinant antibodies with their targets were detected by enzyme-linked immunosorbent assay; and Western blotting suggested that linear rather than conformational epitopes were recognised. Binding of the recombinant antibodies to surface-layer proteins and their components showed strain specificity, with good recognition of proteins from C. difficile 630. However, no reaction was observed for strain R20291-a representative of the 027 ribotype. Binding of the recombinant antibodies to C. difficile M120 extracts indicated that a component of a surface-layer protein of this strain might possess immunoglobulin-binding activities. The recombinant antibodies against FliC and FliD proteins were able to inhibit bacterial motility. Copyright © 2016. Published by Elsevier Editora Ltda.

Novel amphiphilic poly(dimethylsiloxane) based polyurethane networks tethered with carboxybetaine and their combined antibacterial and anti-adhesive property

NASA Astrophysics Data System (ADS)

Jiang, Jingxian; Fu, Yuchen; Zhang, Qinghua; Zhan, Xiaoli; Chen, Fengqiu

2017-08-01

The traditional nonfouling materials are powerless against bacterial cells attachment, while the hydrophobic bactericidal surfaces always suffer from nonspecific protein adsorption and dead bacterial cells accumulation. Here, amphiphilic polyurethane (PU) networks modified with poly(dimethylsiloxane) (PDMS) and cationic carboxybetaine diol through simple crosslinking reaction were developed, which had an antibacterial efficiency of 97.7%. Thereafter, the hydrolysis of carboxybetaine ester into zwitterionic groups brought about anti-adhesive properties against bacteria and proteins. The surface chemical composition and wettability performance of the PU network surfaces were investigated by attenuated total reflectance Fourier transform infrared spectroscopy (ATR-FTIR), X-ray photoelectron spectroscopy (XPS) and contact angle analysis. The surface distribution of PDMS and zwitterionic segments produced an obvious amphiphilic heterogeneous surface, which was demonstrated by atomic force microscopy (AFM). Enzyme-linked immunosorbent assays (ELISA) were used to test the nonspecific protein adsorption behaviors. With the advantages of the transition from excellent bactericidal performance to anti-adhesion and the combination of fouling resistance and fouling release property, the designed PDMS-based amphiphilic PU network shows great application potential in biomedical devices and marine facilities.

Stereoselective hydrogenation of olefins using rhodium-substituted carbonic anhydrase--a new reductase.

PubMed

Jing, Qing; Okrasa, Krzysztof; Kazlauskas, Romas J

2009-01-01

One useful synthetic reaction missing from nature's toolbox is the direct hydrogenation of substrates using hydrogen. Instead nature uses cofactors like NADH to reduce organic substrates, which adds complexity and cost to these reductions. To create an enzyme that can directly reduce organic substrates with hydrogen, researchers have combined metal hydrogenation catalysts with proteins. One approach is an indirect link where a ligand is linked to a protein and the metal binds to the ligand. Another approach is direct linking of the metal to protein, but nonspecific binding of the metal limits this approach. Herein, we report a direct hydrogenation of olefins catalyzed by rhodium(I) bound to carbonic anhydrase (CA-[Rh]). We minimized nonspecific binding of rhodium by replacing histidine residues on the protein surface using site-directed mutagenesis or by chemically modifying the histidine residues. Hydrogenation catalyzed by CA-[Rh] is slightly slower than for uncomplexed rhodium(I), but the protein environment induces stereoselectivity favoring cis- over trans-stilbene by about 20:1. This enzyme is the first cofactor-independent reductase that reduces organic molecules using hydrogen. This catalyst is a good starting point to create variants with tailored reactivity and selectivity. This strategy to insert transition metals in the active site of metalloenzymes opens opportunities to a wider range of enzyme-catalyzed reactions.

Amyloidogenic Regions and Interaction Surfaces Overlap in Globular Proteins Related to Conformational Diseases

PubMed Central

Castillo, Virginia; Ventura, Salvador

2009-01-01

Protein aggregation underlies a wide range of human disorders. The polypeptides involved in these pathologies might be intrinsically unstructured or display a defined 3D-structure. Little is known about how globular proteins aggregate into toxic assemblies under physiological conditions, where they display an initially folded conformation. Protein aggregation is, however, always initiated by the establishment of anomalous protein-protein interactions. Therefore, in the present work, we have explored the extent to which protein interaction surfaces and aggregation-prone regions overlap in globular proteins associated with conformational diseases. Computational analysis of the native complexes formed by these proteins shows that aggregation-prone regions do frequently overlap with protein interfaces. The spatial coincidence of interaction sites and aggregating regions suggests that the formation of functional complexes and the aggregation of their individual subunits might compete in the cell. Accordingly, single mutations affecting complex interface or stability usually result in the formation of toxic aggregates. It is suggested that the stabilization of existing interfaces in multimeric proteins or the formation of new complexes in monomeric polypeptides might become effective strategies to prevent disease-linked aggregation of globular proteins. PMID:19696882

Acrolein Microspheres Are Bonded To Large-Area Substrates

NASA Technical Reports Server (NTRS)

Rembaum, Alan; Yen, Richard C. K.

1988-01-01

Reactive cross-linked microspheres produced under influence of ionizing radiation in aqueous solutions of unsaturated aldehydes, such as acrolein, with sodium dodecyl sulfate. Diameters of spheres depend on concentrations of ingredients. If polystyrene, polymethylmethacrylate, or polypropylene object immersed in solution during irradiation, microspheres become attached to surface. Resulting modified surface has grainy coating with reactivity similar to free microspheres. Aldehyde-substituted-functional microspheres react under mild conditions with number of organic reagents and with most proteins. Microsphere-coated macrospheres or films used to immobilize high concentrations of proteins, enzymes, hormones, viruses, cells, and large number of organic compounds. Applications include separation techniques, clinical diagnostic tests, catalytic processes, and battery separators.

Oligosaccharide ligands for NKR-P1 protein activate NK cells and cytotoxicity

NASA Astrophysics Data System (ADS)

Bezouška, Karel; Yuen, Chun-Ting; O'Brien, Jacqui; Childs, Robert A.; Chai, Wengang; Lawson, Alexander M.; Drbal, Karel; Fišerová, Anna; Posíšil, Miloslav; Feizi, Ten

1994-11-01

A diversity of high-affinity Oligosaccharide ligands are identified for NKR-P1, a membrane protein on natural killer (NK) cells which contains an extracellular Ca2+-dependent lectin domain. Interactions of such oligosaccharides on the target cell surface with NKR-P1 on the killer cell surface are crucial both for target cell recognition and for delivery of stimulatory or inhibitory signals linked to the NK cytolytic machinery. NK-resistant tumour cells are rendered susceptible by preincubation with liposomes expressing NKR-P1 ligands, suggesting that purging of tumour or virally infected cells in vivo may be a therapeutic possibility.

Cross-linking of surface Ig receptors on murine B lymphocytes stimulates the expression of nuclear tetradecanoyl phorbol acetate-response element-binding proteins

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

Chiles, T.C.; Liu, J.L.; Rothstein, T.L.

1991-03-15

Cross-linking of sIg on primary B lymphocytes leads to increased nuclear DNA-binding activity specific for the tetradecanoyl phorbol acetate-response element (TRE), as judged by gel mobility shift assays. Stimulation of B cells to enter S phase of the cell cycle by treatment with the combination of phorbol ester plus calcium ionophore also stimulated nuclear TRE-binding activity within 2 h, with maximal expression at 4 h; however, phorbol ester and calcium ionophore were not as effective in stimulating binding activity when examined separately. Stimulated nuclear expression of TRE-binding activity appears to require protein synthesis. Fos- and Jun/AP-1-related proteins participate directly inmore » the identified nucleoprotein complex, as shown by the ability of c-fos- and c-jun-specific antisera to either alter or completely abolish electrophoretic migration of the complex in native gels. Further, UV photo-cross-linking studies identified two major TRE-binding protein species, whose sizes correspond to TRE-binding proteins derived from HeLa cell nuclear extracts. The results suggest that in primary B cells nuclear TRE-binding activity represents a downstream signaling event that occurs subsequent to changes in protein kinase C activity and intracellular Ca2+ but that can be triggered physiologically through sIg.« less

Latex-protein complexes from an acute phase recombinant antigen of Toxoplasma gondii for the diagnosis of recently acquired toxoplasmosis.

PubMed

Peretti, Leandro E; Gonzalez, Verónica D G; Marcipar, Iván S; Gugliotta, Luis M

2014-08-01

The synthesis and characterization of latex-protein complexes (LPC), from the acute phase recombinant antigen P35 (P35Ag) of Toxoplasma gondii and "core-shell" carboxylated or polystyrene (PS) latexes (of different sizes and charge densities) are considered, with the aim of producing immunoagglutination reagents able to detect recently acquired toxoplasmosis. Physical adsorption (PA) and chemical coupling (CC) of P35Ag onto latex particles at different pH were investigated. Greater amounts of adsorbed protein were obtained on PS latexes than on carboxylated latexes, indicating that hydrophobic forces govern the interactions between the protein and the particle surface. In the CC experiments, the highest amount of bound protein was obtained at pH 6, near the isoelectric point of the protein (IP=6.27). At this pH, it decreased both the repulsion between particle surface and protein, and the repulsion between neighboring molecules. The LPC were characterized and the antigenicity of the P35Ag protein coupled on the particles surface was evaluated by Enzyme-Linked ImmunoSorbent Assay (ELISA). Results from ELISA showed that the P35Ag coupled to the latex particles surface was not affected during the particles sensitization by PA and CC and the produced LPC were able to recognize specific anti-P35Ag antibodies present in the acute phase of the disease. Copyright © 2014 Elsevier B.V. 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

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.

Clusterin Facilitates Exchange of Glycosyl Phosphatidylinositol-Linked SPAM1 Between Reproductive Luminal Fluids and Mouse and Human Sperm Membranes1

PubMed Central

Griffiths, Genevieve S.; Galileo, Deni S.; Aravindan, Rolands G.; Martin-DeLeon, Patricia A.

2009-01-01

Glycosyl phosphatidylinositol (GPI)-linked proteins, which are involved in post-testicular maturation of sperm and have a role in fertilization, are acquired on the sperm surface from both vesicular and membrane-free soluble fractions of epididymal luminal fluid (LF) and uterine LF. Herein, we investigate the mechanism of uptake of these proteins from the soluble fraction of LFs using sperm adhesion molecule 1 (SPAM1) as a model. Ultracentrifugation and native Western blot analysis of the soluble fraction revealed that SPAM1 is present in low-molecular-weight (monomeric) and high-molecular-weight (oligomeric) complexes. The latter are incapable of transferring SPAM1 and may serve to produce monomers. Monomers are stabilized by hydrophobic interactions with clusterin (CLU), a lipid carrier that is abundantly expressed in LFs. We show that CLU is involved in the transfer of SPAM1 monomers, whose delivery was decreased by anti-CLU antibody under normal and apolipoprotein-enhanced conditions. Coimmunoprecipitation revealed an intimate association of CLU with SPAM1. Both plasma and recombinant CLU had a dose-related effect on transfer efficiency: high concentrations reduced and low concentrations enhanced delivery of SPAM1 to human and mouse sperm membranes, reflecting physiological states in the epididymal tract. We propose a lipid exchange model (akin to the lipid-poor model for cholesterol efflux) for the delivery of GPI-linked proteins to sperm membranes via CLU. Our investigation defines specific conditions for membrane-free GPI-linked protein transfer in vitro and could lead to technology for improving fertility or treating sperm pathology by the addition of relevant GPI-linked proteins critical for successful fertilization in humans and domestic animals. PMID:19357365

Succinimidyl Ester Surface Chemistry: Implications of the Competition between Aminolysis and Hydrolysis on Covalent Protein Immobilization

PubMed Central

2015-01-01

N-Hydroxysuccinimide (NHS) ester terminal groups are commonly used to covalently couple amine-containing biomolecules (e.g., proteins and peptides) to surfaces via amide linkages. This one-step aminolysis is often performed in buffered aqueous solutions near physiological pH (pH 6 to pH 9). Under these conditions, the hydrolysis of the ester group competes with the amidization process, potentially degrading the efficiency of the coupling chemistry. The work herein examines the efficiency of covalent protein immobilization in borate buffer (50 mM, pH 8.50) using the thiolate monolayer formed by the chemisorption of dithiobis (succinimidyl propionate) (DSP) on gold films. The structure and reactivity of these adlayers are assessed via infrared spectroscopy (IR), X-ray photoelectron spectroscopy (XPS), electrochemical reductive desorption, and contact angle measurements. The hydrolysis of the DSP-based monolayer is proposed to follow a reaction mechanism with an initial nucleation step, in contrast to a simple pseudo first-order reaction rate law for the entire reaction, indicating a strong dependence of the interfacial reaction on the packing and presence of defects in the adlayer. This interpretation is used in the subsequent analysis of IR-ERS kinetic plots which give a heterogeneous aminolysis rate constant, ka, that is over 3 orders of magnitude lower than that of the heterogeneous hydrolysis rate constant, kh. More importantly, a projection of these heterogeneous kinetic rates to protein immobilization suggests that under coupling conditions in which low protein concentrations and buffers of near physiological pH are used, proteins are more likely physically adsorbed rather than covalently linked. This result is paramount for biosensors that use NHS chemistry for protein immobilization due to effects that may arise from noncovalently linked proteins. PMID:25317495

Turnip Yellow Mosaic Virus

NASA Technical Reports Server (NTRS)

2000-01-01

The bumpy exterior of the turnip yellow mosaic virus (TYMV) protein coat, or capsid, was defined in detail by Dr. Alexander McPherson of the University of California, Irvin using proteins crystallized in space for analysis on Earth. TYMV is an icosahedral virus constructed from 180 copies of the same protein arranged into 12 clusters of five proteins (pentamers), and 20 clusters of six proteins (hexamers). The final TYMV structure led to the unexpected hypothesis that the virus releases its RNA by essentially chemical-mechanical means. Most viruses have fairly flat coats, but in TYNV, the fold in each protein, called the jellyroll, is clustered at the points where the protein pentamers and hexamers join. The jellyrolls are almost standing on end, producing a bumpy surface with knobs at all of the pentamers and hexamers. At the inside surface of the pentamers is a void that is not present at the hexamers. The coating had been seen in early stuties of TYMV, but McPherson's atomic structure shows much more detail. The inside surface is strikingly, and unexpectedly, different than the outside. While the pentamers contain a central void on the inside, the hexameric units contain peptides linked to each other, forming a ring or, more accurately, rings to fill the void. Credit: Dr. Alexander McPherson, University of California, Irvine

Microgravity

NASA Image and Video Library

2000-05-01

The bumpy exterior of the turnip yellow mosaic virus (TYMV) protein coat, or capsid, was defined in detail by Dr. Alexander McPherson of the University of California, Irvin using proteins crystallized in space for analysis on Earth. TYMV is an icosahedral virus constructed from 180 copies of the same protein arranged into 12 clusters of five proteins (pentamers), and 20 clusters of six proteins (hexamers). The final TYMV structure led to the unexpected hypothesis that the virus releases its RNA by essentially chemical-mechanical means. Most viruses have fairly flat coats, but in TYNV, the fold in each protein, called the jellyroll, is clustered at the points where the protein pentamers and hexamers join. The jellyrolls are almost standing on end, producing a bumpy surface with knobs at all of the pentamers and hexamers. At the inside surface of the pentamers is a void that is not present at the hexamers. The coating had been seen in early stuties of TYMV, but McPherson's atomic structure shows much more detail. The inside surface is strikingly, and unexpectedly, different than the outside. While the pentamers contain a central void on the inside, the hexameric units contain peptides linked to each other, forming a ring or, more accurately, rings to fill the void. Credit: Dr. Alexander McPherson, University of California, Irvine

The Application of Ligand-Mapping Molecular Dynamics Simulations to the Rational Design of Peptidic Modulators of Protein-Protein Interactions.

PubMed

Tan, Yaw Sing; Spring, David R; Abell, Chris; Verma, Chandra S

2015-07-14

A computational ligand-mapping approach to detect protein surface pockets that interact with hydrophobic moieties is presented. In this method, we incorporated benzene molecules into explicit solvent molecular dynamics simulations of various protein targets. The benzene molecules successfully identified the binding locations of hydrophobic hot-spot residues and all-hydrocarbon cross-links from known peptidic ligands. They also unveiled cryptic binding sites that are occluded by side chains and the protein backbone. Our results demonstrate that ligand-mapping molecular dynamics simulations hold immense promise to guide the rational design of peptidic modulators of protein-protein interactions, including that of stapled peptides, which show promise as an exciting new class of cell-penetrating therapeutic molecules.

Colloid Surface Chemistry Critically Affects Multiple Particle Tracking Measurements of Biomaterials

PubMed Central

Valentine, M. T.; Perlman, Z. E.; Gardel, M. L.; Shin, J. H.; Matsudaira, P.; Mitchison, T. J.; Weitz, D. A.

2004-01-01

Characterization of the properties of complex biomaterials using microrheological techniques has the promise of providing fundamental insights into their biomechanical functions; however, precise interpretations of such measurements are hindered by inadequate characterization of the interactions between tracers and the networks they probe. We here show that colloid surface chemistry can profoundly affect multiple particle tracking measurements of networks of fibrin, entangled F-actin solutions, and networks of cross-linked F-actin. We present a simple protocol to render the surface of colloidal probe particles protein-resistant by grafting short amine-terminated methoxy-poly(ethylene glycol) to the surface of carboxylated microspheres. We demonstrate that these poly(ethylene glycol)-coated tracers adsorb significantly less protein than particles coated with bovine serum albumin or unmodified probe particles. We establish that varying particle surface chemistry selectively tunes the sensitivity of the particles to different physical properties of their microenvironments. Specifically, particles that are weakly bound to a heterogeneous network are sensitive to changes in network stiffness, whereas protein-resistant tracers measure changes in the viscosity of the fluid and in the network microstructure. We demonstrate experimentally that two-particle microrheology analysis significantly reduces differences arising from tracer surface chemistry, indicating that modifications of network properties near the particle do not introduce large-scale heterogeneities. Our results establish that controlling colloid-protein interactions is crucial to the successful application of multiple particle tracking techniques to reconstituted protein networks, cytoplasm, and cells. PMID:15189896

Characterization of stable, electroactive protein cage/synthetic polymer multilayer thin films prepared by layer-by-layer assembly

NASA Astrophysics Data System (ADS)

Uto, Koichiro; Yamamoto, Kazuya; Kishimoto, Naoko; Muraoka, Masahiro; Aoyagi, Takao; Yamashita, Ichiro

2013-04-01

We have fabricated electroactive multilayer thin films containing ferritin protein cages. The multilayer thin films were prepared on a solid substrate by the alternate electrostatic adsorption of (apo)ferritin and poly( N-isopropylacrylamide- co-2-carboxyisopropylacrylamide) (NIPAAm- co-CIPAAm) in pH 3.5 acetate buffer solution. The assembly process was monitored using a quartz crystal microbalance. The (apo)ferritin/poly(NIPAAm- co-CIPAAm) multilayer thin films were then cross-linked using a water-soluble carbodiimide, 1-[3-(dimethylamino)propyl]-3-ethylcarbodiimide. The cross-linked films were stable under a variety of conditions. The surface morphology and thickness of the multilayer thin films were characterized by atomic force microscopy, and the ferritin iron cores were observed by scanning electron microscopy to confirm the assembly mechanism. Cyclic voltammetry measurements showed different electrochemical properties for the cross-linked ferritin and apoferritin multilayer thin films, and the effect of stability of the multilayer film on its electrochemical properties was also examined. Our method for constructing multilayer films containing protein cages is expected to be useful in building more complex functional inorganic nanostructures.

The creation of a biomimetic interface between boron-doped diamond and immobilized proteins.

PubMed

Hoffmann, René; Kriele, Armin; Obloh, Harald; Tokuda, Norio; Smirnov, Waldemar; Yang, Nianjun; Nebel, Christoph E

2011-10-01

Immobilization of proteins on a solid electrode is to date done by chemical cross-linking or by addition of an adjustable intermediate. In this paper we introduce a concept where a solid with variable surface properties is optimized to mediate binding of the electron-transfer protein Cytochrome c (Cyt c) by mimicking the natural binding environment. It is shown that, as a carbon-based material, boron-doped diamond can be adjusted by simple electrochemical surface treatments to the specific biochemical requirements of Cyt c. The structure and functionality of passively adsorbed Cyt c on variously terminated diamond surfaces were characterized in detail using a combination of electrochemical techniques and atomic force microscopy with single-molecule resolution. Partially oxidized diamond allowed stable immobilization of Cyt c together with high electron transfer activity, driven by a combination of electrostatic and hydrophobic interactions. This surface mimics the natural binding partner, where coarse orientation is governed by electrostatic interaction of the protein's dipole and hydrophobic interactions assist in formation of the electron transfer complex. The optimized surface mediated electron transfer kinetics around 100 times faster than those reported for other solids and even faster kinetics than on self-assembled monolayers of alkanethiols. Copyright © 2011 Elsevier Ltd. All rights reserved.

Tuning cell adhesive properties via layer-by-layer assembly of chitosan and alginate

PubMed Central

Silva, Joana M.; García, José R.; Reis, Rui L.; García, Andrés J.; Mano, João F.

2017-01-01

Understanding the mechanisms controlling cell-multilayer film interactions is crucial to the successful engineering of these coatings for biotechnological and biomedical applications. Herein, we present a strategy to tune the cell adhesive properties of multilayers based on marine polysaccharides with and without cross-linking and/or coating with extracellular matrix proteins. Chemical cross-linking of multilayers improved mechanical properties of the coatings but also elicited changes in surface chemistry that alter the adhesion of human umbilical vein endothelial cells. We evaluated a strategy to decouple the mechanical and chemical properties of these films, enabling the transition from cell-adhesive to cell-resistant multilayers. Addition of chitosan/alginate multilayers on top of cross-linked films decreased endothelial cell adhesion, spreading, and proliferation to similar levels as uncross-linked films. Our findings highlight the key role of surface chemistry in cell-multilayer film interactions, and these engineered nanocoatings represent a tunable model of cell adhesive and non-adhesive multilayered films. PMID:28126597

Analysis of Nidogen-1/Laminin γ1 Interaction by Cross-Linking, Mass Spectrometry, and Computational Modeling Reveals Multiple Binding Modes

PubMed Central

Lössl, Philip; Kölbel, Knut; Tänzler, Dirk; Nannemann, David; Ihling, Christian H.; Keller, Manuel V.; Schneider, Marian; Zaucke, Frank; Meiler, Jens; Sinz, Andrea

2014-01-01

We describe the detailed structural investigation of nidogen-1/laminin γ1 complexes using full-length nidogen-1 and a number of laminin γ1 variants. The interactions of nidogen-1 with laminin variants γ1 LEb2–4, γ1 LEb2–4 N836D, γ1 short arm, and γ1 short arm N836D were investigated by applying a combination of (photo-)chemical cross-linking, high-resolution mass spectrometry, and computational modeling. In addition, surface plasmon resonance and ELISA studies were used to determine kinetic constants of the nidogen-1/laminin γ1 interaction. Two complementary cross-linking strategies were pursued to analyze solution structures of laminin γ1 variants and nidogen-1. The majority of distance information was obtained with the homobifunctional amine-reactive cross-linker bis(sulfosuccinimidyl)glutarate. In a second approach, UV-induced cross-linking was performed after incorporation of the diazirine-containing unnatural amino acids photo-leucine and photo-methionine into laminin γ1 LEb2–4, laminin γ1 short arm, and nidogen-1. Our results indicate that Asn-836 within laminin γ1 LEb3 domain is not essential for complex formation. Cross-links between laminin γ1 short arm and nidogen-1 were found in all protein regions, evidencing several additional contact regions apart from the known interaction site. Computational modeling based on the cross-linking constraints indicates the existence of a conformational ensemble of both the individual proteins and the nidogen-1/laminin γ1 complex. This finding implies different modes of interaction resulting in several distinct protein-protein interfaces. PMID:25387007

Enzymic cross-linkage of monomeric extensin precursors in vitro. [Lycopersicon esculentum

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

Everdeen, D.S.; Kiefer, S.; Willard, J.J.

Rapidly growing tomato (Lycopersicon esculentum) cell suspension cultures contain transiently high levels of cell surface, salt-elutable, monomeric precursors to the covalently cross-linked extensin network of the primary cell wall. Thus, the authors purified a highly soluble monomeric extensin substrate from rapidly growing cells, and devised a soluble in vitro cross-linking assay based on Superose-6 fast protein liquid chromatography separation, which resolved extensin monomers from the newly formed oligomers within 25 minutes. Salt elution of slowly growing (early stationary phase) cells yielded little or no extensin monomers but did give a highly active enzymic preparation that specifically cross-linked extensin monomers inmore » the presence of hydrogen peroxide, judging from: (a) a decrease in the extensin monomer peak on fast protein liquid chromatography gel filtration, (b) appearance of oligomeric peaks, and (c) direct electron microscopical observation of the cross-linked oligomers. The cross-linking reaction had a broad pH optimum between 5.5 and 6.5. An approach to substrate saturation of the enzyme required extensin monomer concentrations of 20 to 40 milligrams per milliliter. Preincubation with catalase completely inhibited the cross-linking reaction, which was highly dependent on hydrogen peroxide and optimal at 15 to 50 micromolar. They therefore identified the cross-linking activity as extensin peroxidase.« less

In-depth comparative analysis of malaria parasite genomes reveals protein-coding genes linked to human disease in Plasmodium falciparum genome.

PubMed

Liu, Xuewu; Wang, Yuanyuan; Liang, Jiao; Wang, Luojun; Qin, Na; Zhao, Ya; Zhao, Gang

2018-05-02

Plasmodium falciparum is the most virulent malaria parasite capable of parasitizing human erythrocytes. The identification of genes related to this capability can enhance our understanding of the molecular mechanisms underlying human malaria and lead to the development of new therapeutic strategies for malaria control. With the availability of several malaria parasite genome sequences, performing computational analysis is now a practical strategy to identify genes contributing to this disease. Here, we developed and used a virtual genome method to assign 33,314 genes from three human malaria parasites, namely, P. falciparum, P. knowlesi and P. vivax, and three rodent malaria parasites, namely, P. berghei, P. chabaudi and P. yoelii, to 4605 clusters. Each cluster consisted of genes whose protein sequences were significantly similar and was considered as a virtual gene. Comparing the enriched values of all clusters in human malaria parasites with those in rodent malaria parasites revealed 115 P. falciparum genes putatively responsible for parasitizing human erythrocytes. These genes are mainly located in the chromosome internal regions and participate in many biological processes, including membrane protein trafficking and thiamine biosynthesis. Meanwhile, 289 P. berghei genes were included in the rodent parasite-enriched clusters. Most are located in subtelomeric regions and encode erythrocyte surface proteins. Comparing cluster values in P. falciparum with those in P. vivax and P. knowlesi revealed 493 candidate genes linked to virulence. Some of them encode proteins present on the erythrocyte surface and participate in cytoadhesion, virulence factor trafficking, or erythrocyte invasion, but many genes with unknown function were also identified. Cerebral malaria is characterized by accumulation of infected erythrocytes at trophozoite stage in brain microvascular. To discover cerebral malaria-related genes, fast Fourier transformation (FFT) was introduced to extract genes highly transcribed at the trophozoite stage. Finally, 55 candidate genes were identified. Considering that parasite-infected erythrocyte surface protein 2 (PIESP2) contains gap-junction-related Neuromodulin_N domain and that anti-PIESP2 might provide protection against malaria, we chose PIESP2 for further experimental study. Our analysis revealed a limited number of genes linked to human disease in P. falciparum genome. These genes could be interesting targets for further functional characterization.

DRoP: a water analysis program identifies Ras-GTP-specific pathway of communication between membrane-interacting regions and the active site.

PubMed

Kearney, Bradley M; Johnson, Christian W; Roberts, Daniel M; Swartz, Paul; Mattos, Carla

2014-02-06

Ras GTPase mediates several cellular signal transduction pathways and is found mutated in a large number of cancers. It is active in the GTP-bound state, where it interacts with effector proteins, and at rest in the GDP-bound state. The catalytic domain is tethered to the membrane, with which it interacts in a nucleotide-dependent manner. Here we present the program Detection of Related Solvent Positions (DRoP) for crystallographic water analysis on protein surfaces and use it to study Ras. DRoP reads and superimposes multiple Protein Data Bank coordinates, transfers symmetry-related water molecules to the position closest to the protein surface, and ranks the waters according to how well conserved and tightly clustered they are in the set of structures. Coloring according to this rank allows visualization of the results. The effector-binding region of Ras is hydrated with highly conserved water molecules at the interface between the P-loop, switch I, and switch II, as well as at the Raf-RBD binding pocket. Furthermore, we discovered a new conserved water-mediated H-bonding network present in Ras-GTP, but not in Ras-GDP, that links the nucleotide sensor residues R161 and R164 on helix 5 to the active site. The double mutant RasN85A/N86A, where the final link between helix 5 and the nucleotide is not possible, is a severely impaired enzyme, while the single mutant RasN86A, with partial connection to the active site, has a wild-type hydrolysis rate. DRoP was instrumental in determining the water-mediated connectivity networks that link two lobes of the catalytic domain in Ras. Copyright © 2013 Elsevier Ltd. All rights reserved.

Ribosome-Inactivating Proteins: From Plant Defense to Tumor Attack

PubMed Central

de Virgilio, Maddalena; Lombardi, Alessio; Caliandro, Rocco; Fabbrini, Maria Serena

2010-01-01

Ribosome-inactivating proteins (RIPs) are EC3.2.32.22 N-glycosidases that recognize a universally conserved stem-loop structure in 23S/25S/28S rRNA, depurinating a single adenine (A4324 in rat) and irreversibly blocking protein translation, leading finally to cell death of intoxicated mammalian cells. Ricin, the plant RIP prototype that comprises a catalytic A subunit linked to a galactose-binding lectin B subunit to allow cell surface binding and toxin entry in most mammalian cells, shows a potency in the picomolar range. The most promising way to exploit plant RIPs as weapons against cancer cells is either by designing molecules in which the toxic domains are linked to selective tumor targeting domains or directly delivered as suicide genes for cancer gene therapy. Here, we will provide a comprehensive picture of plant RIPs and discuss successful designs and features of chimeric molecules having therapeutic potential. PMID:22069572

Krebs cycle metabolon: structural evidence of substrate channeling revealed by cross-linking and mass spectrometry.

PubMed

Wu, Fei; Minteer, Shelley

2015-02-02

It has been hypothesized that the high metabolic flux in the mitochondria is due to the self-assembly of enzyme supercomplexes (called metabolons) that channel substrates from one enzyme to another, but there has been no experimental confirmation of this structure or the channeling. A structural investigation of enzyme organization within the Krebs cycle metabolon was accomplished by in vivo cross-linking and mass spectrometry. Eight Krebs cycle enzyme components were isolated upon chemical fixation, and interfacial residues between mitochondrial malate dehydrogenase, citrate synthase, and aconitase were identified. Using constraint protein docking, a low-resolution structure for the three-enzyme complex was achieved, as well as the two-fold symmetric octamer. Surface analysis showed formation of electrostatic channeling upon protein-protein association, which is the first structural evidence of substrate channeling in the Krebs cycle metabolon. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Unexpected Role for a Serine/Threonine-Rich Domain in the Candida albicans Iff Protein Family▿†

PubMed Central

Boisramé, Anita; Cornu, Amandine; Da Costa, Grégory; Richard, Mathias L.

2011-01-01

Glycosylphosphatidylinositol (GPI)-anchored proteins are an important class of cell wall proteins in Candida albicans because of their localization and their function, even if more than half of them have no characterized homolog in the databases. In this study, we focused on the IFF protein family, investigating their exposure on the cell surface and the sequences that determine their subcellular localization. Protein localization and surface exposure were monitored by the addition of a V5 tag on all members of the family. The data obtained using the complete proteins showed for Iff3 (or -9), Iff5, Iff6, and Iff8 a covalent linkage to the β-1,6-glucan network but, remarkably, showed that Iff2/Hyr3 was linked through disulfide bridges or NaOH-labile bonds. However, since some proteins of the Iff family were undetectable, we designed chimeric constructions using the last 60 amino acids of these proteins to test the localization signal. These constructions showed a β-1,6-glucan linkage for Iff1/Rbr3, Iff2/Hyr3, Iff4 and Iff7/Hyr4 C-terminal–Iff5 fusion proteins, and a membrane localization for the Iff10/Flo9 C terminus-Iff5 fusion protein. Immunofluorescence analyses coupled to these cell fraction data confirmed the importance of the length of the central serine/threonine-rich region for cell surface exposure. Further analysis of the Iff2/Hyr3 linkage to the cell surface showed for the first time that a serine/threonine central region of a GPI-anchored protein may be responsible for the disulfide and the NaOH bonds to the glucan and glycoproteins network and may also override the signal of the proximal ω site region. PMID:21841123

Sperm Lysozyme-Like Protein 1 (SLLP1), an intra-acrosomal oolemmal-binding sperm protein, reveals filamentous organization in protein crystal form

PubMed Central

Zheng, Heping; Mandal, Arabinda; Shumilin, Igor A.; Chordia, Mahendra D.; Panneerdoss, Subbarayalu; Herr, John C.; Minor, Wladek

2016-01-01

Sperm Lysozyme-Like Protein 1 (SLLP1) is one of the lysozyme-like proteins predominantly expressed in mammalian testes that lacks bacteriolytic activity, localizes in the sperm acrosome, and exhibits high affinity for an oolemmal receptor, SAS1B. The crystal structure of mouse SLLP1 (mSLLP1) was determined at 2.15Å resolution. mSLLP1 monomer adopts a structural fold similar to that of chicken/mouse lysozymes retaining all four canonical disulfide bonds. mSLLP1 is distinct from c-lysozyme by substituting two essential catalytic residues (E35T/D52N), exhibiting different surface charge distribution, and by forming helical filaments approximately 75Å in diameter with a 25Å central pore comprised of six monomers per helix turn repeating every 33Å. Cross-species alignment of all reported SLLP1 sequences revealed a set of invariant surface regions comprising a characteristic fingerprint uniquely identifying SLLP1 from other c-lysozyme family members. The fingerprint surface regions reside around the lips of the putative glycan binding groove including three polar residues (Y33/E46/H113). A flexible salt bridge (E46-R61) was observed covering the glycan binding groove. The conservation of these regions may be linked to their involvement in oolemmal protein binding. Interaction between SLLP1 monomer and its oolemmal receptor SAS1B was modeled using protein-protein docking algorithms, utilizing the SLLP1 fingerprint regions along with the SAS1B conserved surface regions. This computational model revealed complementarity between the conserved SLLP1/SAS1B interacting surfaces supporting the experimentally-observed SLLP1/SAS1B interaction involved in fertilization. PMID:26198801

Sperm Lysozyme-Like Protein 1 (SLLP1), an intra-acrosomal oolemmal-binding sperm protein, reveals filamentous organization in protein crystal form.

PubMed

Zheng, H; Mandal, A; Shumilin, I A; Chordia, M D; Panneerdoss, S; Herr, J C; Minor, W

2015-07-01

Sperm lysozyme-like protein 1 (SLLP1) is one of the lysozyme-like proteins predominantly expressed in mammalian testes that lacks bacteriolytic activity, localizes in the sperm acrosome, and exhibits high affinity for an oolemmal receptor, SAS1B. The crystal structure of mouse SLLP1 (mSLLP1) was determined at 2.15 Å resolution. mSLLP1 monomer adopts a structural fold similar to that of chicken/mouse lysozymes retaining all four canonical disulfide bonds. mSLLP1 is distinct from c-lysozyme by substituting two essential catalytic residues (E35T/D52N), exhibiting different surface charge distribution, and by forming helical filaments approximately 75 Å in diameter with a 25 Å central pore comprised of six monomers per helix turn repeating every 33 Å. Cross-species alignment of all reported SLLP1 sequences revealed a set of invariant surface regions comprising a characteristic fingerprint uniquely identifying SLLP1 from other c-lysozyme family members. The fingerprint surface regions reside around the lips of the putative glycan-binding groove including three polar residues (Y33/E46/H113). A flexible salt bridge (E46-R61) was observed covering the glycan-binding groove. The conservation of these regions may be linked to their involvement in oolemmal protein binding. Interaction between SLLP1 monomer and its oolemmal receptor SAS1B was modeled using protein-protein docking algorithms, utilizing the SLLP1 fingerprint regions along with the SAS1B conserved surface regions. This computational model revealed complementarity between the conserved SLLP1/SAS1B interacting surfaces supporting the experimentally observed SLLP1/SAS1B interaction involved in fertilization. © 2015 American Society of Andrology and European Academy of Andrology.

A novel water-based process produces eco-friendly bio-adhesive made from green cross-linked soybean soluble polysaccharide and soy protein.

PubMed

Yuan, Cheng; Chen, Mingsong; Luo, Jing; Li, Xiaona; Gao, Qiang; Li, Jianzhang

2017-08-01

In this study, an eco-friendly soy protein adhesive was developed that utilized two components from soybean meal without addition of any toxic material. A plant-based, water-soluble and inexpensive soybean soluble polysaccharide was used as the novel renewable material to combine with soy protein to produce a soy protein adhesive. Three-plywood was fabricated with the resulting adhesive, and its wet shear strength was measured. The results showed the wet shear strength of plywood bonded by the adhesive reached 0.99MPa, meeting the water resistance requirement for interior use panels. This improvement was attributed to the following reasons: (1) Combination of cross-linked soybean soluble polysaccharide and soy protein formed an interpenetrating network structure, improving the thermal stability and water resistance of the cured adhesive. (2) Adding CL-SSPS decreased the adhesive viscosity to 15.14Pas, which increased the amount of the adhesive that penetrate the wood's surface and formed more interlocks. Copyright © 2017 Elsevier Ltd. All rights reserved.

Molecular modeling of fibronectin adsorption on topographically nanostructured rutile (110) surfaces

NASA Astrophysics Data System (ADS)

Guo, Chuangqiang; Wu, Chunya; Chen, Mingjun; Zheng, Ting; Chen, Ni; Cummings, Peter T.

2016-10-01

To investigate the topographical dependency of protein adsorption, molecular dynamics simulations were employed to describe the adsorption behavior of the tenth type-III module of fibronectin (FN-III10) on nanostructured rutile (110) surfaces. The results indicated that the residence time of adsorbed FN-III10 largely relied on its binding mode (direct or indirect) with the substrate and the region for protein migration on the periphery (protrusion) or in the interior (cavity or groove) of nanostructures. In the direct binding mode, FN-III10 molecules were found to be 'trapped' at the anchoring sites of rutile surface, or even penetrate deep into the interior of nanostructures, regardless of the presented geometrical features. In the indirect binding mode, FN-III10 molecules were indirectly connected to the substrate via a hydrogen-bond network (linking FN-III10 and interfacial hydrations). The facets created by nanostructures, which exerted restraints on protein migration, were suggested to play an important role in the stability of indirect FN-III10-rutile binding. However, a doubly unfavorable situation - indirect FN-III10-rutile connections bridged by a handful of mediating waters and few constraints on movement of protein provided by nanostructures - would result in an early desorption of protein.

Binding of heparin to plasma proteins and endothelial surfaces is inhibited by covalent linkage to antithrombin.

PubMed

Chan, Anthony K C; Paredes, Nethnapha; Thong, Bruce; Chindemi, Paul; Paes, Bosco; Berry, Leslie R; Monagle, Paul

2004-05-01

Unfractionated heparin (UFH) and low molecular weight heparin (LMWH) are used for prophylaxis and treatment of thrombosis. However, UFH has a short plasma half-life and variable anticoagulant response in vivo due to plasma or vessel wall protein binding and LMWH has a decreased ability to inactivate thrombin, the pivotal enzyme in the coagulation cascade. Covalent linkage of antithrombin to heparin gave a complex (ATH) with superior anticoagulant activity compared to UFH and LMWH, and longer intravenous half-life compared to UFH. We found that plasma proteins bound more to UFH than ATH, and least to LMWH. Also, UFH bound significantly more to endothelial cells than ATH, with 100% of UFH and 94% of ATH binding being on the cell surface and the remainder was endocytosed. Competition studies with UFH confirmed that ATH binding was likely through its heparin moiety. These findings suggest that differences in plasma protein and endothelial cell binding may be due to available heparin chain length. Although ATH is polydisperse, the covalently-linked antithrombin may shield a portion of the heparin chain from association with plasma or endothelial cell surface proteins. This model is consistent with ATH's better bioavailability and more predictable dose response.

Surface Functionalization of Exosomes Using Click Chemistry

PubMed Central

2015-01-01

A method for conjugation of ligands to the surface of exosomes was developed using click chemistry. Copper-catalyzed azide alkyne cycloaddition (click chemistry) is ideal for biocojugation of small molecules and macromolecules to the surface of exosomes, due to fast reaction times, high specificity, and compatibility in aqueous buffers. Exosomes cross-linked with alkyne groups using carbodiimide chemistry were conjugated to a model azide, azide-fluor 545. Conjugation had no effect on the size of exosomes, nor was there any change in the extent of exosome adherence/internalization with recipient cells, suggesting the reaction conditions were mild on exosome structure and function. We further investigated the extent of exosomal protein modification with alkyne groups. Using liposomes with surface alkyne groups of a similar size and concentration to exosomes, we estimated that approximately 1.5 alkyne groups were present for every 150 kDa of exosomal protein. PMID:25220352

Protein crystals as scanned probes for recognition atomic force microscopy.

PubMed

Wickremasinghe, Nissanka S; Hafner, Jason H

2005-12-01

Lysozyme crystal growth has been localized at the tip of a conventional silicon nitride cantilever through seeded nucleation. After cross-linking with glutaraldehyde, lysozyme protein crystal tips image gold nanoparticles and grating standards with a resolution comparable to that of conventional tips. Force spectra between the lysozyme crystal tips and surfaces covered with antilysozyme reveal an adhesion force that drops significantly upon blocking with free lysozyme, thus confirming that lysozyme crystal tips can detect molecular recognition interactions.

Dextran hydrogel coated surface plasmon resonance imaging (SPRi) sensor for sensitive and label-free detection of small molecule drugs

NASA Astrophysics Data System (ADS)

Li, Shaopeng; Yang, Mo; Zhou, Wenfei; Johnston, Trevor G.; Wang, Rui; Zhu, Jinsong

2015-11-01

The label-free and sensitive detection of small molecule drugs on SPRi is still a challenging task, mainly due to the limited surface immobilization capacity of the sensor. In this research, a dextran hydrogel-coated gold sensor chip for SPRi was successfully fabricated via photo-cross-linking for enhanced surface immobilization capacity. The density of the dextran hydrogel was optimized for protein immobilization and sensitive small molecule detection. The protein immobilization capacity of the hydrogel was 10 times greater than a bare gold surface, and 20 times greater than an 11-mercaptoundecanoic acid (MUA) surface. Such a drastic improvement in immobilization capacity allowed the SPRi sensor to detect adequate response signals when probing small molecule binding events. The binding signal of 4 nM liquid-phase biotin to streptavidin immobilized on the dextran surface reached 435 RU, while no response was observed on bare gold or MUA surfaces. The dextran hydrogel-coated SPRi sensor was also applied in a kinetic study of the binding between an immunosuppressive drug (FK506) and its target protein (FKBP12) in a high-throughput microarray format. The measured binding affinity was shown to be consistent with reported literature values, and a detection limit of 0.5 nM was achieved.

Comparative analysis of Staphylococcus epidermidis strains utilizing quantitative and cell surface shaving proteomics.

PubMed

Solis, Nestor; Cain, Joel A; Cordwell, Stuart J

2016-01-01

Staphylococcus epidermidis is an opportunistic pathogen that is an emerging risk factor in hospitals worldwide and is often difficult to eradicate as virulent strains produce a protective biofilm matrix. We utilized cell shaving proteomics to profile surface-exposed proteins from two fully genome sequenced S. epidermidis strains: the avirulent, non-biofilm forming ATCC12228 and the virulent, strongly adherent biofilm forming ATCC35984 (RP62A). A false positive control strategy was employed to calculate the probabilities of proteins being truly surface-exposed. A total of 78 surface-exposed proteins were identified, of which only 19 proteins were common to ATCC12228 and RP62A, and which thus represents the core surfaceome. S. epidermidis RP62A displayed additional proteins involved in biofilm formation (cell wall-associated Bhp and intercellular adhesion protein IcaB), surface antigenicity, peptidoglycan biosynthesis and antibiotic resistance. We concurrently profiled whole cell proteomes of the two strains using iTRAQ quantitation and LC-MS/MS. A total of 1610 proteins were confidently identified (representing 64% of the theoretical S. epidermidis proteome). One hundred and ninety one proteins were differentially abundant between strains. Proteins associated with RP62A were clustered into functions including Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-mediated defense, sulfate assimilation, antibiotic resistance and biofilm formation. Validation of the sulfate assimilation and cysteine/methionine biosynthesis pathways showed RP62A contained elevated levels (~25% increase) of methionine that are likely linked to biofilm formation. Cell shaving and quantitative proteomics identified proteins associated with a biofilm-forming, virulent strain of S. epidermidis (RP62A). These proteins show RP62A maintains an active CRISPR-mediated defense, as well as heightened antibiotic resistance in comparison to a non-virulent, non-biofilm forming strain. Increased abundances of sulfate assimilation proteins lead to elevated intracellular methionine. Proteins and their exposed peptides identified on the surface of S. epidermidis RP62A may be useful vaccine antigens in clinical settings if administered in at-risk patients prior to surgical implantations. Copyright © 2015 Elsevier B.V. All rights reserved.

Affinity modulation of small-molecule ligands by borrowing endogenous protein surfaces

PubMed Central

Briesewitz, Roger; Ray, Gregory T.; Wandless, Thomas J.; Crabtree, Gerald R.

1999-01-01

A general strategy is described for improving the binding properties of small-molecule ligands to protein targets. A bifunctional molecule is created by chemically linking a ligand of interest to another small molecule that binds tightly to a second protein. When the ligand of interest is presented to the target protein by the second protein, additional protein–protein interactions outside of the ligand-binding sites serve either to increase or decrease the affinity of the binding event. We have applied this approach to an intractable target, the SH2 domain, and demonstrate a 3-fold enhancement over the natural peptide. This approach provides a way to modulate the potency and specificity of biologically active compounds. PMID:10051576

Investigation of molybdenum-crosslinker interfaces for affinity based electrochemical biosensing applications

NASA Astrophysics Data System (ADS)

Kamakoti, Vikramshankar; Shanmugam, Nandhinee Radha; Tanak, Ambalika Sanjeev; Jagannath, Badrinath; Prasad, Shalini

2018-04-01

Molybdenum (Mo) has been investigated for implementation as an electrode material for affinity based biosensing towards devloping flexibe electronic biosensors. Treatment of the native oxide of molybdenum was investigated through two surface treatment strategies namely thiol and carbodiimide crosslinking methods. The binding interaction between cross-linker molecules and Mo electrode surface has been characterized using Fourier Transform Infrared Spectroscopy (FTIR), X-ray photoelectron spectroscopy (XPS) and optical microscopy. The efficacy of treatment of Mo with its native oxide using carbodiimide cross linking methodology was established. The carbodiimide cross-linking chemistry was found to possess better surface coverage and binding affinity with Molybdenum electrode surface when compared to thiol cross-linking chemistry.Electrochemical characterization of Mo electrode using Electrochemical Impedance Spectroscopy (EIS) and Cyclic Voltametry (CV) techniques was performed to evaluate the effect of ionic properties of solution buffer on the Mo electrode's performance. Affinity based biosensing of C-Reactive Protein (CRP) has been demonstrated on a flexible nanoporous polymeric substrate with detection threshold of 100 pg/ml in synthetic urine buffer medium. The biosensor has been evaluated to be developed as a dipstick based point of care device for detection of biomarkers in urine.

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.

Arginine- and lysine-specific polymers for protein recognition and immobilization.

PubMed

Renner, Christian; Piehler, Jacob; Schrader, Thomas

2006-01-18

Free radical polymerization of methacrylamide-based bisphosphonates turns weak arginine binders into powerful polymeric protein receptors. Dansyl-labeled homo- and copolymers with excellent water solubility are accessible through a simple copolymerization protocol. Modeling studies point to a striking structural difference between the stiff rodlike densely packed homopolymer 1 and the flexible copolymer 2 with spatially separated bisphosphonate units. Fluorescence titrations in buffered aqueous solution (pH = 7.0) confirm the superior affinity of the homopolymer toward oligoarginine peptides reaching nanomolar K(D) values for the Tat peptide. Basic proteins are bound almost equally well by 1 and 2 with micromolar affinities, with the latter producing much more soluble complexes. The Arg selectivity of the monomer is transferred to the polymer, which binds Arg-rich proteins 1 order of magnitude tighter than lysine-rich pendants of comparable pI, size, and (Arg/Lys vs Glu/Asp) ratio. Noncovalent deposition of both polymers on glass substrates via polyethyleneimine layers results in new materials suitable for peptide and protein immobilization. RIfS measurements allow calculation of association constants K(a) as well as dissociation kinetics k(D). They generally confirm the trends already found in free solution. Close inspection of electrostatic potential surfaces suggest that basic domains favor protein binding on the flat surface. The high specificity of the bisphosphonate polymers toward basic proteins is demonstrated by comparison with polyvinyl sulfate, which has almost no effect in RIfS experiments. Thus, copolymerization of few different comonomer units without cross-linking enables surface recognition of basic proteins in free solution as well as their effective immobilization on surfaces.

Ca2+ Binding to EF Hands 1 and 3 Is Essential for the Interaction of Apoptosis-Linked Gene-2 with Alix/AIP1 in Ocular Melanoma†

PubMed Central

Subramanian, Lalita; Polans, Arthur S.; Walker, Teresa M.; van Ginkel, Paul R.; Bhattacharya, Saswati; Dellaria, Julia M.; Crabb, John W.; Cox, Jos; Durussel, Isabelle; Palczewski, Krzysztof

2005-01-01

Apoptosis-linked gene-2 (ALG-2) encodes a 22 kDa Ca2+-binding protein of the penta EF-hand family that is required for programmed cell death in response to various apoptotic agents. Here, we demonstrate that ALG-2 mRNA and protein are down-regulated in human uveal melanoma cells compared to their progenitor cells, normal melanocytes. The down regulation of ALG-2 may provide melanoma cells with a selective advantage. ALG-2 and its putative target molecule, Alix/AIP1, are localized primarily in the cytoplasm of melanocytes and melanoma cells independent of the intracellular Ca2+ concentration or the activation of apoptosis. Cross-linking and analytical centrifugation studies support a single-species dimer conformation of ALG-2, also independent of Ca2+ concentration. However, binding of Ca2+ to both EF-1 and EF-3 is necessary for ALG-2 interaction with Alix/AIP1 as demonstrated using surface plasmon resonance spectroscopy. Mutations in EF-5 result in reduced target interaction without alteration in Ca2+ affinity. The addition of N-terminal ALG-2 peptides, residues 1–22 or residues 7–17, does not alter the interaction of ALG-2 or an N-terminal deletion mutant of ALG-2 with Alix/AIP1, as might be expected from a model derived from the crystal structure of ALG-2. Fluorescence studies of ALG-2 demonstrate that an increase in surface hydrophobicity is primarily due to Ca2+ binding to EF-3, while Ca2+ binding to EF-1 has little effect on surface exposure of hydrophobic residues. Together, these data indicate that gross surface hydrophobicity changes are insufficient for target recognition. PMID:15366927

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.

The leukocyte common antigen (CD45): a putative receptor-linked protein tyrosine phosphatase.

PubMed Central

Charbonneau, H; Tonks, N K; Walsh, K A; Fischer, E H

1988-01-01

A major protein tyrosine phosphatase (PTPase 1B) has been isolated in essentially homogeneous form from the soluble and particulate fractions of human placenta. Unexpectedly, partial amino acid sequences displayed no homology with the primary structures of the protein Ser/Thr phosphatases deduced from cDNA clones. However, the sequence is strikingly similar to the tandem C-terminal homologous domains of the leukocyte common antigen (CD45). A 157-residue segment of PTPase 1B displayed 40% and 33% sequence identity with corresponding regions from cytoplasmic domains I and II of human CD45. Similar degrees of identity have been observed among the catalytic domains of families of regulatory proteins such as protein kinases and cyclic nucleotide phosphodiesterases. On this basis, it is proposed that the CD45 family has protein tyrosine phosphatase activity and may represent a set of cell-surface receptors involved in signal transduction. This suggests that the repertoire of signal transduction mechanisms may include the direct control of an intracellular protein tyrosine phosphatase, offering the possibility of a regulatory balance with those protein tyrosine kinases that act at the internal surface of the membrane. Images PMID:2845400

Raising the shields: PCR in the presence of metallic surfaces protected by tailor-made coatings.

PubMed

Scherag, Frank D; Brandstetter, Thomas; Rühe, Jürgen

2014-10-01

The implementation of PCR reactions in the presence of metallic surfaces is interesting for the generation of novel bioanalytical devices, because metals exhibit high mechanical stability, good thermal conductivity, and flexibility during deformation. However, metallic substrates are usually non-compatible with enzymatic reactions such as PCR due to poisoning of the active center of the enzyme or nonspecific adsorption of the enzymeto the metal surface, which could result in protein denaturation. We present a method for the generation of polymer coatings on metallic surfaces which are designed to minimize protein adsorption and also prevent the release of metal ions. These coatings consist of three layers covalently linked to each other; a self-assembled monolayer to promote adhesion, a photochemically generated barrier layer and a photochemically generated hydrogel. The coatings can be deposited onto aluminum, stainless steel, gold and copper surfaces. We compare PCR efficiencies in the presence of bare metallic surfaces with those of surfaces treated with the novel coating system. Copyright © 2014 Elsevier B.V. All rights reserved.

New malaria vaccine candidates based on the Plasmodium vivax Merozoite Surface Protein-1 and the TLR-5 agonist Salmonella Typhimurium FliC flagellin.

PubMed

Bargieri, Daniel Y; Rosa, Daniela S; Braga, Catarina J M; Carvalho, Bruna O; Costa, Fabio T M; Espíndola, Noeli Maria; Vaz, Adelaide José; Soares, Irene S; Ferreira, Luis C S; Rodrigues, Mauricio M

2008-11-11

The present study evaluated the immunogenicity of new malaria vaccine formulations based on the 19kDa C-terminal fragment of Plasmodium vivax Merozoite Surface Protein-1 (MSP1(19)) and the Salmonella enterica serovar Typhimurium flagellin (FliC), a Toll-like receptor 5 (TLR5) agonist. FliC was used as an adjuvant either admixed or genetically linked to the P. vivax MSP1(19) and administered to C57BL/6 mice via parenteral (s.c.) or mucosal (i.n.) routes. The recombinant fusion protein preserved MSP1(19) epitopes recognized by sera collected from P. vivax infected humans and TLR5 agonist activity. Mice parenterally immunized with recombinant P. vivax MSP1(19) in the presence of FliC, either admixed or genetically linked, elicited strong and long-lasting MSP1(19)-specific systemic antibody responses with a prevailing IgG1 subclass response. Incorporation of another TLR agonist, CpG ODN 1826, resulted in a more balanced response, as evaluated by the IgG1/IgG2c ratio, and higher cell-mediated immune response measured by interferon-gamma secretion. Finally, we show that MSP1(19)-specific antibodies recognized the native protein expressed on the surface of P. vivax parasites harvested from infected humans. The present report proposes a new class of malaria vaccine formulation based on the use of malarial antigens and the innate immunity agonist FliC. It contains intrinsic adjuvant properties and enhanced ability to induce specific humoral and cellular immune responses when administered alone or in combination with other adjuvants.

Ligand-specific regulation of the extracellular surface of a G-protein-coupled receptor

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

Bokoch, Michael P.; Zou, Yaozhong; Rasmussen, Søren G.F.

G-protein-coupled receptors (GPCRs) are seven-transmembrane proteins that mediate most cellular responses to hormones and neurotransmitters. They are the largest group of therapeutic targets for a broad spectrum of diseases. Recent crystal structures of GPCRs have revealed structural conservation extending from the orthosteric ligand-binding site in the transmembrane core to the cytoplasmic G-protein-coupling domains. In contrast, the extracellular surface (ECS) of GPCRs is remarkably diverse and is therefore an ideal target for the discovery of subtype-selective drugs. However, little is known about the functional role of the ECS in receptor activation, or about conformational coupling of this surface to the nativemore » ligand-binding pocket. Here we use NMR spectroscopy to investigate ligand-specific conformational changes around a central structural feature in the ECS of the {beta}{sub 2} adrenergic receptor: a salt bridge linking extracellular loops 2 and 3. Small-molecule drugs that bind within the transmembrane core and exhibit different efficacies towards G-protein activation (agonist, neutral antagonist and inverse agonist) also stabilize distinct conformations of the ECS. We thereby demonstrate conformational coupling between the ECS and the orthosteric binding site, showing that drugs targeting this diverse surface could function as allosteric modulators with high subtype selectivity. Moreover, these studies provide a new insight into the dynamic behaviour of GPCRs not addressable by static, inactive-state crystal structures.« less

Role of surface layer collagen binding protein from indigenous Lactobacillus plantarum 91 in adhesion and its anti-adhesion potential against gut pathogen.

PubMed

Yadav, Ashok Kumar; Tyagi, Ashish; Kaushik, Jai Kumar; Saklani, Asha Chandola; Grover, Sunita; Batish, Virender Kumar

2013-12-14

Human feacal isolates were ascertain as genus Lactobacillus using specific primer LbLMA1/R16-1 and further identified as Lactobacillus plantarum with species specific primers Lpl-3/Lpl-2. 25 L. plantarum strains were further assessed for hydrophobicity following the microbial adhesion to hydrocarbons (MATH) method and colonization potentials based on their adherence to immobilized human collagen type-1. Surface proteins were isolated from selected L. plantarum 91(Lp91) strain. The purified collagen binding protein (Cbp) protein was assessed for its anti-adhesion activity against enteric Escherichia coli 0157:H7 pathogen on immobilized collagen. Four L. plantarum strains displayed high degree of hydrophobicity and significant adhesion to collagen. A 72 kDa protein was purified which reduced 59.71% adhesion of E. coli 0157:H7 on immobilized collagen as compared to control well during adhesion assay. Cbp protein is the major influencing factor in inhibition of E. coli 0157:H7 adhesion with extracellular matrix (ECM) components. Hydrophobicity and adhesion potential are closely linked attributes precipitating in better colonization potential of the lactobacillus strains. Cbp is substantiated as a crucial surface protein contributing in adhesion of lactobacillus strains. The study can very well be the platform for commercialization of indigenous probiotic strain once their functional attributes are clinically explored. Copyright © 2013 Elsevier GmbH. All rights reserved.

Epididymosomes: transfer of fertility-modulating proteins to the sperm surface

PubMed Central

Martin-DeLeon, Patricia A

2015-01-01

A variety of glycosylphosphatidylinositol (GPI)-linked proteins are acquired on spermatozoa from epididymal luminal fluids (ELF) during sperm maturation. These proteins serve roles in immunoprotection and in key steps of fertilization such as capacitation, acrosomal exocytosis and sperm-egg interactions. Their acquisition on sperm cells is mediated both by membrane vesicles (epididymosomes, EP) which were first reported to dock on the sperm surface, and by lipid carriers which facilitate the transfer of proteins associated with the membrane-free fraction of ELF. While the nonvesicular fraction is more efficient, both pathways are dependent on hydrophobic interactions between the GPI-anchor and the external lipid layer of the sperm surface. More recently proteomic and hypothesis-driven studies have shown that EP from several mammals carry transmembrane (TM) proteins, including plasma membrane Ca2+-ATPase 4 (PMCA4). Synthesized in the testis, PMCA4 is an essential protein and the major Ca2+ efflux pump in murine spermatozoa. Delivery of PMCA4 to spermatozoa from bovine and mouse EP during epididymal maturation and in vitro suggests that the docking of EP on the sperm surface precedes fusion, and experimental evidence supports a fusogenic mechanism for TM proteins. Fusion is facilitated by CD9, which generates fusion–competent sites on membranes. On the basis of knowledge of PMCA4's interacting partners a number of TM and membrane-associated proteins have been identified or are predicted to be present, in the epididymosomal cargo deliverable to spermatozoa. These Ca2+-dependent proteins, undetected in proteomic studies, play essential roles in sperm motility and fertility, and their detection highlights the usefulness of the hypothesis-driven approach. PMID:26112481

Epididymosomes: transfer of fertility-modulating proteins to the sperm surface.

PubMed

Martin-DeLeon, Patricia A

2015-01-01

A variety of glycosylphosphatidylinositol (GPI)-linked proteins are acquired on spermatozoa from epididymal luminal fluids (ELF) during sperm maturation. These proteins serve roles in immunoprotection and in key steps of fertilization such as capacitation, acrosomal exocytosis and sperm-egg interactions. Their acquisition on sperm cells is mediated both by membrane vesicles (epididymosomes, EP) which were first reported to dock on the sperm surface, and by lipid carriers which facilitate the transfer of proteins associated with the membrane-free fraction of ELF. While the nonvesicular fraction is more efficient, both pathways are dependent on hydrophobic interactions between the GPI-anchor and the external lipid layer of the sperm surface. More recently proteomic and hypothesis-driven studies have shown that EP from several mammals carry transmembrane (TM) proteins, including plasma membrane Ca 2 + -ATPase 4 (PMCA4). Synthesized in the testis, PMCA4 is an essential protein and the major Ca 2 + efflux pump in murine spermatozoa. Delivery of PMCA4 to spermatozoa from bovine and mouse EP during epididymal maturation and in vitro suggests that the docking of EP on the sperm surface precedes fusion, and experimental evidence supports a fusogenic mechanism for TM proteins. Fusion is facilitated by CD9, which generates fusion-competent sites on membranes. On the basis of knowledge of PMCA4's interacting partners a number of TM and membrane-associated proteins have been identified or are predicted to be present, in the epididymosomal cargo deliverable to spermatozoa. These Ca 2 + -dependent proteins, undetected in proteomic studies, play essential roles in sperm motility and fertility, and their detection highlights the usefulness of the hypothesis-driven approach.

Comparison of analytical protein separation characteristics for three amine-based capillary-channeled polymer (C-CP) stationary phases.

PubMed

Jiang, Liuwei; Marcus, R Kenneth

2016-02-01

Capillary-channeled polymer (C-CP) fiber stationary phases are finding utility in the realms of protein analytics as well as downstream processing. We have recently described the modification of poly(ethylene terephthalate) (PET) C-CP fibers to affect amine-rich phases for the weak anion-exchange (WAX) separation of proteins. Polyethylenimine (PEI) is covalently coupled to the PET surface, with subsequent cross-linking imparted by treatment with 1,4-butanediol diglycidyl ether (BUDGE). These modifications yield vastly improved dynamic binding capacities over the unmodified fibers. We have also previously employed native (unmodified) nylon 6 C-CP fibers as weak anion/cation-exchange (mixed-mode) and hydrophobic interaction chromatography (HIC) phases for protein separations. Polyamide, nylon 6, consists of amide groups along the polymer backbone, with primary amines and carboxylic acid end groups. The analytical separation characteristics of these three amine-based C-CP fiber phases are compared here. Each of the C-CP fiber columns in this study was shown to be able to separate a bovine serum albumin/hemoglobin/lysozyme mixture at high mobile phase linear velocity (∼70 mm s(-1)) but with different elution characteristics. These differences reflect the types of protein-surface interactions that are occurring, based on the active group composition of the fiber surfaces. This study provides important fundamental understanding for the development of surface-modified C-CP fiber columns for protein separation.

Unprecedented Ionization Processes in Mass Spectrometry Provide Missing Link between ESI and MALDI.

PubMed

Trimpin, Sarah; Lee, Chuping; Weidner, Steffen M; El-Baba, Tarick J; Lutomski, Corinne A; Inutan, Ellen D; Foley, Casey D; Ni, Chi-Kung; McEwen, Charles N

2018-03-05

In the field of mass spectrometry, producing intact, highly-charged protein ions from surfaces is a conundrum with significant potential payoff in application areas ranging from biomedical to clinical research. Here, we report on the ability to form intact, highly-charged protein ions on high vacuum time-of-flight mass spectrometers in the linear and reflectron modes achievable using experimental conditions that allow effective matrix removal from both the sample surfaces and from the charged clusters formed by the laser ablation event. The charge states are the highest reported on high vacuum mass spectrometers, yet they remain at only around a third of the highest charge obtained using laser ablation with a suitable matrix at atmospheric pressure. Other than physical instrument modifications, the key to forming abundant and stable highly-charged ions appears to be the volatility of the matrix used. Cumulative results suggest mechanistic links between the ionization process reported here and traditional ionization methods of electrospray ionization and matrix-assisted laser desorption/ionization. © 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

The Identification and Functional Characterization of WxL Proteins from Enterococcus faecium Reveal Surface Proteins Involved in Extracellular Matrix Interactions

PubMed Central

Galloway-Peña, Jessica R.; Liang, Xiaowen; Singh, Kavindra V.; Yadav, Puja; Chang, Chungyu; La Rosa, Sabina Leanti; Shelburne, Samuel; Ton-That, Hung; Höök, Magnus

2014-01-01

The WxL domain recently has been identified as a novel cell wall binding domain found in numerous predicted proteins within multiple Gram-positive bacterial species. However, little is known about the function of proteins containing this novel domain. Here, we identify and characterize 6 Enterococcus faecium proteins containing the WxL domain which, by reverse transcription-PCR (RT-PCR) and genomic analyses, are located in three similarly organized operons, deemed WxL loci A, B, and C. Western blotting, electron microscopy, and enzyme-linked immunosorbent assays (ELISAs) determined that genes of WxL loci A and C encode antigenic, cell surface proteins exposed at higher levels in clinical isolates than in commensal isolates. Secondary structural analyses of locus A recombinant WxL domain-containing proteins found they are rich in β-sheet structure and disordered segments. Using Biacore analyses, we discovered that recombinant WxL proteins from locus A bind human extracellular matrix proteins, specifically type I collagen and fibronectin. Proteins encoded by locus A also were found to bind to each other, suggesting a novel cell surface complex. Furthermore, bile salt survival assays and animal models using a mutant from which all three WxL loci were deleted revealed the involvement of WxL operons in bile salt stress and endocarditis pathogenesis. In summary, these studies extend our understanding of proteins containing the WxL domain and their potential impact on colonization and virulence in E. faecium and possibly other Gram-positive bacterial species. PMID:25512313

Glycomic characterization of basal tears and changes with diabetes and diabetic retinopathy.

PubMed

Nguyen-Khuong, Terry; Everest-Dass, Arun V; Kautto, Liisa; Zhao, Zhenjun; Willcox, Mark D P; Packer, Nicolle H

2015-03-01

As a secreted fluid, the state of tear glycosylation is particularly important in the role of immunity of the ocular surface. Tears are a valuable source of non-invasive biomarkers for disease and there are continued efforts to characterize their components thoroughly. In this study, a small volume of basal tears (5 μL) was collected from healthy controls, patients with diabetes without retinopathy and patients with diabetes and retinopathy. The detailed N- and O-linked tear protein glycome was characterized and the relative abundance of each structure determined. Of the 50 N-linked glycans found, 89% were complex with 50% containing a bisecting N-acetylglucosamine, 65% containing a core fucose whilst 33% were sialylated. Of the 8 O-linked glycans detected, 3 were of cores 1 and 5 of core 2 type, with a majority of them being sialylated (90%). Additionally, these glycan structures were profiled across the three diabetic disease groups. Whilst the higher abundant structures did not alter across the three groups, only five low abundance N-linked glycans and 1 O-linked glycan did alter with the onset of diabetes mellitus and diabetic retinopathy (DR). These results suggest the conservation of glycan types on basal tear proteins between individuals and point to only small changes in glycan expression on the proteins in tears with the development of diabetes and DR. © The Author 2014. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

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.

The cDNA sequence of mouse Pgp-1 and homology to human CD44 cell surface antigen and proteoglycan core/link proteins.

PubMed

Wolffe, E J; Gause, W C; Pelfrey, C M; Holland, S M; Steinberg, A D; August, J T

1990-01-05

We describe the isolation and sequencing of a cDNA encoding mouse Pgp-1. An oligonucleotide probe corresponding to the NH2-terminal sequence of the purified protein was synthesized by the polymerase chain reaction and used to screen a mouse macrophage lambda gt11 library. A cDNA clone with an insert of 1.2 kilobases was selected and sequenced. In Northern blot analysis, only cells expressing Pgp-1 contained mRNA species that hybridized with this Pgp-1 cDNA. The nucleotide sequence of the cDNA has a single open reading frame that yields a protein-coding sequence of 1076 base pairs followed by a 132-base pair 3'-untranslated sequence that includes a putative polyadenylation signal but no poly(A) tail. The translated sequence comprises a 13-amino acid signal peptide followed by a polypeptide core of 345 residues corresponding to an Mr of 37,800. Portions of the deduced amino acid sequence were identical to those obtained by amino acid sequence analysis from the purified glycoprotein, confirming that the cDNA encodes Pgp-1. The predicted structure of Pgp-1 includes an NH2-terminal extracellular domain (residues 14-265), a transmembrane domain (residues 266-286), and a cytoplasmic tail (residues 287-358). Portions of the mouse Pgp-1 sequence are highly similar to that of the human CD44 cell surface glycoprotein implicated in cell adhesion. The protein also shows sequence similarity to the proteoglycan tandem repeat sequences found in cartilage link protein and cartilage proteoglycan core protein which are thought to be involved in binding to hyaluronic acid.

Near-atomic cryo-EM structure of PRC1 bound to the microtubule.

PubMed

Kellogg, Elizabeth H; Howes, Stuart; Ti, Shih-Chieh; Ramírez-Aportela, Erney; Kapoor, Tarun M; Chacón, Pablo; Nogales, Eva

2016-08-23

Proteins that associate with microtubules (MTs) are crucial to generate MT arrays and establish different cellular architectures. One example is PRC1 (protein regulator of cytokinesis 1), which cross-links antiparallel MTs and is essential for the completion of mitosis and cytokinesis. Here we describe a 4-Å-resolution cryo-EM structure of monomeric PRC1 bound to MTs. Residues in the spectrin domain of PRC1 contacting the MT are highly conserved and interact with the same pocket recognized by kinesin. We additionally found that PRC1 promotes MT assembly even in the presence of the MT stabilizer taxol. Interestingly, the angle of the spectrin domain on the MT surface corresponds to the previously observed cross-bridge angle between MTs cross-linked by full-length, dimeric PRC1. This finding, together with molecular dynamic simulations describing the intrinsic flexibility of PRC1, suggests that the MT-spectrin domain interface determines the geometry of the MT arrays cross-linked by PRC1.

Near-atomic cryo-EM structure of PRC1 bound to the microtubule

PubMed Central

Kellogg, Elizabeth H.; Howes, Stuart; Ti, Shih-Chieh; Ramírez-Aportela, Erney; Kapoor, Tarun M.; Chacón, Pablo; Nogales, Eva

2016-01-01

Proteins that associate with microtubules (MTs) are crucial to generate MT arrays and establish different cellular architectures. One example is PRC1 (protein regulator of cytokinesis 1), which cross-links antiparallel MTs and is essential for the completion of mitosis and cytokinesis. Here we describe a 4-Å–resolution cryo-EM structure of monomeric PRC1 bound to MTs. Residues in the spectrin domain of PRC1 contacting the MT are highly conserved and interact with the same pocket recognized by kinesin. We additionally found that PRC1 promotes MT assembly even in the presence of the MT stabilizer taxol. Interestingly, the angle of the spectrin domain on the MT surface corresponds to the previously observed cross-bridge angle between MTs cross-linked by full-length, dimeric PRC1. This finding, together with molecular dynamic simulations describing the intrinsic flexibility of PRC1, suggests that the MT–spectrin domain interface determines the geometry of the MT arrays cross-linked by PRC1. PMID:27493215

Structures of the major capsid proteins of the human Karolinska Institutet and Washington University polyomaviruses.

PubMed

Neu, Ursula; Wang, Jianbo; Macejak, Dennis; Garcea, Robert L; Stehle, Thilo

2011-07-01

The Karolinska Institutet and Washington University polyomaviruses (KIPyV and WUPyV, respectively) are recently discovered human viruses that infect the respiratory tract. Although they have not yet been linked to disease, they are prevalent in populations worldwide, with initial infection occurring in early childhood. Polyomavirus capsids consist of 72 pentamers of the major capsid protein viral protein 1 (VP1), which determines antigenicity and receptor specificity. The WUPyV and KIPyV VP1 proteins are distant in evolution from VP1 proteins of known structure such as simian virus 40 or murine polyomavirus. We present here the crystal structures of unassembled recombinant WUPyV and KIPyV VP1 pentamers at resolutions of 2.9 and 2.55 Å, respectively. The WUPyV and KIPyV VP1 core structures fold into the same β-sandwich that is a hallmark of all polyomavirus VP1 proteins crystallized to date. However, differences in sequence translate into profoundly different surface loop structures in KIPyV and WUPyV VP1 proteins. Such loop structures have not been observed for other polyomaviruses, and they provide initial clues about the possible interactions of these viruses with cell surface receptors.

CLAG3 Self-Associates in Malaria Parasites and Quantitatively Determines Nutrient Uptake Channels at the Host Membrane

PubMed Central

Gupta, Ankit; Balabaskaran-Nina, Praveen; Nguitragool, Wang; Saggu, Gagandeep S.; Schureck, Marc A.

2018-01-01

ABSTRACT Malaria parasites increase host erythrocyte permeability to ions and nutrients via a broad-selectivity channel known as the plasmodial surface anion channel (PSAC), linked to parasite-encoded CLAG3 and two associated proteins. These proteins lack the multiple transmembrane domains typically present in channel-forming proteins, raising doubts about their precise roles. Using the virulent human Plasmodium falciparum parasite, we report that CLAG3 undergoes self-association and that this protein’s expression determines channel phenotype quantitatively. We overcame epigenetic silencing of clag3 paralogs and engineered parasites that express two CLAG3 isoforms simultaneously. Stoichiometric expression of these isoforms yielded intermediate channel phenotypes, in agreement with observed trafficking of both proteins to the host membrane. Coimmunoprecipitation and surface labeling revealed formation of CLAG3 oligomers. In vitro selections applied to these transfectant lines yielded distinct mutants with correlated changes in channel activity. These findings support involvement of the identified oligomers in PSAC formation and parasite nutrient acquisition. PMID:29739907

Roles for SH2 and SH3 domains in Lyn kinase association with activated FcepsilonRI in RBL mast cells revealed by patterned surface analysis.

PubMed

Hammond, Stephanie; Wagenknecht-Wiesner, Alice; Veatch, Sarah L; Holowka, David; Baird, Barbara

2009-10-01

In mast cells, antigen-mediated cross-linking of IgE bound to its high-affinity surface receptor, FcepsilonRI, initiates a signaling cascade that culminates in degranulation and release of allergic mediators. Antigen-patterned surfaces, in which the antigen is deposited in micron-sized features on a silicon substrate, were used to examine the spatial relationship between clustered IgE-FcepsilonRI complexes and Lyn, the signal-initiating tyrosine kinase. RBL mast cells expressing wild-type Lyn-EGFP showed co-redistribution of this protein with clustered IgE receptors on antigen-patterned surfaces, whereas Lyn-EGFP containing an inhibitory point mutation in its SH2 domain did not significantly accumulate with the patterned antigen, and Lyn-EGFP with an inhibitory point mutation in its SH3 domain exhibited reduced interactions. Our results using antigen-patterned surfaces and quantitative cross-correlation image analysis reveal that both the SH2 and SH3 domains contribute to interactions between Lyn kinase and cross-linked IgE receptors in stimulated mast cells.

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.

Pili and flagella biology, structure, and biotechnological applications.

PubMed

Van Gerven, Nani; Waksman, Gabriel; Remaut, Han

2011-01-01

Bacteria and Archaea expose on their outer surfaces a variety of thread-like proteinaceous organelles with which they interact with their environments. These structures are repetitive assemblies of covalently or non-covalently linked protein subunits, organized into filamentous polymers known as pili ("hair"), flagella ("whips") or injectisomes ("needles"). They serve different roles in cell motility, adhesion and host invasion, protein and DNA secretion and uptake, conductance, or cellular encapsulation. Here we describe the functional, morphological and genetic diversity of these bacterial filamentous protein structures. The organized, multi-copy build-up and/or the natural function of pili and flagella have lead to their biotechnological application as display and secretion tools, as therapeutic targets or as molecular motors. We review the documented and potential technological exploitation of bacterial surface filaments in light of their structural and functional traits. Copyright © 2011 Elsevier Inc. All rights reserved.

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.

Cross-Link Guided Molecular Modeling with ROSETTA

PubMed Central

Leitner, Alexander; Rosenberger, George; Aebersold, Ruedi; Malmström, Lars

2013-01-01

Chemical cross-links identified by mass spectrometry generate distance restraints that reveal low-resolution structural information on proteins and protein complexes. The technology to reliably generate such data has become mature and robust enough to shift the focus to the question of how these distance restraints can be best integrated into molecular modeling calculations. Here, we introduce three workflows for incorporating distance restraints generated by chemical cross-linking and mass spectrometry into ROSETTA protocols for comparative and de novo modeling and protein-protein docking. We demonstrate that the cross-link validation and visualization software Xwalk facilitates successful cross-link data integration. Besides the protocols we introduce XLdb, a database of chemical cross-links from 14 different publications with 506 intra-protein and 62 inter-protein cross-links, where each cross-link can be mapped on an experimental structure from the Protein Data Bank. Finally, we demonstrate on a protein-protein docking reference data set the impact of virtual cross-links on protein docking calculations and show that an inter-protein cross-link can reduce on average the RMSD of a docking prediction by 5.0 Å. The methods and results presented here provide guidelines for the effective integration of chemical cross-link data in molecular modeling calculations and should advance the structural analysis of particularly large and transient protein complexes via hybrid structural biology methods. PMID:24069194

Genetic diversity of Plasmodium falciparum merozoite surface protein-1 block 2 in sites of contrasting altitudes and malaria endemicities in the Mount Cameroon region.

PubMed

Wanji, Samuel; Kengne-Ouafo, Arnaud J; Eyong, Ebanga E Joan; Kimbi, Helen K; Tendongfor, Nicholas; Ndamukong-Nyanga, Judith L; Nana-Djeunga, Hugues C; Bourguinat, Catherine; Sofeu-Feugaing, David D; Charvet, Claude L

2012-05-01

The present study analyzed the relationship between the genetic diversity of Plasmodium falciparum and parasitologic/entomologic indices in the Mount Cameroon region by using merozoite surface protein 1 as a genetic marker. Blood samples were collected from asymptomatic children from three altitude zones (high, intermediate, and low). Parasitologic and entomologic indices were determined by microscopy and landing catch mosquito collection/circumsporozoite protein-enzyme-linked immunosorbent assay, respectively. A total of 142 randomly selected P. falciparum-positive blood samples were genotyped by using a nested polymerase chain reaction-based technique. K-1 polymerase chain reaction products were also sequenced. As opposed to high altitude, the highest malaria prevalence (70.65%) and entomologic inoculation rate (2.43 infective/bites/night) were recorded at a low altitude site. Seven (18.91%), 22 (36.66%), and 19 (42.22%) samples from high, intermediate, and low altitudes, respectively, contained multiclonal infections. A new K-1 polymorphism was identified. This study shows a positive non-linear association between low/intermediate altitude (high malaria transmission) and an increase in P. falciparum merozoite surface protein 1 block 2 polymorphisms.

Mapping HA-tagged protein at the surface of living cells by atomic force microscopy.

PubMed

Formosa, C; Lachaize, V; Galés, C; Rols, M P; Martin-Yken, H; François, J M; Duval, R E; Dague, E

2015-01-01

Single-molecule force spectroscopy using atomic force microscopy (AFM) is more and more used to detect and map receptors, enzymes, adhesins, or any other molecules at the surface of living cells. To be specific, this technique requires antibodies or ligands covalently attached to the AFM tip that can specifically interact with the protein of interest. Unfortunately, specific antibodies are usually lacking (low affinity and specificity) or are expensive to produce (monoclonal antibodies). An alternative strategy is to tag the protein of interest with a peptide that can be recognized with high specificity and affinity with commercially available antibodies. In this context, we chose to work with the human influenza hemagglutinin (HA) tag (YPYDVPDYA) and labeled two proteins: covalently linked cell wall protein 12 (Ccw12) involved in cell wall remodeling in the yeast Saccharomyces cerevisiae and the β2-adrenergic receptor (β2-AR), a G protein-coupled receptor (GPCR) in higher eukaryotes. We first described the interaction between HA antibodies, immobilized on AFM tips, and HA epitopes, immobilized on epoxy glass slides. Using our system, we then investigated the distribution of Ccw12 proteins over the cell surface of the yeast S. cerevisiae. We were able to find the tagged protein on the surface of mating yeasts, at the tip of the mating projections. Finally, we could unfold multimers of β2-AR from the membrane of living transfected chinese hamster ovary cells. This result is in agreement with GPCR oligomerization in living cell membranes and opens the door to the study of the influence of GPCR ligands on the oligomerization process. Copyright © 2014 John Wiley & Sons, Ltd.

The Structure of the Poxvirus A33 Protein Reveals a Dimer of Unique C-Type Lectin-Like Domains

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

Su, Hua-Poo; Singh, Kavita; Gittis, Apostolos G.

2010-11-03

The current vaccine against smallpox is an infectious form of vaccinia virus that has significant side effects. Alternative vaccine approaches using recombinant viral proteins are being developed. A target of subunit vaccine strategies is the poxvirus protein A33, a conserved protein in the Chordopoxvirinae subfamily of Poxviridae that is expressed on the outer viral envelope. Here we have determined the structure of the A33 ectodomain of vaccinia virus. The structure revealed C-type lectin-like domains (CTLDs) that occur as dimers in A33 crystals with five different crystal lattices. Comparison of the A33 dimer models shows that the A33 monomers have amore » degree of flexibility in position within the dimer. Structural comparisons show that the A33 monomer is a close match to the Link module class of CTLDs but that the A33 dimer is most similar to the natural killer (NK)-cell receptor class of CTLDs. Structural data on Link modules and NK-cell receptor-ligand complexes suggest a surface of A33 that could interact with viral or host ligands. The dimer interface is well conserved in all known A33 sequences, indicating an important role for the A33 dimer. The structure indicates how previously described A33 mutations disrupt protein folding and locates the positions of N-linked glycosylations and the epitope of a protective antibody.« less

Label-Free Quantitative Immunoassay of Fibrinogen in Alzheimer Disease Patient Plasma Using Fiber Optical Surface Plasmon Resonance

NASA Astrophysics Data System (ADS)

Kim, Jisoo; Kim, SeJin; Nguyen, Tan Tai; Lee, Renee; Li, Tiehua; Yun, Changhyun; Ham, Youngeun; An, Seong Soo A.; Ju, Heongkyu

2016-05-01

We present a real-time quantitative immunoassay to detect fibrinogen in the blood plasma of Alzheimer's disease patients using multimode fiber optical sensors in which surface plasmon resonance (SPR) was employed. Nanometer-thick bimetals including silver and aluminum were coated onto the core surface of the clad-free part (5 cm long) of the fiber for SPR excitation at the He-Ne laser wavelength of 632.8 nm. The histidine-tagged peptide was then coated on the metal surface to immobilize the fibrinogen antibody for the selective capture of fibrinogen among the proteins in the patient blood plasma. The SPR fiber optical sensor enabled quantitative detection of concentrations of fibrinogen from the different human patient blood at a detection limit of ˜20 ng/ml. We also observed a correlation in the fibrinogen concentration measurement between enzyme-linked immunosorbent assay and our SPR fiber-based sensors. This suggests that the presented SPR fiber-based sensors that do not rely on the use of labels such as fluorophores can be used for a real-time quantitative assay of a specific protein such as fibrinogen in a human blood that is known to contain many other kinds of proteins together.

Conservative mutation Met8 --> Leu affects the folding process and structural stability of squash trypsin inhibitor CMTI-I.

PubMed Central

Zhukov, I.; Jaroszewski, L.; Bierzyński, A.

2000-01-01

Protein molecules can accommodate a large number of mutations without noticeable effects on their stability and folding kinetics. On the other hand, some mutations can have quite strong effects on protein conformational properties. Such mutations either destabilize secondary structures, e.g., alpha-helices, are incompatible with close packing of protein hydrophobic cores, or lead to disruption of some specific interactions such as disulfide cross links, salt bridges, hydrogen bonds, or aromatic-aromatic contacts. The Met8 --> Leu mutation in CMTI-I results in significant destabilization of the protein structure. This effect could hardly be expected since the mutation is highly conservative, and the side chain of residue 8 is situated on the protein surface. We show that the protein destabilization is caused by rearrangement of a hydrophobic cluster formed by side chains of residues 8, Ile6, and Leu17 that leads to partial breaking of a hydrogen bond formed by the amide group of Leu17 with water and to a reduction of a hydrophobic surface buried within the cluster. The mutation perturbs also the protein folding. In aerobic conditions the reduced wild-type protein folds effectively into its native structure, whereas more then 75% of the mutant molecules are trapped in various misfolded species. The main conclusion of this work is that conservative mutations of hydrophobic residues can destabilize a protein structure even if these residues are situated on the protein surface and partially accessible to water. Structural rearrangement of small hydrophobic clusters formed by such residues can lead to local changes in protein hydration, and consequently, can affect considerably protein stability and folding process. PMID:10716179

Modeling Protein Excited-state Structures from "Over-length" Chemical Cross-links.

PubMed

Ding, Yue-He; Gong, Zhou; Dong, Xu; Liu, Kan; Liu, Zhu; Liu, Chao; He, Si-Min; Dong, Meng-Qiu; Tang, Chun

2017-01-27

Chemical cross-linking coupled with mass spectroscopy (CXMS) provides proximity information for the cross-linked residues and is used increasingly for modeling protein structures. However, experimentally identified cross-links are sometimes incompatible with the known structure of a protein, as the distance calculated between the cross-linked residues far exceeds the maximum length of the cross-linker. The discrepancies may persist even after eliminating potentially false cross-links and excluding intermolecular ones. Thus the "over-length" cross-links may arise from alternative excited-state conformation of the protein. Here we present a method and associated software DynaXL for visualizing the ensemble structures of multidomain proteins based on intramolecular cross-links identified by mass spectrometry with high confidence. Representing the cross-linkers and cross-linking reactions explicitly, we show that the protein excited-state structure can be modeled with as few as two over-length cross-links. We demonstrate the generality of our method with three systems: calmodulin, enzyme I, and glutamine-binding protein, and we show that these proteins alternate between different conformations for interacting with other proteins and ligands. Taken together, the over-length chemical cross-links contain valuable information about protein dynamics, and our findings here illustrate the relationship between dynamic domain movement and protein function. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

TMEM2: A missing link in hyaluronan catabolism identified?

PubMed

Yamaguchi, Yu; Yamamoto, Hayato; Tobisawa, Yuki; Irie, Fumitoshi

2018-03-27

Hyaluronan (HA) is a glycosaminoglycan (GAG) composed of repeating disaccharide units of glucuronic acid and N-acetylglucosamine. HA is an extremely long, unbranched polymer, which often exceeds 10 6  Da and sometimes reaches 10 7  Da. A feature that epitomizes HA is its rapid turnover; one-third of the total body HA is turned over daily. The current model of HA catabolism postulates that high-molecular weight HA in the extracellular space is first cleaved into smaller fragments by a hyaluronidase(s) that resides at the cell surface, followed by internalization of fragments and their degradation into monosaccharides in lysosomes. Over the last decade, considerable research has shown that the HYAL family of hyaluronidases plays significant roles in HA catabolism. Nonetheless, the identity of a hyaluronidase responsible for the initial step of HA cleavage on the cell surface remains elusive, as biochemical and enzymological properties of HYAL proteins are not entirely consistent with those expected of cell surface hyaluronidases. Recent identification of transmembrane 2 (TMEM2) as a cell surface protein that possesses potent hyaluronidase activity suggests that it may be the "missing" cell surface hyaluronidase, and that novel models of HA catabolism should include this protein. Copyright © 2017 International Society of Matrix Biology. Published by Elsevier B.V. All rights reserved.

X-linked juvenile retinoschisis: Clinical diagnosis, genetic analysis, and molecular mechanisms

PubMed Central

Molday, Robert S.; Kellner, Ulrich; Weber, Bernhard H.F.

2012-01-01

X-linked juvenile retinoschisis (XLRS, MIM 312700) is a common early onset macular degeneration in males characterized by mild to severe loss in visual acuity, splitting of retinal layers, and a reduction in the b-wave of the electroretinogram (ERG). The RS1 gene (MIM 300839) associated with the disease encodes retinoschisin, a 224 amino acid protein containing a discoidin domain as the major structural unit, an N-terminal cleavable signal sequence, and regions responsible for subunit oligomerization. Retinoschisin is secreted from retinal cells as a disulphide-linked homo-octameric complex which binds to the surface of photoreceptors and bipolar cells to help maintain the integrity of the retina. Over 190 disease-causing mutations in the RS1 gene are known with most mutations occurring as non-synonymous changes in the discoidin domain. Cell expression studies have shown that disease-associated missense mutations in the discoidin domain cause severe protein misfolding and retention in the endoplasmic reticulum, mutations in the signal sequence result in aberrant protein synthesis, and mutations in regions flanking the discoidin domain cause defective disulphide-linked subunit assembly, all of which produce a non-functional protein. Knockout mice deficient in retinoschisin have been generated and shown to display most of the characteristic features found in XLRS patients. Recombinant adeno-associated virus (rAAV) mediated delivery of the normal RS1 gene to the retina of young knockout mice result in long term retinoschisin expression and rescue of retinal structure and function providing a ‘proof of concept’ that gene therapy may be an effective treatment for XLRS. PMID:22245536

X-linked juvenile retinoschisis: clinical diagnosis, genetic analysis, and molecular mechanisms.

PubMed

Molday, Robert S; Kellner, Ulrich; Weber, Bernhard H F

2012-05-01

X-linked juvenile retinoschisis (XLRS, MIM 312700) is a common early onset macular degeneration in males characterized by mild to severe loss in visual acuity, splitting of retinal layers, and a reduction in the b-wave of the electroretinogram (ERG). The RS1 gene (MIM 300839) associated with the disease encodes retinoschisin, a 224 amino acid protein containing a discoidin domain as the major structural unit, an N-terminal cleavable signal sequence, and regions responsible for subunit oligomerization. Retinoschisin is secreted from retinal cells as a disulphide-linked homo-octameric complex which binds to the surface of photoreceptors and bipolar cells to help maintain the integrity of the retina. Over 190 disease-causing mutations in the RS1 gene are known with most mutations occurring as non-synonymous changes in the discoidin domain. Cell expression studies have shown that disease-associated missense mutations in the discoidin domain cause severe protein misfolding and retention in the endoplasmic reticulum, mutations in the signal sequence result in aberrant protein synthesis, and mutations in regions flanking the discoidin domain cause defective disulphide-linked subunit assembly, all of which produce a non-functional protein. Knockout mice deficient in retinoschisin have been generated and shown to display most of the characteristic features found in XLRS patients. Recombinant adeno-associated virus (rAAV) mediated delivery of the normal RS1 gene to the retina of young knockout mice result in long-term retinoschisin expression and rescue of retinal structure and function providing a 'proof of concept' that gene therapy may be an effective treatment for XLRS. Copyright © 2012 Elsevier Ltd. All rights reserved.

Model studies of diffusion-controlled (2-hydroxyethyl methacrylate) HEMA hydrogel membranes for controlled release of proteins

NASA Astrophysics Data System (ADS)

Appawu, Jennifer A. M.

This thesis project consisted of three main components that were connected by roots in chemical analysis for studies in tissue engineering. The first part focused on characterizing the structural parameters of synthetic cross-linked poly (2-hydroxyethyl methacrylate) (Poly(HEMA) hydrogel membranes to determine optimal formulations for clinical studies. Poly(HEMA) membranes were loaded with Keratincocyte Growth Factor (KGF) for controlled release studies. Protein loading and release kinetics were determined with fluorescence spectroscopy. The spatial distribution of a protein in the membrane was determined using Time-of-Flight Secondary Ion Mass Spectrometry (ToF-SIMS). The last part of the project focused on determining the biological effects of the polymer membranes in-vitro with a model cell line and a pilot in-vivo animal study. Based on the components completed in this project, five chapters are included in this dissertation document and are summarized below. A new protocol was developed using fluorescence spectroscopy that measured the rate of protein diffusion into cross-linked polymer membranes by measuring the change in the fluorescence intensity of the protein solution. This technique was also able to detect a conformational change that occurs within protein when KGF was imbibed within these cross-linked polymer membranes. ToF-SIMS chemical imaging and 3D depth profiling was used to determine the spatial distribution of KGF protein in frozen-hydrated HEMA hydrogel membranes. The 3D depth profiles showed that the KGF protein was aggregated in bright spots that indicated that KGF was not spatially homogenous on the surface and through the depth profiles. 3D depth profiles of the membranes studied at various times during release studies show that areas with aggregated proteins were retained during release, and at times with maximum release. The interpretation of the bright regions is that the KGf protein interacted with the cross-linked network of the hydrogel membranes, making it not available for release. The in-vitro biological experiments with the HaCaT cell line showed that the HEMA hydrogels were capable of sustaining cell viability, proliferation, and adhesion through cell adhesion and wounding experiments. The pilot in-vivo animal study also revealed that KGF protein had retained its pharmacological activity. The study also showed that the KGF protein enhanced the rate of wound closure.

Study of intermolecular contacts in proteins and oligomer interfaces and preliminary investigations into the design and production of nanomaterials from proteins

NASA Astrophysics Data System (ADS)

Iyer, Ganesh Hariharan

The first part of this research involved a study of the nature and extent of nonbonded interactions at crystal and oligomer interfaces. A survey was compiled of several characteristics of intersubunit contacts in 58 different oligomeric proteins, and of the intermolecular contacts in 223 protein crystal structures. Routines written in "S" language were utilized for the generation of the observed and expected contacts. The information in the Protein Data Bank (PDB) was extracted using the database management system, Protein Knowledge Base (PKB). Potentials of mean force for atom-atom contacts and residue-residue contacts were derived by comparison of the number of observed interactions with the number expected by mass action. Preference association matrices and log-linear analyses were applied to determine the different factors that could contribute to the overall interactions at the interfaces of oligomers and crystals. Surface patches at oligomer and crystal interfaces were also studied to further investigate the origin of the differences in their stabilities. Total number of atoms in contact and the secondary structure elements involved are similar in the two types of interfaces. Crystal contacts result from more numerous interactions by polar residues, compared with a tendency toward nonpolar amino acid prominent in oligomer interfaces. Contact potentials indicate that hydrophobic interactions at oligomer interfaces favor aromatic amino acids and methionine over aliphatic amino acids; and that crystal contacts form in such a way as to avoid inclusion of hydrophobic interactions. The second part involved the development of a new class of biomaterials from two-dimensional arrays of ordered proteins. Point mutations were planned to introduce cysteine residues at appropriate locations to enable cross-linking at the molecular interface within given crystallographic planes. Crystallization and subsequent cross-linking of the modified protein would lead to the formation of arrays on subsequent dissociation of the crystal. Novel protein architectures can be generated from these cross-linked nanostructures. Experiments with model protein, maltose-binding protein (MBP) were performed to develop purification, cross-linking and crystallization techniques. The long-term goal of this project is to apply the experience gained with MBP to the fabrication of nanomaterials from other, application-specific proteins for ultrafiltration and microelectronic devices.

Nanocellulose-based biosensors: design, preparation, and activity of peptide-linked cotton cellulose nanocrystals having fluorimetric and colorimetric elastase detection sensitivity

USDA-ARS?s Scientific Manuscript database

Nanocrystalline cellulose is an amphiphilic, high surface area material that can be easily functionalized and is biocom-patible and eco-friendly. It has been used singularly and in combination with other nanomaterials to optimize biosensor design. The attachment of peptides and proteins to nanocryst...

Structural Insights into Cargo Recognition by the Yeast PTS1 Receptor*

PubMed Central

Hagen, Stefanie; Drepper, Friedel; Fischer, Sven; Fodor, Krisztian; Passon, Daniel; Platta, Harald W.; Zenn, Michael; Schliebs, Wolfgang; Girzalsky, Wolfgang; Wilmanns, Matthias; Warscheid, Bettina; Erdmann, Ralf

2015-01-01

The peroxisomal matrix protein import is facilitated by cycling import receptors that shuttle between the cytosol and the peroxisomal membrane. The import receptor Pex5p mediates the import of proteins harboring a peroxisomal targeting signal of type I (PTS1). Purified recombinant Pex5p forms a dimeric complex with the PTS1-protein Pcs60p in vitro with a KD of 0.19 μm. To analyze the structural basis for receptor-cargo recognition, the PTS1 and adjacent amino acids of Pcs60p were systematically scanned for Pex5p binding by an in vitro site-directed photo-cross-linking approach. The cross-linked binding regions of the receptor were subsequently identified by high resolution mass spectrometry. Most cross-links were found with TPR6, TPR7, as well as the 7C-loop of Pex5p. Surface plasmon resonance analysis revealed a bivalent interaction mode for Pex5p and Pcs60p. Interestingly, Pcs60p lacking its C-terminal tripeptide sequence was efficiently cross-linked to the same regions of Pex5p. The KD value of the interaction of truncated Pcs60p and Pex5p was in the range of 7.7 μm. Isothermal titration calorimetry and surface plasmon resonance measurements revealed a monovalent binding mode for the interaction of Pex5p and Pcs60p lacking the PTS1. Our data indicate that Pcs60p contains a second contact site for its receptor Pex5p, beyond the C-terminal tripeptide. The physiological relevance of the ancillary binding region was supported by in vivo import studies. The bivalent binding mode might be explained by a two-step concept as follows: first, cargo recognition and initial tethering by the PTS1-receptor Pex5p; second, lock-in of receptor and cargo. PMID:26359497

NSP-CAS Protein Complexes: Emerging Signaling Modules in Cancer.

PubMed

Wallez, Yann; Mace, Peter D; Pasquale, Elena B; Riedl, Stefan J

2012-05-01

The CAS (CRK-associated substrate) family of adaptor proteins comprises 4 members, which share a conserved modular domain structure that enables multiple protein-protein interactions, leading to the assembly of intracellular signaling platforms. Besides their physiological role in signal transduction downstream of a variety of cell surface receptors, CAS proteins are also critical for oncogenic transformation and cancer cell malignancy through associations with a variety of regulatory proteins and downstream effectors. Among the regulatory partners, the 3 recently identified adaptor proteins constituting the NSP (novel SH2-containing protein) family avidly bind to the conserved carboxy-terminal focal adhesion-targeting (FAT) domain of CAS proteins. NSP proteins use an anomalous nucleotide exchange factor domain that lacks catalytic activity to form NSP-CAS signaling modules. Additionally, the NSP SH2 domain can link NSP-CAS signaling assemblies to tyrosine-phosphorylated cell surface receptors. NSP proteins can potentiate CAS function by affecting key CAS attributes such as expression levels, phosphorylation state, and subcellular localization, leading to effects on cell adhesion, migration, and invasion as well as cell growth. The consequences of these activities are well exemplified by the role that members of both families play in promoting breast cancer cell invasiveness and resistance to antiestrogens. In this review, we discuss the intriguing interplay between the NSP and CAS families, with a particular focus on cancer signaling networks.

Structural Aspects of N-Glycosylations and the C-terminal Region in Human Glypican-1*

PubMed Central

Awad, Wael; Adamczyk, Barbara; Örnros, Jessica; Karlsson, Niclas G.; Mani, Katrin; Logan, Derek T.

2015-01-01

Glypicans are multifunctional cell surface proteoglycans involved in several important cellular signaling pathways. Glypican-1 (Gpc1) is the predominant heparan sulfate proteoglycan in the developing and adult human brain. The two N-linked glycans and the C-terminal domain that attach the core protein to the cell membrane are not resolved in the Gpc1 crystal structure. Therefore, we have studied Gpc1 using crystallography, small angle x-ray scattering, and chromatographic approaches to elucidate the composition, structure, and function of the N-glycans and the C terminus and also the topology of Gpc1 with respect to the membrane. The C terminus is shown to be highly flexible in solution, but it orients the core protein transverse to the membrane, directing a surface evolutionarily conserved in Gpc1 orthologs toward the membrane, where it may interact with signaling molecules and/or membrane receptors on the cell surface, or even the enzymes involved in heparan sulfate substitution in the Golgi apparatus. Furthermore, the N-glycans are shown to extend the protein stability and lifetime by protection against proteolysis and aggregation. PMID:26203194

Role of N-linked oligosaccharides in the biosynthetic processing of the cystic fibrosis membrane conductance regulator

PubMed Central

Chang, Xiu-bao; Mengos, April; Hou, Yue-xian; Cui, Liying; Jensen, Timothy J.; Aleksandrov, Andrei; Riordan, John R.; Gentzsch, Martina

2009-01-01

Summary The epithelial chloride channel CFTR is a glycoprotein that is modified by two N-linked oligosaccharides. The most common mutant CFTR protein in patients with cystic fibrosis, ΔF508, is misfolded and retained by ER quality control. As oligosaccharide moieties of glycoproteins are known to mediate interactions with ER lectin chaperones, we investigated the role of N-linked glycosylation in the processing of wild-type and ΔF508 CFTR. We found that N-glycosylation and ER lectin interactions are not major determinants of trafficking of wild-type and ΔF508 from the ER to the plasma membrane. Unglycosylated CFTR, generated by removal of glycosylation sites or treatment of cells with the N-glycosylation inhibitor tunicamycin, did not bind calnexin, but did traffic to the cell surface and exhibited chloride channel activity. Most importantly, unglycosylated Δ F508 CFTR still could not escape quality control in the early secretory pathway and remained associated with the ER. However, the absence of N-linked oligosaccharides did reduce the stability of wild-type CFTR, causing significantly more-rapid turnover in post-ER compartments. Surprisingly, the individual N-linked carbohydrates do not play equivalent roles and modulate the fate of the wild-type protein in different ways in its early biosynthetic pathway. PMID:18682497

Role of N-linked oligosaccharides in the biosynthetic processing of the cystic fibrosis membrane conductance regulator.

PubMed

Chang, Xiu-Bao; Mengos, April; Hou, Yue-Xian; Cui, Liying; Jensen, Timothy J; Aleksandrov, Andrei; Riordan, John R; Gentzsch, Martina

2008-09-01

The epithelial chloride channel CFTR is a glycoprotein that is modified by two N-linked oligosaccharides. The most common mutant CFTR protein in patients with cystic fibrosis, DeltaF508, is misfolded and retained by ER quality control. As oligosaccharide moieties of glycoproteins are known to mediate interactions with ER lectin chaperones, we investigated the role of N-linked glycosylation in the processing of wild-type and DeltaF508 CFTR. We found that N-glycosylation and ER lectin interactions are not major determinants of trafficking of wild-type and DeltaF508 from the ER to the plasma membrane. Unglycosylated CFTR, generated by removal of glycosylation sites or treatment of cells with the N-glycosylation inhibitor tunicamycin, did not bind calnexin, but did traffic to the cell surface and exhibited chloride channel activity. Most importantly, unglycosylated DeltaF508 CFTR still could not escape quality control in the early secretory pathway and remained associated with the ER. However, the absence of N-linked oligosaccharides did reduce the stability of wild-type CFTR, causing significantly more-rapid turnover in post-ER compartments. Surprisingly, the individual N-linked carbohydrates do not play equivalent roles and modulate the fate of the wild-type protein in different ways in its early biosynthetic pathway.

Surface vimentin is critical for the cell entry of SARS-CoV.

PubMed

Yu, Yvonne Ting-Chun; Chien, Ssu-Chia; Chen, I-Yin; Lai, Chia-Tsen; Tsay, Yeou-Guang; Chang, Shin C; Chang, Ming-Fu

2016-01-22

Severe acute respiratory syndrome coronavirus (SARS-CoV) caused a global panic due to its high morbidity and mortality during 2002 and 2003. Soon after the deadly disease outbreak, the angiotensin-converting enzyme 2 (ACE2) was identified as a functional cellular receptor in vitro and in vivo for SARS-CoV spike protein. However, ACE2 solely is not sufficient to allow host cells to become susceptible to SARS-CoV infection, and other host factors may be involved in SARS-CoV spike protein-ACE2 complex. A host intracellular filamentous cytoskeletal protein vimentin was identified by immunoprecipitation and LC-MS/MS analysis following chemical cross-linking on Vero E6 cells that were pre-incubated with the SARS-CoV spike protein. Moreover, flow cytometry data demonstrated an increase of the cell surface vimentin level by 16.5 % after SARS-CoV permissive Vero E6 cells were treated with SARS-CoV virus-like particles (VLPs). A direct interaction between SARS-CoV spike protein and host surface vimentin was further confirmed by far-Western blotting. In addition, antibody neutralization assay and shRNA knockdown experiments indicated a vital role of vimentin in cell binding and uptake of SARS-CoV VLPs and the viral spike protein. A direct interaction between vimentin and SARS-CoV spike protein during viral entry was observed. Vimentin is a putative anti-viral drug target for preventing/reducing the susceptibility to SARS-CoV infection.

A novel approach for application of nylon membranes in the biosensing domain

NASA Astrophysics Data System (ADS)

Farahmand, Elham; Ibrahim, Fatimah; Hosseini, Samira; Rothan, Hussin A.; Yusof, Rohana; Koole, Leo H.; Djordjevic, Ivan

2015-10-01

In this paper we report the polymer-coated microporous nylon membranes and their application as platforms for protein immobilization and subsequent detection of the dengue virus (DV) in blood serum. Protein recognition experiments were performed with enzyme-linked immunosorbent assay (ELISA). The polymers used for coatings were synthesized by free-radical polymerization reaction between methyl methacrylate (MMA) and methacrylic acid (MAA) in different concentrations. The MAA monomer has carefully been chosen to generate polymers with pendant carboxyl (-COOH) groups, which also exist on polymer surfaces. A high degree of control over surface-exposed -COOH groups has been achieved through variation of monomers concentration in polymerization reaction. The general aspect of this work relies on the dengue antibody (Ab) immobilization on surface -COOH groups via physical attachment or covalent immobilization. Prior to Ab immobilization and ELISA experiment, polymer-coated nylon samples were analyzed in detail for their physical properties by atomic force microscopy (AFM), scanning electron microscopy (SEM), and water-in-air contact angle (WCA) measurements. Membranes were further analyzed by Fourier transform infrared spectroscopy (FTIR) in order to establish the relationship between wettability, porosity, and surface roughness with chemical composition and concentration of -COOH groups on the coating's surface. Optimized coatings have shown high sensitivity towards dengue Ab molecules, revealing fundamental aspect of polymer-protein interfaces as a function of surface -COOH groups' concentration.

Impact Mediated Loading Cytoplasmic Loading of Macromolecules into Adherent Cells

NASA Technical Reports Server (NTRS)

Clarke, Mark S. F.; Feeback, Daniel L.; Vanderburg, Charles R.

2003-01-01

The advent of modern molecular biology, including the development of gene array technologies, has resulted in an explosion of information concerning the specific genes activated during normal cellular development, as well as those associated with a variety of pathological conditions. These techniques have served as a highly efficient, broacI.-based screening approach for those specific genes involved. in regulating normal cellular physiology and identifying candidate genes directly associated with the etiology of specific disease states. However, this approach provides information at the transcriptional' level only and does not necessarily indicate . that the gene in question is in fact translated ito a protein, or whether or not post-translational modification of the protein occurs. The critical importance of post-translational modification (i.e. phosphorylation, glycosylation, sialyation, etc.) to protein function has been recognized with regard to a number of proteins involved in a variety of important disease states. For example, altered glycosylation of beta-amyloid precursor protein results in an increase in the amount of beta-amyloid peptide generated and hence secreted as insoluble extracellular amyloid deposits (Georgopoulou, McLaughlin et al. 2001; Walter, Fluhrer et al. 2001), a pathological hal1nark of Alzheimer's disease. Abnormal phosphorylaion of synapsin I has been linked to alterations in synaptic vesicle trafficking leading to defective neurotransmission in Huntington's disease (Lievens, Woodman et al. 2002). Altered phosphorylation of the TAU protein involved in microtubule function has been linked to a number of neurodegenative diseases such as Alzheimer's disease (Billingsley and Kincaid 1997; Sanchez, Alvarez-Tllada et a1. 2001). Aberrant siaIyation of cell/I surface antigens has been detected in a number of different tumor cell types and has been linked to the acquisition of a neoplastic phenotype (Sell 1990), while improper' sia1yation of sodium channels in cardiac tissue has been linked to heart failure (Ufret-Vincenty, Baro et al. 2001; Fozzard and Kyle 2002).

Use of protein cross-linking and radiolytic footprinting to elucidate PsbP and PsbQ interactions within higher plant Photosystem II

DOE PAGES

Mummadisetti, Manjula P.; Frankel, Laurie K.; Bellamy, Henry D.; ...

2014-10-27

We used protein cross-linking and radiolytic footprinting coupled with high-resolution mass spectrometry to examine the structure of PsbP and PsbQ when they are bound to Photosystem II, in this paper. In its bound state, the N-terminal 15-amino-acid residue domain of PsbP, which is unresolved in current crystal structures, interacts with domains in the C terminus of the protein. These interactions may serve to stabilize the structure of the N terminus and may facilitate PsbP binding and function. These interactions place strong structural constraints on the organization of PsbP when associated with the Photosystem II complex. Additionally, amino acid residues inmore » the structurally unresolved loop 3A domain of PsbP ( 90K– 107V), 93Y and 96K, are in close proximity (≤11.4 Å) to the N-terminal 1E residue of PsbQ. Our findings are the first, to our knowledge, to identify a putative region of interaction between these two components. Cross-linked domains within PsbQ were also identified, indicating that two PsbQ molecules can interact in higher plants in a manner similar to that observed by Liu et al. [(2014) Proc Natl Acad Sci 111(12):4638–4643] in cyanobacterial Photosystem II. Furthermore, this interaction is consistent with either intra-Photosystem II dimer or inter-Photosystem II dimer models in higher plants. Finally, OH• produced by synchrotron radiolysis of water was used to oxidatively modify surface residues on PsbP and PsbQ. Finally, domains on the surface of both protein subunits were resistant to modification, indicating that they were shielded from water and appear to define buried regions that are in contact with other Photosystem II components.« less

Purification of functional baculovirus particles from silkworm larval hemolymph and their use as nanoparticles for the detection of human prorenin receptor (PRR) binding

PubMed Central

2011-01-01

Background Baculovirus, which has a width of 40 nm and a length of 250-300 nm, can display functional peptides, receptors and antigens on its surface by their fusion with a baculovirus envelop protein, GP64. In addition, some transmembrane proteins can be displayed without GP64 fusion, using the native transmembrane domains of the baculovirus. We used this functionality to display human prorenin receptor fused with GFPuv (GFPuv-hPRR) on the surface of silkworm Bombyx mori nucleopolyhedrovirus (BmNPV) and then tested whether these baculovirus particles could be used to detect protein-protein interactions. Results BmNPV displaying GFPuv-hPRR (BmNPV-GFPuv-hPRR) was purified from hemolymph by using Sephacryl S-1000 column chromatography in the presence of 0.01% Triton X-100. Its recovery was 86% and the final baculovirus particles number was 4.98 × 108 pfu. Based on the results of enzyme-linked immunosorbent assay (ELISA), 3.1% of the total proteins in BmNPV-GFPuv-hPRR were GFPuv-hPRR. This value was similar to that calculated from the result of western blot by a densitometry (2.7%). To determine whether BmNPV-GFPuv-hPRR particles were bound to human prorenin, ELISA results were compared with those from ELISAs using protease negative BmNPV displaying β1,3-N-acetylglucosaminyltransferase 2 fused with the gene encoding GFPuv (GGT2) (BmNPV-CP--GGT2) particles, which do not display hPRR on their surfaces. Conclusion The display of on the surface of the BmNPV particles will be useful for the detection of protein-protein interactions and the screening of inhibitors and drugs in their roles as nanobioparticles. PMID:21635720

Cross-linking of cell surface amyloid precursor protein leads to increased β-amyloid peptide production in hippocampal neurons: implications for Alzheimer's disease.

PubMed

Lefort, Roger; Pozueta, Julio; Shelanski, Michael

2012-08-01

The accumulation of the β-amyloid peptide (Aβ) in Alzheimer's disease (AD) is thought to play a causative role in triggering synaptic dysfunction in neurons, leading to their eventual demise through apoptosis. Aβ is produced and secreted upon sequential cleavage of the amyloid precursor protein (APP) by β-secretases and γ-secretases. However, while Aβ levels have been shown to be increased in the brains of AD patients, little is known about how the cleavage of APP and the subsequent generation of Aβ is influenced, or whether the cleavage process changes over time. It has been proposed that Aβ can bind APP and promote amyloidogenic processing of APP, further enhancing Aβ production. Proof of this idea has remained elusive because a clear mechanism has not been identified, and the promiscuous nature of Aβ binding complicates the task of demonstrating the idea. To work around these problems, we used an antibody-mediated approach to bind and cross-link cell-surface APP in cultured rat primary hippocampal neurons. Here we show that cross-linking of APP is sufficient to raise the levels of Aβ in viable neurons with a concomitant increase in the levels of the β-secretase BACE1. This appears to occur as a result of a sorting defect that stems from the caspase-3-mediated inactivation of a key sorting adaptor protein, namely GGA3, which prevents the lysosomal degradation of BACE1. Together, our data suggest the occurrence of a positive pathogenic feedback loop involving Aβ and APP in affected neurons possibly allowing Aβ to spread to nearby healthy neurons.

Dock mediates Scar- and WASp-dependent actin polymerization through interaction with cell adhesion molecules in founder cells and fusion-competent myoblasts.

PubMed

Kaipa, Balasankara Reddy; Shao, Huanjie; Schäfer, Gritt; Trinkewitz, Tatjana; Groth, Verena; Liu, Jianqi; Beck, Lothar; Bogdan, Sven; Abmayr, Susan M; Önel, Susanne-Filiz

2013-01-01

The formation of the larval body wall musculature of Drosophila depends on the asymmetric fusion of two myoblast types, founder cells (FCs) and fusion-competent myoblasts (FCMs). Recent studies have established an essential function of Arp2/3-based actin polymerization during myoblast fusion, formation of a dense actin focus at the site of fusion in FCMs, and a thin sheath of actin in FCs and/or growing muscles. The formation of these actin structures depends on recognition and adhesion of myoblasts that is mediated by cell surface receptors of the immunoglobulin superfamily. However, the connection of the cell surface receptors with Arp2/3-based actin polymerization is poorly understood. To date only the SH2-SH3 adaptor protein Crk has been suggested to link cell adhesion with Arp2/3-based actin polymerization in FCMs. Here, we propose that the SH2-SH3 adaptor protein Dock, like Crk, links cell adhesion with actin polymerization. We show that Dock is expressed in FCs and FCMs and colocalizes with the cell adhesion proteins Sns and Duf at cell-cell contact points. Biochemical data in this study indicate that different domains of Dock are involved in binding the cell adhesion molecules Duf, Rst, Sns and Hbs. We emphasize the importance of these interactions by quantifying the enhanced myoblast fusion defects in duf dock, sns dock and hbs dock double mutants. Additionally, we show that Dock interacts biochemically and genetically with Drosophila Scar, Vrp1 and WASp. Based on these data, we propose that Dock links cell adhesion in FCs and FCMs with either Scar- or Vrp1-WASp-dependent Arp2/3 activation.

CMC-modified cellulose biointerface for antibody conjugation.

PubMed

Orelma, Hannes; Teerinen, Tuija; Johansson, Leena-Sisko; Holappa, Susanna; Laine, Janne

2012-04-09

In this Article, we present a new strategy for preparing an antihemoglobin biointerface on cellulose. The preparation method is based on functionalization of the cellulose surface by the irreversible adsorption of CMC, followed by covalent linking of antibodies to CMC. This would provide the means for affordable and stable cellulose-based biointerfaces for immunoassays. The preparation and characterization of the biointerface were studied on Langmuir-Schaefer cellulose model surfaces in real time using the quartz crystal microbalance with dissipation and surface plasmon resonance techniques. The stable attachment of antihemoglobin to adsorbed CMC was achieved, and a linear calibration of hemoglobin was obtained. CMC modification was also observed to prevent nonspecific protein adsorption. The antihemoglobin-CMC surface regenerated well, enabling repeated immunodetection cycles of hemoglobin on the same surface.

GPU-enabled molecular dynamics simulations of ankyrin kinase complex

NASA Astrophysics Data System (ADS)

Gautam, Vertika; Chong, Wei Lim; Wisitponchai, Tanchanok; Nimmanpipug, Piyarat; Zain, Sharifuddin M.; Rahman, Noorsaadah Abd.; Tayapiwatana, Chatchai; Lee, Vannajan Sanghiran

2014-10-01

The ankyrin repeat (AR) protein can be used as a versatile scaffold for protein-protein interactions. It has been found that the heterotrimeric complex between integrin-linked kinase (ILK), PINCH, and parvin is an essential signaling platform, serving as a convergence point for integrin and growth-factor signaling and regulating cell adhesion, spreading, and migration. Using ILK-AR with high affinity for the PINCH1 as our model system, we explored a structure-based computational protocol to probe and characterize binding affinity hot spots at protein-protein interfaces. In this study, the long time scale dynamics simulations with GPU accelerated molecular dynamics (MD) simulations in AMBER12 have been performed to locate the hot spots of protein-protein interaction by the analysis of the Molecular Mechanics-Poisson-Boltzmann Surface Area/Generalized Born Solvent Area (MM-PBSA/GBSA) of the MD trajectories. Our calculations suggest good binding affinity of the complex and also the residues critical in the binding.

Structural characterization of IgG1 mAb aggregates and particles generated under various stress conditions.

PubMed

Telikepalli, Srivalli N; Kumru, Ozan S; Kalonia, Cavan; Esfandiary, Reza; Joshi, Sangeeta B; Middaugh, C Russell; Volkin, David B

2014-03-01

IgG1 mAb solutions were prepared with and without sodium chloride and subjected to different environmental stresses. Formation of aggregates and particles of varying size was monitored by a combination of size-exclusion chromatography, Nanoparticle Tracking Analysis, Micro-flow Imaging (MFI), turbidity, and visual assessments. Stirring and heating induced the highest concentration of particles. In general, the presence of NaCl enhanced this effect. The morphology of the particles formed from mAb samples exposed to different stresses was analyzed from transmission electron microscopy and MFI images. Shaking samples without NaCl generated the most fibrillar particles, whereas stirring created largely spherical particles. The composition of the particles was evaluated for covalent cross-linking by SDS-PAGE, overall secondary structure by FTIR microscopy, and surface apolarity by extrinsic fluorescence spectroscopy. Freeze-thaw and shaking led to particles containing protein with native-like secondary structure. Heating and stirring produced IgG1-containing aggregates and particles with some non-native disulfide cross-links, varying levels of intermolecular beta sheet content, and increased surface hydrophobicity. These results highlight the importance of evaluating protein particle morphology and composition, in addition to particle number and size distributions, to better understand the effect of solution conditions and environmental stresses on the formation of protein particles in mAb solutions. © 2014 Wiley Periodicals, Inc. and the American Pharmacists Association.

Structural basis for the stabilization of the complement alternative pathway C3 convertase by properdin

PubMed Central

Alcorlo, Martín; Tortajada, Agustín; Rodríguez de Córdoba, Santiago; Llorca, Oscar

2013-01-01

Complement is an essential component of innate immunity. Its activation results in the assembly of unstable protease complexes, denominated C3/C5 convertases, leading to inflammation and lysis. Regulatory proteins inactivate C3/C5 convertases on host surfaces to avoid collateral tissue damage. On pathogen surfaces, properdin stabilizes C3/C5 convertases to efficiently fight infection. How properdin performs this function is, however, unclear. Using electron microscopy we show that the N- and C-terminal ends of adjacent monomers in properdin oligomers conform a curly vertex that holds together the AP convertase, interacting with both the C345C and vWA domains of C3b and Bb, respectively. Properdin also promotes a large displacement of the TED (thioester-containing domain) and CUB (complement protein subcomponents C1r/C1s, urchin embryonic growth factor and bone morphogenetic protein 1) domains of C3b, which likely impairs C3-convertase inactivation by regulatory proteins. The combined effect of molecular cross-linking and structural reorganization increases stability of the C3 convertase and facilitates recruitment of fluid-phase C3 convertase to the cell surfaces. Our model explains how properdin mediates the assembly of stabilized C3/C5-convertase clusters, which helps to localize complement amplification to pathogen surfaces. PMID:23901101

Forward Genetic Dissection of Biofilm Development by Fusobacterium nucleatum: Novel Functions of Cell Division Proteins FtsX and EnvC.

PubMed

Wu, Chenggang; Al Mamun, Abu Amar Mohamed; Luong, Truc Thanh; Hu, Bo; Gu, Jianhua; Lee, Ju Huck; D'Amore, Melissa; Das, Asis; Ton-That, Hung

2018-04-24

Fusobacterium nucleatum is a key member of the human oral biofilm. It is also implicated in preterm birth and colorectal cancer. To facilitate basic studies of fusobacterial virulence, we describe here a versatile transposon mutagenesis procedure and a pilot screen for mutants defective in biofilm formation. Out of 10 independent biofilm-defective mutants isolated, the affected genes included the homologs of the Escherichia coli cell division proteins FtsX and EnvC, the electron transport protein RnfA, and four proteins with unknown functions. Next, a facile new gene deletion method demonstrated that nonpolar, in-frame deletion of ftsX or envC produces viable bacteria that are highly filamentous due to defective cell division. Transmission electron and cryo-electron microscopy revealed that the Δ ftsX and Δ envC mutant cells remain joined with apparent constriction, and scanning electron microscopy (EM) uncovered a smooth cell surface without the microfolds present in wild-type cells. FtsX and EnvC proteins interact with each other as well as a common set of interacting partners, many with unknown function. Last, biofilm development is altered when cell division is blocked by MinC overproduction; however, unlike the phenotypes of Δ ftsX and Δ envC mutants, a weakly adherent biofilm is formed, and the wild-type rugged cell surface is maintained. Therefore, FtsX and EnvC may perform novel functions in Fusobacterium cell biology. This is the first report of an unbiased approach to uncover genetic determinants of fusobacterial biofilm development. It points to an intriguing link among cytokinesis, cell surface dynamics, and biofilm formation, whose molecular underpinnings remain to be elucidated. IMPORTANCE Little is known about the virulence mechanisms and associated factors in F. nucleatum , due mainly to the lack of convenient genetic tools for this organism. We employed two efficient genetic strategies to identify F. nucleatum biofilm-defective mutants, revealing FtsX and EnvC among seven biofilm-associated factors. Electron microscopy established cell division defects of the Δ ftsX and Δ envC mutants, accompanied with a smooth cell surface, unlike the microfold, rugged appearance of wild-type bacteria. Proteomic studies demonstrated that FtsX and EnvC interact with each other as well as a set of common and unique interacting proteins, many with unknown functions. Importantly, blocking cell division by MinC overproduction led to formation of a weakly adherent biofilm, without alteration of the wild-type cell surface. Thus, this work links cell division and surface dynamics to biofilm development and lays a foundation for future genetic and biochemical investigations of basic cellular processes in this clinically significant pathogen. Copyright © 2018 Wu et al.

Combining Amine-Reactive Cross-Linkers and Photo-Reactive Amino Acids for 3D-Structure Analysis of Proteins and Protein Complexes.

PubMed

Lössl, Philip; Sinz, Andrea

2016-01-01

During the last 15 years, the combination of chemical cross-linking and high-resolution mass spectrometry (MS) has matured into an alternative approach for analyzing 3D-structures of proteins and protein complexes. Using the distance constraints imposed by the cross-links, models of the protein or protein complex under investigation can be created. The majority of cross-linking studies are currently conducted with homobifunctional amine-reactive cross-linkers. We extend this "traditional" cross-linking/MS strategy by adding complementary photo-cross-linking data. For this, the diazirine-containing unnatural amino acids photo-leucine and photo-methionine are incorporated into the proteins and cross-link formation is induced by UV-A irradiation. The advantage of the photo-cross-linking strategy is that it is not restricted to lysine residues and that hydrophobic regions in proteins can be targeted, which is advantageous for investigating membrane proteins. We consider the strategy of combining cross-linkers with orthogonal reactivities and distances to be ideally suited for maximizing the amount of structural information that can be gained from a cross-linking experiment.

Mapping the distribution of packing topologies within protein interiors shows predominant preference for specific packing motifs

PubMed Central

2011-01-01

Background Mapping protein primary sequences to their three dimensional folds referred to as the 'second genetic code' remains an unsolved scientific problem. A crucial part of the problem concerns the geometrical specificity in side chain association leading to densely packed protein cores, a hallmark of correctly folded native structures. Thus, any model of packing within proteins should constitute an indispensable component of protein folding and design. Results In this study an attempt has been made to find, characterize and classify recurring patterns in the packing of side chain atoms within a protein which sustains its native fold. The interaction of side chain atoms within the protein core has been represented as a contact network based on the surface complementarity and overlap between associating side chain surfaces. Some network topologies definitely appear to be preferred and they have been termed 'packing motifs', analogous to super secondary structures in proteins. Study of the distribution of these motifs reveals the ubiquitous presence of typical smaller graphs, which appear to get linked or coalesce to give larger graphs, reminiscent of the nucleation-condensation model in protein folding. One such frequently occurring motif, also envisaged as the unit of clustering, the three residue clique was invariably found in regions of dense packing. Finally, topological measures based on surface contact networks appeared to be effective in discriminating sequences native to a specific fold amongst a set of decoys. Conclusions Out of innumerable topological possibilities, only a finite number of specific packing motifs are actually realized in proteins. This small number of motifs could serve as a basis set in the construction of larger networks. Of these, the triplet clique exhibits distinct preference both in terms of composition and geometry. PMID:21605466

A glycosylphosphatidylinositol anchor is required for membrane localization but dispensable for cell wall association of chitin deacetylase 2 in Cryptococcus neoformans.

PubMed

Gilbert, Nicole M; Baker, Lorina G; Specht, Charles A; Lodge, Jennifer K

2012-01-01

Cell wall proteins (CWPs) mediate important cellular processes in fungi, including adhesion, invasion, biofilm formation, and flocculation. The current model of fungal cell wall organization includes a major class of CWPs covalently bound to β-1,6-glucan via a remnant of a glycosylphosphatidylinositol (GPI) anchor. This model was established by studies of ascomycetes more than a decade ago, and relatively little work has been done with other fungi, although the presumption has been that proteins identified in the cell wall which contain a predicted GPI anchor are covalently linked to cell wall glucans. The pathogenic basidiomycete Cryptococcus neoformans encodes >50 putatively GPI-anchored proteins, some of which have been identified in the cell wall. One of these proteins is chitin deacetylase 2 (Cda2), an enzyme responsible for converting chitin to chitosan, a cell wall polymer recently established as a virulence factor for C. neoformans infection of mammalian hosts. Using a combination of biochemistry, molecular biology, and genetics, we show that Cda2 is GPI anchored to membranes but noncovalently associated with the cell wall by means independent of both its GPI anchor and β-1,6-glucan. We also show that Cda2 produces chitosan when localized to the plasma membrane, but association with the cell wall is not essential for this process, thereby providing insight into the mechanism of chitosan biosynthesis. These results increase our understanding of the surface of C. neoformans and provide models of cell walls likely applicable to other undercharacterized basidiomycete pathogenic fungi. The surface of a pathogenic microbe is a major interface with its host. In fungi, the outer surface consists of a complex matrix known as the cell wall, which includes polysaccharides, proteins, and other molecules. The mammalian host recognizes many of these surface molecules and mounts appropriate responses to combat the microbial infection. Cryptococcus neoformans is a serious fungal pathogen that kills over 600,000 people annually. It converts most of its chitin, a cell wall polysaccharide, to chitosan, which is necessary for virulence. Chitin deacetylase enzymes have been identified in the cell wall, and our studies were undertaken to understand how the deacetylase is linked to the wall and where it has activity. Our results have implications for the current model of chitosan biosynthesis and further challenge the paradigm of covalent linkages between cell wall proteins and polysaccharides through a lipid modification of the protein.

Synthesis of Protein Bioconjugates via Cysteine-maleimide Chemistry.

PubMed

Mason, Alexander F; Thordarson, Pall

2016-07-20

The chemical linking or bioconjugation of proteins to fluorescent dyes, drugs, polymers and other proteins has a broad range of applications, such as the development of antibody drug conjugates (ADCs) and nanomedicine, fluorescent microscopy and systems chemistry. For many of these applications, specificity of the bioconjugation method used is of prime concern. The Michael addition of maleimides with cysteine(s) on the target proteins is highly selective and proceeds rapidly under mild conditions, making it one of the most popular methods for protein bioconjugation. We demonstrate here the modification of the only surface-accessible cysteine residue on yeast cytochrome c with a ruthenium(II) bisterpyridine maleimide. The protein bioconjugation is verified by gel electrophoresis and purified by aqueous-based fast protein liquid chromatography in 27% yield of isolated protein material. Structural characterization with MALDI-TOF MS and UV-Vis is then used to verify that the bioconjugation is successful. The protocol shown here is easily applicable to other cysteine - maleimide coupling of proteins to other proteins, dyes, drugs or polymers.

Facile Method for the Site-Specific, Covalent Attachment of full-length IgG onto Nanoparticles

PubMed Central

Hui, James Zhe; Al Zaki, Ajlan; Cheng, Zhiliang; Popik, Vladimir; Zhang, Hongtao; Luning Prak, Eline T.

2014-01-01

Antibodies, most commonly IgGs, have been widely used as targeting ligands in research and therapeutic applications due to their wide array of targets, high specificity and proven efficacy. Many of these applications require antibodies to be conjugated onto surfaces (e.g. nanoparticles and microplates); however, most conventional bioconjugation techniques exhibit low crosslinking efficiencies, reduced functionality due to non-site-specific labeling and random surface orientation, and/or require protein engineering (e.g. cysteine handles), which can be technically challenging. To overcome these limitations, we have recombinantly expressed Protein Z, which binds the Fc region of IgG, with an UV active non-natural amino acid benzoylphenyalanine (BPA) within its binding domain. Upon exposure to long wavelength UV light, the BPA is activated and forms a covalent link between the Protein Z and the bound Fc region of IgG. This technology was combined with expressed protein ligation (EPL), which allowed for the introduction of a fluorophore and click chemistry-compatible azide group onto the C-terminus of Protein Z during the recombinant protein purification step. This enabled crosslinked-Protein Z-IgG complexes to be efficiently and site-specifically attached to aza-dibenzycyclooctyne-modified nanoparticles, via copper-free click chemistry. PMID:24729432

Facile method for the site-specific, covalent attachment of full-length IgG onto nanoparticles.

PubMed

Hui, James Zhe; Al Zaki, Ajlan; Cheng, Zhiliang; Popik, Vladimir; Zhang, Hongtao; Luning Prak, Eline T; Tsourkas, Andrew

2014-08-27

Antibodies, most commonly IgGs, have been widely used as targeting ligands in research and therapeutic applications due to their wide array of targets, high specificity and proven efficacy. Many of these applications require antibodies to be conjugated onto surfaces (e.g. nanoparticles and microplates); however, most conventional bioconjugation techniques exhibit low crosslinking efficiencies, reduced functionality due to non-site-specific labeling and random surface orientation, and/or require protein engineering (e.g. cysteine handles), which can be technically challenging. To overcome these limitations, we have recombinantly expressed Protein Z, which binds the Fc region of IgG, with an UV active non-natural amino acid benzoylphenyalanine (BPA) within its binding domain. Upon exposure to long wavelength UV light, the BPA is activated and forms a covalent link between the Protein Z and the bound Fc region of IgG. This technology was combined with expressed protein ligation (EPL), which allowed for the introduction of a fluorophore and click chemistry-compatible azide group onto the C-terminus of Protein Z during the recombinant protein purification step. This enabled the crosslinked-Protein Z-IgG complexes to be efficiently and site-specifically attached to aza-dibenzocyclooctyne-modified nanoparticles, via copper-free click chemistry. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

The Saccharomyces cerevisiae DPM1 gene encoding dolichol-phosphate-mannose synthase is able to complement a glycosylation-defective mammalian cell line.

PubMed Central

Beck, P J; Orlean, P; Albright, C; Robbins, P W; Gething, M J; Sambrook, J F

1990-01-01

The Saccharomyces cerevisiae DPM1 gene product, dolichol-phosphate-mannose (Dol-P-Man) synthase, is involved in the coupled processes of synthesis and membrane translocation of Dol-P-Man. Dol-P-Man is the lipid-linked sugar donor of the last four mannose residues that are added to the core oligosaccharide transferred to protein during N-linked glycosylation in the endoplasmic reticulum. We present evidence that the S. cerevisiae gene DPM1, when stably transfected into a mutant Chinese hamster ovary cell line, B4-2-1, is able to correct the glycosylation defect of the cells. Evidence for complementation includes (i) fluorescence-activated cell sorter analysis of differential lectin binding to cell surface glycoproteins, (ii) restoration of Dol-P-Man synthase enzymatic activity in crude cell lysates, (iii) isolation and high-performance liquid chromatography fractionation of the lipid-linked oligosaccharides synthesized in the transfected and control cell lines, and (iv) the restoration of endoglycosidase H sensitivity to the oligosaccharides transferred to a specific glycoprotein synthesized in the DPM1 CHO transfectants. Indirect immunofluorescence with a primary antibody directed against the DPM1 protein shows a reticular staining pattern of protein localization in transfected hamster and monkey cell lines. Images PMID:2201896

Large heat capacity change in a protein-monovalent cation interaction.

PubMed

Guinto, E R; Di Cera, E

1996-07-09

Current views about protein-ligand interactions state that electrostatic forces drive the binding of charged species and that burial of hydrophobic and polar surfaces controls the heat capacity change associated with the reaction. For the interaction of a protein with a monovalent cation the electrostatic components are expected to be significant due to the ionic nature of the ligand, whereas the heat capacity change is expected to be small due to the size of the surface area involved in the recognition event. The physiologically important interaction of Na+ with thrombin was studied over the temperature range from 5 to 45 degrees C and the ionic strength range from 50 to 800 mM. These measurements reveal an unanticipated result that bears quite generally on studies of molecular recognition and protein folding. Binding of Na+ to thrombin is characterized by a modest dependence on ionic strength but a large and negative heat capacity change of -1.1 +/- 0.1 kcal mol-1 K-1. The small electrostatic coupling can be explained in terms of a minimal perturbation of the ionic atmosphere of the protein upon Na+ binding. The large heat capacity change, however, is difficult to reconcile with current views on the origin of this effect from surface area changes or large folding transitions coupled to binding. It is proposed that this change is linked to burial of a large cluster of water molecules in the Na+ binding pocket upon Na+ binding. Due to their reduced mobility and highly ordered structure, water molecules sequestered in the interior of a protein must have a lower heat capacity compared to those on the surface of a protein or in the bulk solvent. Hence, a binding or folding event where water molecules are buried may result in significant heat capacity changes independent of changes in exposed hydrophobic surface or coupled conformational transitions.

Pickering emulsions stabilized by whey protein nanoparticles prepared by thermal cross-linking.

PubMed

Wu, Jiande; Shi, Mengxuan; Li, Wei; Zhao, Luhai; Wang, Ze; Yan, Xinzhong; Norde, Willem; Li, Yuan

2015-03-01

A Pickering (o/w) emulsion was formed and stabilized by whey protein isolate nanoparticles (WPI NPs). Those WPI NPs were prepared by thermal cross-linking of denatured WPI proteins within w/o emulsion droplets at 80°C for 15 min. During heating of w/o emulsions containing 10% (w/v) WPI proteins in the water phase, the emulsions displayed turbid-transparent-turbid phase transitions, which is ascribed to the change in the size of the protein-containing water droplets caused by thermal cross-linking between denatured protein molecules. The transparent stage indicated the formation of WPI NPs. WPI NPs of different sizes were obtained by varying the mixing speed. WPI NPs of 200-500 nm were selected to prepare o/w Pickering emulsions because of their good stability against coalescence. By Confocal Laser Scanning Microscopy, it was observed that WPI NPs were closely packed and distributed at the surface of the emulsion droplets. By measuring water contact angles of WPI NPs films, it was found that under most conditions WPI NPs present good partial wetting properties, but that at the isoelectric point (pI) and high ionic strength the particles become more hydrophobic, resulting in less stable Pickering emulsion. Thus, at pH above and below the pI of WPI NPs and low to moderate ionic strengths (1-10 mM), and with a WPI NPs concentration of 2% (w/v), a stable Pickering emulsion can be obtained. The results may provide useful information for applications of WPI NPs in environmentally friendly and food grade applications, notably in food, pharmaceutical and cosmetic products. Copyright © 2015 Elsevier B.V. All rights reserved.

Immobilization of cross linked Col-I-OPN bone matrix protein on aminolysed PCL surfaces enhances initial biocompatibility of human adipogenic mesenchymal stem cells (hADMSC)

NASA Astrophysics Data System (ADS)

Kim, Young-Hee; Jyoti, Md. Anirban; Song, Ho-Yeon

2014-06-01

In bone tissue engineering surface modification is considered as one of the important ways of fabricating successful biocompatible material. Addition of biologically active functionality on the surfaces has been tried for improving the overall biocompatibility of the system. In this study poly-ɛ-caprolactone film surfaces have been modified through aminolysis and immobilization process. Collagen type I (COL-I) and osteopontin (OPN), which play an important role in osteogenesis, was immobilized onto PCL films followed by aminolysis treatment using 1,6-hexanediamine. Characterization of animolysed and immobilized surfaces were done by a number techniques using scanning electron microscopy (SEM), FT-IR, XPS, ninhydrin staining, SDS-PAGE and confocal microscopy and compared between the modified and un-modified surfaces. Results of the successive experiments showed that aminolysis treatment was homogeneously achieved which helped to entrap or immobilize Col-I-OPN proteins on surfaces of PCL film. In vitro studies with human adipogenic mesenchymal stem cells (hADMSC) also confirmed the attachment and proliferation of cells was better in modified PCL surfaces than the unmodified surfaces. SEM, confocal microscopy and MTT assay showed a significant increase in cell spreading, attachment and proliferations on the biofunctionalized surfaces compared to the unmodified PCL surfaces at all-time points indicating the success of surface biofunctionalization.

Cloning and characterization of a G protein-activated human phosphoinositide-3 kinase.

PubMed

Stoyanov, B; Volinia, S; Hanck, T; Rubio, I; Loubtchenkov, M; Malek, D; Stoyanova, S; Vanhaesebroeck, B; Dhand, R; Nürnberg, B

1995-08-04

Phosphoinositide-3 kinase activity is implicated in diverse cellular responses triggered by mammalian cell surface receptors and in the regulation of protein sorting in yeast. Receptors with intrinsic and associated tyrosine kinase activity recruit heterodimeric phosphoinositide-3 kinases that consist of p110 catalytic subunits and p85 adaptor molecules containing Src homology 2 (SH2) domains. A phosphoinositide-3 kinase isotype, p110 gamma, was cloned and characterized. The p110 gamma enzyme was activated in vitro by both the alpha and beta gamma subunits of heterotrimeric guanosine triphosphate (GTP)-binding proteins (G proteins) and did not interact with p85. A potential pleckstrin homology domain is located near its amino terminus. The p110 gamma isotype may link signaling through G protein-coupled receptors to the generation of phosphoinositide second messengers phosphorylated in the D-3 position.

Bullied no more:when and how DNA shoves proteins around

PubMed Central

Pettitt, B. Montgomery; Sumners, De Witt L.; Harris, Sarah A.; Zechiedrich, Lynn

2016-01-01

The predominant protein-centric perspective in protein–DNA-binding studies assumes that the protein drives the interaction. Research focuses on protein structural motifs, electrostatic surfaces and contact potentials, while DNA is often ignored as a passive polymer to be manipulated. Recent studies of DNA topology, the supercoiling, knotting, and linking of the helices, have shown that DNA has the capability to be an active participant in its transactions. DNA topology-induced structural and geometric changes can drive, or at least strongly influence, the interactions between protein and DNA. Deformations of the B-form structure arise from both the considerable elastic energy arising from supercoiling and from the electrostatic energy. Here, we discuss how these energies are harnessed for topology-driven, sequence-specific deformations that can allow DNA to direct its own metabolism. PMID:22850561

BAR domain proteins regulate Rho GTPase signaling.

PubMed

Aspenström, Pontus

2014-01-01

BAR proteins comprise a heterogeneous group of multi-domain proteins with diverse biological functions. The common denominator is the Bin-Amphiphysin-Rvs (BAR) domain that not only confers targeting to lipid bilayers, but also provides scaffolding to mold lipid membranes into concave or convex surfaces. This function of BAR proteins is an important determinant in the dynamic reconstruction of membrane vesicles, as well as of the plasma membrane. Several BAR proteins function as linkers between cytoskeletal regulation and membrane dynamics. These links are provided by direct interactions between BAR proteins and actin-nucleation-promoting factors of the Wiskott-Aldrich syndrome protein family and the Diaphanous-related formins. The Rho GTPases are key factors for orchestration of this intricate interplay. This review describes how BAR proteins regulate the activity of Rho GTPases, as well as how Rho GTPases regulate the function of BAR proteins. This mutual collaboration is a central factor in the regulation of vital cellular processes, such as cell migration, cytokinesis, intracellular transport, endocytosis, and exocytosis.

Structural and Functional Assessment of APOBEC3G Macromolecular Complexes

PubMed Central

Polevoda, Bogdan; McDougall, William M.; Bennett, Ryan P.; Salter, Jason D.; Smith, Harold C.

2016-01-01

There are eleven members in the human APOBEC family of proteins that are evolutionarily related through their zinc-dependent cytidine deaminase domains. The human APOBEC gene clusters arose on chromosome 6 and 22 through gene duplication and divergence to where current day APOBEC proteins are functionally diverse and broadly expressed in tissues. APOBEC serve enzymatic and non enzymatic functions in cells. In both cases, formation of higher-order structures driven by APOBEC protein-protein interactions and binding to RNA and/or single stranded DNA are integral to their function. In some circumstances, these interactions are regulatory and modulate APOBEC activities. We are just beginning to understand how macromolecular interactions drive processes such as APOBEC subcellular compartmentalization, formation of holoenzyme complexes, gene targeting, foreign DNA restriction, anti-retroviral activity, formation of ribonucleoprotein particles and APOBEC degradation. Protein-protein and protein-nucleic acid cross-linking methods coupled with mass spectrometry, electrophoretic mobility shift assays, glycerol gradient sedimentation, fluorescence anisotropy and APOBEC deaminase assays are enabling mapping of interacting surfaces that are essential for these functions. The goal of this methods review is through example of our research on APOBEC3G, describe the application of cross-linking methods to characterize and quantify macromolecular interactions and their functional implications. Given the homology in structure and function, it is proposed that these methods will be generally applicable to the discovery process for other APOBEC and RNA and DNA editing and modifying proteins. PMID:26988126

Evolution of an ancient protein function involved in organized multicellularity in animals.

PubMed

Anderson, Douglas P; Whitney, Dustin S; Hanson-Smith, Victor; Woznica, Arielle; Campodonico-Burnett, William; Volkman, Brian F; King, Nicole; Thornton, Joseph W; Prehoda, Kenneth E

2016-01-07

To form and maintain organized tissues, multicellular organisms orient their mitotic spindles relative to neighboring cells. A molecular complex scaffolded by the GK protein-interaction domain (GKPID) mediates spindle orientation in diverse animal taxa by linking microtubule motor proteins to a marker protein on the cell cortex localized by external cues. Here we illuminate how this complex evolved and commandeered control of spindle orientation from a more ancient mechanism. The complex was assembled through a series of molecular exploitation events, one of which - the evolution of GKPID's capacity to bind the cortical marker protein - can be recapitulated by reintroducing a single historical substitution into the reconstructed ancestral GKPID. This change revealed and repurposed an ancient molecular surface that previously had a radically different function. We show how the physical simplicity of this binding interface enabled the evolution of a new protein function now essential to the biological complexity of many animals.

Mixed-Isotope Labeling with LC-IMS-MS for Characterization of Protein–Protein Interactions by Chemical Cross-Linking

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

Merkley, Eric D.; Baker, Erin S.; Crowell, Kevin L.

2013-02-20

Chemical cross-linking of proteins followed by proteolysis and mass spectrometric analysis of the resulting cross-linked peptides can provide insights into protein structure and protein-protein interactions. However, cross-linked peptides are by necessity of low stoichometry and have different physicochemical properties than linear peptides, routine unambiguous identification of the cross-linked peptides has remained difficult. To address this challenge, we demonstrated the use of liquid chromatography and ion mobility separations coupled with mass spectrometry in combination with a heavy-isotope labeling method. The combination of mixed-isotope cross-linking and ion mobility provided unique and easily interpretable spectral multiplet features for the intermolecular cross-linked peptides. Applicationmore » of the method to two different homodimeric proteins - SrfN, a virulence factor from Salmonella Typhimurium and SO_2176, a protein of unknown function from Shewanella oneidensis- revealed several cross-linked peptides from both proteins that were identified with a low false discovery rate (estimated using a decoy approach). A greater number of cross-linked peptides were identified using ion mobility drift time information in the analysis than when the data were summed across the drift time dimension before analysis. The identified cross-linked peptides migrated more quickly in the ion mobility drift tube than the unmodified peptides.« less

Immunoglobulin subunits of murine B lymphocytes: structure and associations with other membrane proteins.

PubMed Central

Vogel, L; Haustein, D

1989-01-01

The Ig subunit structure of murine B lymphocytes was studied by employing different radiolabelling techniques in combination with chemical cross-linking. The main membrane structure of IgM was a half molecule that was disulphide-linked to proteins with MW 30,000, 45,000 and 55,000, respectively. Small amounts of mu 2L2, microL disulphide-linked to a protein with MW 50,000, and free microL were also detected. The main IgD structures were half molecules disulphide-linked to two proteins with MW 14,000 and two proteins with MW 16,000. Furthermore, IgD half molecules disulphide-linked to a protein with MW 16,000 and free half molecules could be demonstrated. Labelling with hydrophobic reagents showed that all Ig molecules and the protein with MW 50,000, linked to microL, penetrated the lipid bilayer, whereas the other IgM- and IgD-linked proteins probably did not. Additional proteins which were associated exclusively with IgM were detected by chemical cross-linking. These findings offer new possibilities for the investigation of the function(s) of antigen receptors on B cells. Images Figure 1 Figure 2 Figure 4 Figure 5 PMID:2787780

Visualizing Mutation-Specific Differences in the Trafficking-Deficient Phenotype of Kv11.1 Proteins Linked to Long QT Syndrome Type 2.

PubMed

Hall, Allison R; Anderson, Corey L; Smith, Jennifer L; Mirshahi, Tooraj; Elayi, Claude S; January, Craig T; Delisle, Brian P

2018-01-01

KCNH2 encodes the Kv11.1 α-subunit that underlies the rapidly activating delayed-rectifier K + current in the heart. Loss-of-function KCNH2 mutations cause long QT syndrome type 2 (LQT2), and most LQT2-linked missense mutations inhibit the trafficking of Kv11.1 channel protein to the cell surface membrane. Several trafficking-deficient LQT2 mutations (e.g., G601S) generate Kv11.1 proteins that are sequestered in a microtubule-dependent quality control (QC) compartment in the transitional endoplasmic reticulum (ER). We tested the hypothesis that the QC mechanisms that regulate LQT2-linked Kv11.1 protein trafficking are mutation-specific. Confocal imaging analyses of HEK293 cells stably expressing the trafficking-deficient LQT2 mutation F805C showed that, unlike G601S-Kv11.1 protein, F805C-Kv11.1 protein was concentrated in several transitional ER subcompartments. The microtubule depolymerizing drug nocodazole differentially affected G601S- and F805C-Kv11.1 protein immunostaining. Nocodazole caused G601S-Kv11.1 protein to distribute into peripheral reticular structures, and it increased the diffuse immunostaining of F805C-Kv11.1 protein around the transitional ER subcompartments. Proteasome inhibition also affected the immunostaining of G601S- and F805C-Kv11.1 protein differently. Incubating cells in MG132 minimally impacted G601S-Kv11.1 immunostaining, but it dramatically increased the diffuse immunostaining of F805C-Kv11.1 protein in the transitional ER. Similar results were seen after incubating cells in the proteasome inhibitor lactacystin. Differences in the cellular distribution of G601S-Kv11.1 and F805C-Kv11.1 protein persisted in transfected human inducible pluripotent stem cell derived cardiomyocytes. These are the first data to visually demonstrate mutation-specific differences in the trafficking-deficient LQT2 phenotype, and this study has identified a novel way to categorize trafficking-deficient LQT2 mutations based on differences in intracellular retention.

A human antibody against Zika virus crosslinks the E protein to prevent infection

PubMed Central

Hasan, S. Saif; Miller, Andrew; Sapparapu, Gopal; Fernandez, Estefania; Klose, Thomas; Long, Feng; Fokine, Andrei; Porta, Jason C.; Jiang, Wen; Diamond, Michael S.; Crowe Jr., James E.; Kuhn, Richard J.; Rossmann, Michael G.

2017-01-01

The recent Zika virus (ZIKV) epidemic has been linked to unusual and severe clinical manifestations including microcephaly in fetuses of infected pregnant women and Guillian-Barré syndrome in adults. Neutralizing antibodies present a possible therapeutic approach to prevent and control ZIKV infection. Here we present a 6.2 Å resolution three-dimensional cryo-electron microscopy (cryoEM) structure of an infectious ZIKV (strain H/PF/2013, French Polynesia) in complex with the Fab fragment of a highly therapeutic and neutralizing human monoclonal antibody, ZIKV-117. The antibody had been shown to prevent fetal infection and demise in mice. The structure shows that ZIKV-117 Fabs cross-link the monomers within the surface E glycoprotein dimers as well as between neighbouring dimers, thus preventing the reorganization of E protein monomers into fusogenic trimers in the acidic environment of endosomes. PMID:28300075

A human antibody against Zika virus crosslinks the E protein to prevent infection.

PubMed

Hasan, S Saif; Miller, Andrew; Sapparapu, Gopal; Fernandez, Estefania; Klose, Thomas; Long, Feng; Fokine, Andrei; Porta, Jason C; Jiang, Wen; Diamond, Michael S; Crowe, James E; Kuhn, Richard J; Rossmann, Michael G

2017-03-16

The recent Zika virus (ZIKV) epidemic has been linked to unusual and severe clinical manifestations including microcephaly in fetuses of infected pregnant women and Guillian-Barré syndrome in adults. Neutralizing antibodies present a possible therapeutic approach to prevent and control ZIKV infection. Here we present a 6.2 Å resolution three-dimensional cryo-electron microscopy (cryoEM) structure of an infectious ZIKV (strain H/PF/2013, French Polynesia) in complex with the Fab fragment of a highly therapeutic and neutralizing human monoclonal antibody, ZIKV-117. The antibody had been shown to prevent fetal infection and demise in mice. The structure shows that ZIKV-117 Fabs cross-link the monomers within the surface E glycoprotein dimers as well as between neighbouring dimers, thus preventing the reorganization of E protein monomers into fusogenic trimers in the acidic environment of endosomes.

PG1058 Is a Novel Multidomain Protein Component of the Bacterial Type IX Secretion System

PubMed Central

Veith, Paul D.; Butler, Catherine A.; Nor Muhammad, Nor A.; Chen, Yu-Yen; Slakeski, Nada; Peng, Benjamin; Zhang, Lianyi; Dashper, Stuart G.; Cross, Keith J.; Cleal, Steven M.; Moore, Caroline; Reynolds, Eric C.

2016-01-01

Porphyromonas gingivalis utilises the Bacteroidetes-specific type IX secretion system (T9SS) to export proteins across the outer membrane (OM), including virulence factors such as the gingipains. The secreted proteins have a conserved carboxy-terminal domain essential for type IX secretion that is cleaved upon export. In P. gingivalis the T9SS substrates undergo glycosylation with anionic lipopolysaccharide (A-LPS) and are attached to the OM. In this study, comparative analyses of 24 Bacteroidetes genomes identified ten putative novel components of the T9SS in P. gingivalis, one of which was PG1058. Computer modelling of the PG1058 structure predicted a novel N- to C-terminal architecture comprising a tetratricopeptide repeat (TPR) domain, a β-propeller domain, a carboxypeptidase regulatory domain-like fold (CRD) and an OmpA_C-like putative peptidoglycan binding domain. Inactivation of pg1058 in P. gingivalis resulted in loss of both colonial pigmentation and surface-associated proteolytic activity; a phenotype common to T9SS mutants. Immunoblot and LC-MS/MS analyses of subcellular fractions revealed T9SS substrates accumulated within the pg1058 mutant periplasm whilst whole-cell ELISA showed the Kgp gingipain was absent from the cell surface, confirming perturbed T9SS function. Immunoblot, TEM and whole-cell ELISA analyses indicated A-LPS was produced and present on the pg1058 mutant cell surface although it was not linked to T9SS substrate proteins. This indicated that PG1058 is crucial for export of T9SS substrates but not for the translocation of A-LPS. PG1058 is a predicted lipoprotein and was localised to the periplasmic side of the OM using whole-cell ELISA, immunoblot and LC-MS/MS analyses of subcellular fractions. The structural prediction and localisation of PG1058 suggests that it may have a role as an essential scaffold linking the periplasmic and OM components of the T9SS. PMID:27711252

Biochemical characterization of domain-specific glycoproteins of the rat hepatocyte plasma membrane.

PubMed

Bartles, J R; Braiterman, L T; Hubbard, A L

1985-10-15

Seven integral proteins (CE 9, HA 21, HA 116, HA 16, HA 4, HA 201, and HA 301) were isolated from rat hepatocyte plasma membranes by immunoaffinity chromatography on monoclonal antibody-Sepharose. Six of the proteins (all but HA 16) exhibit domain-specific localizations (either bile canalicular or sinusoidal/lateral) about the hepatocyte surface. We identified three of these protein antigens as leucine aminopeptidase (HA 201), dipeptidyl peptidase IV (HA 301), and the asialoglycoprotein receptor (HA 116). We also developed 125I-lectin blotting procedures that, when used in conjunction with chemical and glycosidase treatments, permitted a comparison of the types of oligosaccharides present on the seven proteins. All seven are sialoglycoproteins, based upon the effects of prior neuraminidase and periodate-aniline-cyanoborohydride treatments of blots on labeling by 125I-wheat germ agglutinin. 125I-labeled Ricinus communis agglutinin I and 125I-peanut agglutinin blotting of the desialylated proteins revealed few if any conventional O-linked oligosaccharides, suggesting that the sialyl residues represent termini of N-linked complex-type oligosaccharides. Depending upon the protein, we estimated the presence of 2-26 N-linked oligosaccharides/polypeptide chain from the Mr reductions accompanying chemical or enzymatic deglycosylation. Three of these mature plasma membrane proteins (HA 21, HA 116, and HA 4) have both high mannose-type and complex-type oligosaccharides on every copy of their polypeptide chains. The labeling of these three proteins by 125I-concanavalin A was sensitive to treatment with endoglycosidase H, and each exhibited a quantitative reduction in Mr after the treatment, as assessed independently by 125I-wheat germ agglutinin blotting. At this level of analysis, we were unable to discern differences in the types of oligosaccharides present on these seven glycoproteins that correlate with their patterns of expression within the plasma membrane domains of this polarized epithelial cell.

Quantification of surfactant proteins in tears of patients suffering from dry eye disease compared to healthy subjects.

PubMed

Posa, Andreas; Paulsen, Friedrich; Dietz, Richard; Garreis, Fabian; Sander, Ralph; Schicht, Martin; Sel, Saadettin; Scholz, Michael; Hammer, Christian M; Bräuer, Lars

2018-03-01

To quantify and compare the amounts of surfactant proteins SP-A, SP-B, SP-C and SP-D in the tear fluid collected from patients with dry eye syndrome and from individuals with a healthy ocular surface. Schirmer strips were used to collect tear fluid from both eyes of 241 volunteers (99 men, 142 women; age range: 18-87 years). Dry eye syndrome was diagnosed by ophthalmologists in 125 patients, whereas the healthy control group comprised 116 individuals. The total protein concentration was determined via Bradford assay. The relative concentration of surfactant proteins SP-A through -D was measured by enzyme-linked immuno-sorbent assay (ELISA). The mean relative concentrations of SP-A, SP-C and SP-D were significantly higher in the dry eye group as compared to the healthy controls (p<0.05, one-way ANOVA). SP-B was also detected at a higher concentration in the dry eye group, but the difference to the control group was not statistically significant. The upregulation of SP-A and SP-D in the dry eye group is probably related to these proteins' known antimicrobial and immunomodulatory effects at the ocular surface. It may represent a pathophysiological response to the inflammatory condition of the ocular surface in dry eye. The upregulation of SP-B and SP-C may represent an effort of the lacrimal system to reduce surface tension and thus to counteract the increased tendency of the tear film to tear in dry eye. Copyright © 2017 Elsevier GmbH. All rights reserved.

Periodic protein adsorption at the gold/biotin aqueous solution interface: evidence of kinetics with time delay

NASA Astrophysics Data System (ADS)

Neff, H.; Laborde, H. M.; Lima, A. M. N.

2016-11-01

An oscillatory molecular adsorption pattern of the protein neutravidin from aqueous solution onto gold, in presence of a pre-deposited self assembled mono-molecular biotin film, is reported. Real time surface Plasmon resonance sensing was utilized for evaluation of the adsorption kinetics. Two different fractions were identified: in the initial phase, protein molecules attach irreversibly onto the Biotin ligands beneath towards the jamming limit, forming a neutravidin-biotin fraction. Afterwards, the growth rate exhibits distinct, albeit damped adsorption-desorption oscillations over an extended time span, assigned to a quasi reversibly bound fraction. These findings agree with, and firstly confirm a previously published model, proposing macro-molecular adsorption with time delay. The non-linear dynamic model is applicable to and also resembles non-damped oscillatory binding features of the hetero-catalytic oxidation of carbon monoxide molecules on platinum in the gas phase. An associated surface residence time can be linked to the dynamics and time scale required for self-organization.

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

Characterization of B61, the ligand for the Eck receptor protein-tyrosine kinase.

PubMed

Shao, H; Pandey, A; O'Shea, K S; Seldin, M; Dixit, V M

1995-03-10

B61 was originally described as a novel secreted tumor necrosis factor-alpha-inducible gene product in endothelial cells (Holzman, L. B., Marks, R. M., and Dixit, V. M. (1990) Mol. Cell. Biol. 10, 5830-5838). It was recently discovered that soluble recombinant B61 could serve as a ligand for the Eck receptor protein-tyrosine kinase, a member of the Eph/Eck subfamily of receptor protein-tyrosine kinases (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, R. A., Holst, P. L., Skrine, J. D., Toso, R. J., Zhang, M., Fernandez, E., Trail, G., Yarnum, B., Yarden, Y., Hunter, T., and Fox, G. M. (1994) Nature 368, 558-560). We now show that B61 can also exist as a cell surface glycosylphosphatidyl-inositol-linked protein that is capable of activating the Eck receptor protein-tyrosine kinase, the first such report of a receptor protein-tyrosine kinase ligand that is glycosylphosphatidylinositol-linked. In addition, the expression patterns of B61 and Eck during mouse ontogeny were determined by in situ hybridization. Both were found to be highly expressed in the developing lung and gut, while Eck was preferentially expressed in the thymus. Finally, the gene for B61 was localized to a specific position on mouse chromosome 3 by interspecific back-cross analysis.

Antibody screening identifies 78 putative host proteins involved in Cyprinid herpesvirus 3 infection or propagation in common carp, Cyprinus carpio L.

PubMed Central

Gotesman, M; Soliman, H; El-Matbouli, M

2014-01-01

Cyprinid herpesvirus 3 (CyHV-3) is the aetiological agent of a serious and notifiable disease afflicting common and koi carp, Cyprinus carpio L., termed koi herpesvirus disease (KHVD). Significant progress has been achieved in the last 15 years, since the initial reports surfaced from Germany, USA and Israel of the CyHV-3 virus, in terms of pathology and detection. However, relatively few studies have been carried out in understanding viral replication and propagation. Antibody-based affinity has been used for detection of CyHV-3 in enzyme-linked immunosorbent assay and PCR-based techniques, and immunohistological assays have been used to describe a CyHV-3 membrane protein, termed ORF81. In this study, monoclonal antibodies linked to N-hydroxysuccinimide (NHS)-activated spin columns were used to purify CyHV-3 and host proteins from tissue samples originating in either CyHV-3 symptomatic or asymptomatic fish. The samples were next analysed either by polyacrylamide gel electrophoresis (PAGE) and subsequently by electrospray ionization coupled to mass spectrometry (ESI-MS) or by ESI-MS analysis directly after purification. A total of 78 host proteins and five CyHV-3 proteins were identified in the two analyses. These data can be used to develop novel control methods for CyHV-3, based on pathways or proteins identified in this study. PMID:23347276

Adhesion beyond the interface: Molecular adaptations of the mussel byssus to the intertidal zone

NASA Astrophysics Data System (ADS)

MIller, Dusty Rose

The California mussel, Mytilus californianus, adheres robustly in the high-energy and oxidizing intertidal zone with a fibrous holdfast called the byssus using 3,4-dihydroxyphenyl-L-alanine (Dopa)-containing adhesive mussel foot proteins (mfps). There are many supporting roles to mussel adhesion that are intimately linked and ultimately responsible for mussel byssus's durable and dynamic adhesion. This dissertation explores these supporting mechanisms, including delivery of materials underwater, iron binding, friction, and antioxidant activity. As the outermost covering of the byssus, the cuticle deserves particular attention for its supporting roles to adhesion including the high stiffness and extensibility of the M. californianus byssal cuticle, which make it one of the most energy tolerant materials known. The cuticle's matrix-granule composite structure contributes to its toughness by microcracking between its harder granules and softer matrix. We investigated delivery of cuticular material underwater, cohesion of cuticle proteins, and surface damage mitigation by cuticle protein-based coacervates. To investigate underwater material delivery, we made cuticle matrix mimics by coacervating a key cuticular protein, Mytilus californianus foot protein 1, mfp-1, with hyaluronic acid. These matrix mimics coacervated over a wide range of solution conditions, delivered concentrated material, settled on and coated surfaces underwater. Because the granules are composed of mfp-1 condensed with iron, we used the surface forces apparatus to investigate the effects of iron on the cohesion of mfp-1 from two different species of mussels and found that subtle sequence variations modulate cohesion. Using the coacervate matrix mimics and, modeling the granules as a hard surface (mica), we investigated the wear protection of coacervated mfp-1/HA to mica under frictional shear and found that preventing wear depends critically on the presence of Dopa groups. In addition to cuticle-derived mechanisms for adhesion protection, we also tested for direct chemical mechanisms by tracking redox in the mussel adhesive plaques and found a persistent reservoir of antioxidant activity that can protect Dopa from oxidation. Overall, the mussel byssus represents an excellent model system for understanding adaptive mechanisms of both underwater adhesives and tough materials and I propose in this dissertation that these supporting mechanisms are intimately linked and ultimately responsible for the durable and dynamic underwater adhesion of mussels in the intertidal zone.

Structural and biophysical characterization of an epitope-specific engineered Fab fragment and complexation with membrane proteins: implications for co-crystallization.

PubMed

Johnson, Jennifer L; Entzminger, Kevin C; Hyun, Jeongmin; Kalyoncu, Sibel; Heaner, David P; Morales, Ivan A; Sheppard, Aly; Gumbart, James C; Maynard, Jennifer A; Lieberman, Raquel L

2015-04-01

Crystallization chaperones are attracting increasing interest as a route to crystal growth and structure elucidation of difficult targets such as membrane proteins. While strategies to date have typically employed protein-specific chaperones, a peptide-specific chaperone to crystallize multiple cognate peptide epitope-containing client proteins is envisioned. This would eliminate the target-specific chaperone-production step and streamline the co-crystallization process. Previously, protein engineering and directed evolution were used to generate a single-chain variable (scFv) antibody fragment with affinity for the peptide sequence EYMPME (scFv/EE). This report details the conversion of scFv/EE to an anti-EE Fab format (Fab/EE) followed by its biophysical characterization. The addition of constant chains increased the overall stability and had a negligible impact on the antigen affinity. The 2.0 Å resolution crystal structure of Fab/EE reveals contacts with larger surface areas than those of scFv/EE. Surface plasmon resonance, an enzyme-linked immunosorbent assay, and size-exclusion chromatography were used to assess Fab/EE binding to EE-tagged soluble and membrane test proteins: namely, the β-barrel outer membrane protein intimin and α-helical A2a G protein-coupled receptor (A2aR). Molecular-dynamics simulation of the intimin constructs with and without Fab/EE provides insight into the energetic complexities of the co-crystallization approach.

Protein structure controls the processing of the N-linked oligosaccharides and glycosylphosphatidylinositol glycans of variant surface glycoproteins expressed in bloodstream form Trypanosoma brucei.

PubMed

Zitzmann, N; Mehlert, A; Carrouée, S; Rudd, P M; Ferguson, M A; Carroué, S

2000-03-01

The variant surface glycoproteins (VSGs) of Trypanosoma brucei are a family of homodimeric glycoproteins that adopt similar shapes. An individual trypanosome expresses one VSG at a time in the form of a dense protective mono-layer on the plasma membrane. VSG genes are expressed from one of several polycistronic transcription units (expression sites) that contain several expression site associated genes. We used a transformed trypanosome clone expressing two different VSGs (VSG121 and VSG221) from the same expression site (that of VSG221) to establish whether the genotype of the trypanosome clone or the VSG structure itself controls VSG N-linked oligosaccharide and GPI anchor glycan processing. In-gel release and fluorescent labeling of N-linked oligosaccharides and on-blot fluorescent labeling and release of GPI anchor glycans were employed to compare the carbohydrate structures of VSG121 and VSG221 when expressed individually in wild-type trypanosome clones and when expressed together in the transformed trypanosome clone. The data indicate that the genotype of the trypanosome clone has no effect on the N-linked oligosaccharide structures present on a given VSG variant and only a minor effect on the GPI anchor glycans. The latter is most likely an effect of changes in inter-VSG packing when two VGSs are expressed simultaneously. Thus, N-linked oligosaccharide and GPI anchor processing enzymes appear to be constitutively expressed in bloodstream form African trypanosomes and the tertiary and quaternary structures of the VSG homodimers appear to dictate the processing and glycoform microheterogeneity of surface-expressed VSGs.

Novel applications for glycosylphosphatidylinositol-anchored proteins in pharmaceutical and industrial biotechnology.

PubMed

Müller, Günter

2011-04-01

Glycosylphosphatidylinositol (GPI)-anchored proteins have been regarded as typical cell surface proteins found in most eukaryotic cells from yeast to man. They are embedded in the outer plasma membrane leaflet via a carboxy-terminally linked complex glycolipid GPI structure. The amphiphilic nature of the GPI anchor, its compatibility with the function of the attached protein moiety and the capability of GPI-anchored proteins for spontaneous insertion into and transfer between artificial and cellular membranes initially suggested their potential for biotechnological applications. However, these expectations have been hardly fulfilled so far. Recent developments fuel novel hopes with regard to: (i) Automated online expression, extraction and purification of therapeutic proteins as GPI-anchored proteins based on their preferred accumulation in plasma membrane lipid rafts, (ii) multiplex custom-made protein chips based on GPI-anchored cell wall proteins in yeast, (iii) biomaterials and biosensors with films consisting of sets of distinct GPI-anchored binding-proteins or enzymes for sequential or combinatorial catalysis, and (iv) transport of therapeutic proteins across or into relevant tissue cells, e.g., enterocytes or adipocytes. Latter expectations are based on the demonstrated translocation of GPI-anchored proteins from plasma membrane lipid rafts to cytoplasmic lipid droplets and eventually further into microvesicles which upon release from donor cells transfer their GPI-anchored proteins to acceptor cells. The value of these technologies, which are all based on the interaction of GPI-anchored proteins with membranes and surfaces, for the engineering, production and targeted delivery of biomolecules for a huge variety of therapeutic and biotechnological purposes should become apparent in the near future.

Yeast Surface Display of Two Proteins Previously Shown to Be Protective Against White Spot Syndrome Virus (WSSV) in Shrimp.

PubMed

Ananphongmanee, Vorawit; Srisala, Jiraporn; Sritunyalucksana, Kallaya; Boonchird, Chuenchit

2015-01-01

Cell surface display using the yeasts Saccharomyces cerevisiae and Pichia pastoris has been extensively developed for application in bioindustrial processes. Due to the rigid structure of their cell walls, a number of proteins have been successfully displayed on their cell surfaces. It was previously reported that the viral binding protein Rab7 from the giant tiger shrimp Penaeus monodon (PmRab7) and its binding partner envelope protein VP28 of white spot syndrome virus (WSSV) could independently protect shrimp against WSSV infection. Thus, we aimed to display these two proteins independently on the cell surfaces of 2 yeast clones with the ultimate goal of using a mixture of the two clones as an orally deliverable, antiviral agent to protect shrimp against WSSV infection. PmRab7 and VP28 were modified by N-terminal tagging to the C-terminal half of S. cerevisiae α-agglutinin. DNA fragments, harboring fused-gene expression cassettes under control of an alcohol oxidase I (AOX1) promoter were constructed and used to transform the yeast cells. Immunofluorescence microscopy with antibodies specific to both proteins demonstrated that mutated PmRab7 (mPmRab7) and partial VP28 (pVP28) were localized on the cell surfaces of the respective clones, and fluorescence intensity for each was significantly higher than that of control cells by flow cytometry. Enzyme-linked immunosorbant assay (ELISA) using cells displaying mPmRab7 or pVP28 revealed that the binding of specific antibodies for each was dose-dependent, and could be saturated. In addition, the binding of mPmRab7-expressing cells with free VP28, and vice versa was dose dependent. Binding between the two surface-expressed proteins was confirmed by an assay showing agglutination between cells expressing complementary mPmRab7 and pVP28. In summary, our genetically engineered P. pastoris can display biologically active mPmRab7 and pVP28 and is now ready for evaluation of efficacy in protecting shrimp against WSSV by oral administration.

A mitochondria-anchored isoform of the actin-nucleating spire protein regulates mitochondrial division.

PubMed

Manor, Uri; Bartholomew, Sadie; Golani, Gonen; Christenson, Eric; Kozlov, Michael; Higgs, Henry; Spudich, James; Lippincott-Schwartz, Jennifer

2015-08-25

Mitochondrial division, essential for survival in mammals, is enhanced by an inter-organellar process involving ER tubules encircling and constricting mitochondria. The force for constriction is thought to involve actin polymerization by the ER-anchored isoform of the formin protein inverted formin 2 (INF2). Unknown is the mechanism triggering INF2-mediated actin polymerization at ER-mitochondria intersections. We show that a novel isoform of the formin-binding, actin-nucleating protein Spire, Spire1C, localizes to mitochondria and directly links mitochondria to the actin cytoskeleton and the ER. Spire1C binds INF2 and promotes actin assembly on mitochondrial surfaces. Disrupting either Spire1C actin- or formin-binding activities reduces mitochondrial constriction and division. We propose Spire1C cooperates with INF2 to regulate actin assembly at ER-mitochondrial contacts. Simulations support this model's feasibility and demonstrate polymerizing actin filaments can induce mitochondrial constriction. Thus, Spire1C is optimally positioned to serve as a molecular hub that links mitochondria to actin and the ER for regulation of mitochondrial division.

Concise Review: Cell Surface N-Linked Glycoproteins as Potential Stem Cell Markers and Drug Targets.

PubMed

Boheler, Kenneth R; Gundry, Rebekah L

2017-01-01

Stem cells and their derivatives hold great promise to advance regenerative medicine. Critical to the progression of this field is the identification and utilization of antibody-accessible cell-surface proteins for immunophenotyping and cell sorting-techniques essential for assessment and isolation of defined cell populations with known functional and therapeutic properties. Beyond their utility for cell identification and selection, cell-surface proteins are also major targets for pharmacological intervention. Although comprehensive cell-surface protein maps are highly valuable, they have been difficult to define until recently. In this review, we discuss the application of a contemporary targeted chemoproteomic-based technique for defining the cell-surface proteomes of stem and progenitor cells. In applying this approach to pluripotent stem cells (PSCs), these studies have improved the biological understanding of these cells, led to the enhanced use and development of antibodies suitable for immunophenotyping and sorting, and contributed to the repurposing of existing drugs without the need for high-throughput screening. The utility of this latter approach was first demonstrated with human PSCs (hPSCs) through the identification of small molecules that are selectively toxic to hPSCs and have the potential for eliminating confounding and tumorigenic cells in hPSC-derived progeny destined for research and transplantation. Overall, the cutting-edge technologies reviewed here will accelerate the development of novel cell-surface protein targets for immunophenotyping, new reagents to improve the isolation of therapeutically qualified cells, and pharmacological studies to advance the treatment of intractable diseases amenable to cell-replacement therapies. Stem Cells Translational Medicine 2017;6:131-138. © 2016 The Authors Stem Cells Translational Medicine published by Wiley Periodicals, Inc. on behalf of AlphaMed Press.

Gene expression profiling reveals different molecular patterns in G-protein coupled receptor signaling pathways between early- and late-onset preeclampsia.

PubMed

Liang, Mengmeng; Niu, Jianmin; Zhang, Liang; Deng, Hua; Ma, Jian; Zhou, Weiping; Duan, Dongmei; Zhou, Yuheng; Xu, Huikun; Chen, Longding

2016-04-01

Early-onset preeclampsia and late-onset preeclampsia have been regarded as two different phenotypes with heterogeneous manifestations; To gain insights into the pathogenesis of the two traits, we analyzed the gene expression profiles in preeclamptic placentas. A whole genome-wide microarray was used to determine the gene expression profiles in placental tissues from patients with early-onset (n = 7; <34 weeks), and late-onset (n = 8; >36 weeks) preeclampsia and their controls who delivered preterm (n = 5; <34 weeks) or at term (n = 5; >36 weeks). Genes were termed differentially expressed if they showed a fold-change ≥ 2 and q-value < 0.05. Quantitative real-time reverse transcriptase PCR was used to verify the results. Western blotting was performed to verify the expressions of secreted genes at the protein level. Six hundred twenty-seven genes were differentially expressed in early-compared with late-onset preeclampsia (177 genes were up-regulated and 450 were down-regulated). Gene ontology analysis identified significant alterations in several biological processes; the top two were immune response and cell surface receptor linked signal transduction. Among the cell surface receptor linked signal transduction-related, differentially expressed genes, those involved in the G-protein coupled receptor protein signaling pathway were significantly enriched. G-protein coupled receptor signaling pathway related genes, such as GPR124 and MRGPRF, were both found to be down-regulated in early-onset preeclampsia. The results were consistent with those of western blotting that the abundance of GPR124 was lower in early-onset compared with late-onset preeclampsia. The different gene expression profiles reflect the different levels of transcription regulation between the two conditions and supported the hypothesis that they are separate disease entities. Moreover, the G-protein coupled receptor signaling pathway related genes may contribute to the mechanism underlying early- and late-onset preeclampsia. Copyright © 2016 Elsevier Ltd. All rights reserved.

Nano and Microparticle-Enhanced Immunosensor Approaches for the Detection of Cancer Biomarker Proteins

NASA Astrophysics Data System (ADS)

Mani, Vigneshwaran

Accurate, sensitive, point-of-care multiplexed protein measurements are critical for early disease detection and monitoring, impacting biomarker and drug discovery, and personalized medicine. Significant application involves monitoring panels of proteins in the blood that are biomarkers for diagnosing cancer. However, measurements of biomarker panels in blood or other bodily fluids have been slow to integrate into current practice of cancer diagnostics partly due to the lack of technically simple, low-cost, sensitive, point-of-care multiplexed measurement devices, as well as the lack of rigorously validated protein panels. The present thesis in part addresses these limitations by the development of electrochemical and surface plasmon resonance (SPR) immunosensors utilizing 1mum superparamagnetic labels for accurate detection of prostate cancer biomarker proteins in patient serum samples. Electrochemical discrete immunosensors featuring nanostructured surface with densely packed 5 nm glutathione-coated gold nanoparticles coupled with multi-enzyme magnetic particle (MP) labels enabled measurement of prostate specific antigen (PSA) with a detection limit (DL) of 0.5 pg mL-1 in undiluted serum. Such low DLs are attributed to high surface area, conductivity of nanostructured surface, and multi-enzyme signal amplification. DLs are further improved by utilizing MP bioconjugated with more than 100,000 antibody labels to offline capture proteins from the serum sample matrix, minimizing nonspecific binding of interfering proteins on sensor surface before detection. This approach provided an unprecedented 10 fg DL mL-1 for PSA in undiluted serum using a flow SPR biosensor. Finally electrochemical microfluidic immunoarrays featuring nanostructured surface and offline protein capture by multi-label MPs enabled multiplexed detection of prostate cancer biomarkers PSA and interleukin-6 (IL-6). These approaches provided up to 1000-fold lower DLs compared to commercial bead based assays. The high sensitivity of these approaches will allow monitoring of biomarker levels in diseases states where proteins are in sub pg mL -1 concentrations that are normally challenging to detect using traditional methods such as enzyme linked immunosorbent assays (ELISA). Further emphases will be on SPR-based fundamental studies on binding affinity enhancement of MP conjugates to protein surfaces. In addition, this thesis describes the assembly of glucose/O2 enzymatic biofuel cells for power generation utilizing layer-by-layer films of osmium redox polymers and enzymes. Towards the end, the present thesis describes a simple, low-cost and accurate paper-based electrochemical device fabrication methods and its applications towards monitoring genotoxic activities in the environmental samples.

A Refined Model for the TSG-6 Link Module in Complex with Hyaluronan

PubMed Central

Higman, Victoria A.; Briggs, David C.; Mahoney, David J.; Blundell, Charles D.; Sattelle, Benedict M.; Dyer, Douglas P.; Green, Dixy E.; DeAngelis, Paul L.; Almond, Andrew; Milner, Caroline M.; Day, Anthony J.

2014-01-01

Tumor necrosis factor-stimulated gene-6 (TSG-6) is an inflammation-associated hyaluronan (HA)-binding protein that contributes to remodeling of HA-rich extracellular matrices during inflammatory processes and ovulation. The HA-binding domain of TSG-6 consists solely of a Link module, making it a prototypical member of the superfamily of proteins that interacts with this high molecular weight polysaccharide composed of repeating disaccharides of d-glucuronic acid and N-acetyl-d-glucosamine (GlcNAc). Previously we modeled a complex of the TSG-6 Link module in association with an HA octasaccharide based on the structure of the domain in its HA-bound conformation. Here we have generated a refined model for a HA/Link module complex using novel restraints identified from NMR spectroscopy of the protein in the presence of 10 distinct HA oligosaccharides (from 4- to 8-mers); the model was then tested using unique sugar reagents, i.e. chondroitin/HA hybrid oligomers and an octasaccharide in which a single sugar ring was 13C-labeled. The HA chain was found to make more extensive contacts with the TSG-6 surface than thought previously, such that a d-glucuronic acid ring makes stacking and ionic interactions with a histidine and lysine, respectively. Importantly, this causes the HA to bend around two faces of the Link module (resembling the way that HA binds to CD44), potentially providing a mechanism for how TSG-6 can reorganize HA during inflammation. However, the HA-binding site defined here may not play a role in TSG-6-mediated transfer of heavy chains from inter-α-inhibitor onto HA, a process known to be essential for ovulation. PMID:24403066

GlycoPP: A Webserver for Prediction of N- and O-Glycosites in Prokaryotic Protein Sequences

PubMed Central

Chauhan, Jagat S.; Bhat, Adil H.; Raghava, Gajendra P. S.; Rao, Alka

2012-01-01

Glycosylation is one of the most abundant post-translational modifications (PTMs) required for various structure/function modulations of proteins in a living cell. Although elucidated recently in prokaryotes, this type of PTM is present across all three domains of life. In prokaryotes, two types of protein glycan linkages are more widespread namely, N- linked, where a glycan moiety is attached to the amide group of Asn, and O- linked, where a glycan moiety is attached to the hydroxyl group of Ser/Thr/Tyr. For their biologically ubiquitous nature, significance, and technology applications, the study of prokaryotic glycoproteins is a fast emerging area of research. Here we describe new Support Vector Machine (SVM) based algorithms (models) developed for predicting glycosylated-residues (glycosites) with high accuracy in prokaryotic protein sequences. The models are based on binary profile of patterns, composition profile of patterns, and position-specific scoring matrix profile of patterns as training features. The study employ an extensive dataset of 107 N-linked and 116 O-linked glycosites extracted from 59 experimentally characterized glycoproteins of prokaryotes. This dataset includes validated N-glycosites from phyla Crenarchaeota, Euryarchaeota (domain Archaea), Proteobacteria (domain Bacteria) and validated O-glycosites from phyla Actinobacteria, Bacteroidetes, Firmicutes and Proteobacteria (domain Bacteria). In view of the current understanding that glycosylation occurs on folded proteins in bacteria, hybrid models have been developed using information on predicted secondary structures and accessible surface area in various combinations with training features. Using these models, N-glycosites and O-glycosites could be predicted with an accuracy of 82.71% (MCC 0.65) and 73.71% (MCC 0.48), respectively. An evaluation of the best performing models with 28 independent prokaryotic glycoproteins confirms the suitability of these models in predicting N- and O-glycosites in potential glycoproteins from aforementioned organisms, with reasonably high confidence. A web server GlycoPP, implementing these models is available freely at http:/www.imtech.res.in/raghava/glycopp/. PMID:22808107

Neurodegenerative disease mutations in TREM2 reveal a functional surface and distinct loss-of-function mechanisms

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

Kober, Daniel L.; Alexander-Brett, Jennifer M.; Karch, Celeste M.

Genetic variations in the myeloid immune receptor TREM2 are linked to several neurodegenerative diseases. To determine how TREM2 variants contribute to these diseases, we performed structural and functional studies of wild-type and variant proteins. Our 3.1 Å TREM2 crystal structure revealed that mutations found in Nasu-Hakola disease are buried whereas Alzheimer’s disease risk variants are found on the surface, suggesting that these mutations have distinct effects on TREM2 function. Biophysical and cellular methods indicate that Nasu-Hakola mutations impact protein stability and decrease folded TREM2 surface expression, whereas Alzheimer’s risk variants impact binding to a TREM2 ligand. Additionally, the Alzheimer’s riskmore » variants appear to epitope map a functional surface on TREM2 that is unique within the larger TREM family. These findings provide a guide to structural and functional differences among genetic variants of TREM2, indicating that therapies targeting the TREM2 pathway should be tailored to these genetic and functional differences with patient-specific medicine approaches for neurodegenerative disorders.« less

Genetic Diversity of Plasmodium falciparum Merozoite Surface Protein-1 Block 2 in Sites of Contrasting Altitudes and Malaria Endemicities in the Mount Cameroon Region

PubMed Central

Wanji, Samuel; Kengne-Ouafo, Arnaud J.; Joan Eyong, Ebanga E.; Kimbi, Helen K.; Tendongfor, Nicholas; Ndamukong-Nyanga, Judith L.; Nana-Djeunga, Hugues C.; Bourguinat, Catherine; Sofeu-Feugaing, David D.; Charvet, Claude L.

2012-01-01

The present study analyzed the relationship between the genetic diversity of Plasmodium falciparum and parasitologic/entomologic indices in the Mount Cameroon region by using merozoite surface protein 1 as a genetic marker. Blood samples were collected from asymptomatic children from three altitude zones (high, intermediate, and low). Parasitologic and entomologic indices were determined by microscopy and landing catch mosquito collection/circumsporozoite protein–enzyme-linked immunosorbent assay, respectively. A total of 142 randomly selected P. falciparum-positive blood samples were genotyped by using a nested polymerase chain reaction–based technique. K-1 polymerase chain reaction products were also sequenced. As opposed to high altitude, the highest malaria prevalence (70.65%) and entomologic inoculation rate (2.43 infective/bites/night) were recorded at a low altitude site. Seven (18.91%), 22 (36.66%), and 19 (42.22%) samples from high, intermediate, and low altitudes, respectively, contained multiclonal infections. A new K-1 polymorphism was identified. This study shows a positive non-linear association between low/intermediate altitude (high malaria transmission) and an increase in P. falciparum merozoite surface protein 1 block 2 polymorphisms. PMID:22556072

PPAR-γ and Akt regulate GLUT1 and GLUT3 surface localization during Mycobacterium tuberculosis infection.

PubMed

Dasgupta, Shyamashree; Rai, Ramesh Chandra

2018-03-01

The success of Mycobacterium tuberculosis (Mtb) as a pathogen stems from its ability to manipulate the host macrophage towards increased lipid biogenesis and lipolysis inhibition. Inhibition of lipolysis requires augmented uptake of glucose into the host cell causing an upregulation of the glucose transporters GLUT1 and GLUT3 on the cell surface. Mechanism behind this upregulation of the GLUT proteins during Mtb infection is hitherto unknown and demands intensive investigation in order to understand the pathways linked with governing them. Our endeavor to investigate some of the key proteins that have been found to be affected during Mtb infection led us to investigate host molecular pathways such as Akt and PPAR-γ that remain closely associated with the survival of the bacilli by modulating the localization of glucose transporters GLUT1 and GLUT3.

PEG Molecular Net-Cloth Grafted on Polymeric Substrates and Its Bio-Merits

NASA Astrophysics Data System (ADS)

Zhao, Changwen; Lin, Zhifeng; Yin, Huabing; Ma, Yuhong; Xu, Fujian; Yang, Wantai

2014-05-01

Polymer brushes and hydrogels are sensitive to the environment, which can cause uncontrolled variations on their performance. Herein, for the first time, we report a non-swelling ``PEG molecular net-cloth'' on a solid surface, fabricated using a novel ``visible light induced surface controlled graft cross-linking polymerization'' (VSCGCP) technique. Via this method, we show that 1) the 3D-network structure of the net-cloth can be precisely modulated and its thickness controlled; 2) the PEG net-cloth has excellent resistance to non-specific protein adsorption and cell adhesion; 3) the mild polymerization conditions (i.e. visible light and room temperature) provided an ideal tool for in situ encapsulation of delicate biomolecules such as enzymes; 4) the successive grafting of reactive three-dimensional patterns on the PEG net-cloth enables the creation of protein microarrays with high signal to noise ratio. Importantly, this strategy is applicable to any C-H containing surface, and can be easily tailored for a broad range of applications.

Linking human beta retrovirus infection with primary biliary cirrhosis.

PubMed

Mason, A L; Zhang, G

2010-01-01

Several environmental agents have been linked with primary biliary cirrhosis (PBC) that include bacteria, xenobiotics and viruses. A human beta retrovirus (HBRV) related to mouse mammary tumor virus has been cloned and characterized from patients with PBC. This agent can be detected in the majority of patients' perihepatic lymph nodes by immunochemistry and RT-PCR. The HBRV has recently been isolated in culture and integration sites have been identified in the genome of patients to provide convincing evidence of beta retrovirus infection in patients. Three lines of evidence support a role for the virus in PBC. First, the beta retrovirus is linked with aberrant expression of mitochondrial protein(s) on the biliary epithelium cell (BEC) surface, a disease specific phenotype. Second, the related agent, mouse mammary tumor virus has been linked with autoimmune biliary disease in the NOD.c3c4 mouse model for PBC. In this mouse model, the virus is localized to diseased biliary epithelium that also display aberrant expression of the mitochondrial autoantigens. In translational studies, both patients with PBC and NOD.c3c4 mice demonstrate significant improvement in biliary disease with combination antiviral therapy. An overview of the biological relevance of the beta retrovirus infection in PBC will be discussed in this review. Copyright 2010 Elsevier Masson SAS. All rights reserved.

Crystal Structures of Lys-63-linked tri- and di-ubiquitin Reveal a Highly Extended Chain Architecture

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

Weeks, S.; Grasty, K; Hernandez-Cuebas, L

2009-01-01

The covalent attachment of different types of poly-ubiquitin chains signal different outcomes for the proteins so targeted. For example, a protein modified with Lys-48-linked poly-ubiquitin chains is targeted for proteasomal degradation, whereas Lys-63-linked chains encode nondegradative signals. The structural features that enable these different types of chains to encode different signals have not yet been fully elucidated. We report here the X-ray crystal structures of Lys-63-linked tri- and di-ubiquitin at resolutions of 2.3 and 1.9 {angstrom}, respectively. The tri- and di-ubiquitin species adopt essentially identical structures. In both instances, the ubiquitin chain assumes a highly extended conformation with a left-handedmore » helical twist; the helical chain contains four ubiquitin monomers per turn and has a repeat length of {approx}110 {angstrom}. Interestingly, Lys-48 ubiquitin chains also adopt a left-handed helical structure with a similar repeat length. However, the Lys-63 architecture is much more open than that of Lys-48 chains and exposes much more of the ubiquitin surface for potential recognition events. These new crystal structures are consistent with the results of solution studies of Lys-63 chain conformation, and reveal the structural basis for differential recognition of Lys-63 versus Lys-48 chains.« less

Identification of Surface-Exposed Protein Radicals and A Substrate Oxidation Site in A-Class Dye-Decolorizing Peroxidase from Thermomonospora curvata

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

Shrestha, Ruben; Chen, Xuejie; Ramyar, Kasra X.

Dye-decolorizing peroxidases (DyPs) are a family of heme peroxidases in which a catalytic distal aspartate is involved in H 2O 2 activation to catalyze oxidations under acidic conditions. They have received much attention due to their potential applications in lignin compound degradation and biofuel production from biomass. However, the mode of oxidation in bacterial DyPs remains unknown. We have recently reported that the bacterial TcDyP from Thermomonospora curvata is among the most active DyPs and shows activity toward phenolic lignin model compounds. On the basis of the X-ray crystal structure solved at 1.75 Å, sigmoidal steady-state kinetics with Reactive Bluemore » 19 (RB19), and formation of compound II like product in the absence of reducing substrates observed with stopped-flow spectroscopy and electron paramagnetic resonance (EPR), we hypothesized that the TcDyP catalyzes oxidation of large-size substrates via multiple surface-exposed protein radicals. Among 7 tryptophans and 3 tyrosines in TcDyP consisting of 376 residues for the matured protein, W263, W376, and Y332 were identified as surface-exposed protein radicals. Only the W263 was also characterized as one of the surface-exposed oxidation sites. SDS-PAGE and size-exclusion chromatography demonstrated that W376 represents an off-pathway destination for electron transfer, resulting in the cross-linking of proteins in the absence of substrates. Mutation of W376 improved compound I stability and overall catalytic efficiency toward RB19. While Y332 is highly conserved across all four classes of DyPs, its catalytic function in A-class TcDyP is minimal, possibly due to its extremely small solvent-accessible areas. Identification of surface-exposed protein radicals and substrate oxidation sites is important for understanding the DyP mechanism and modulating its catalytic functions for improved activity on phenolic lignin.« less

Metal-amplified Density Assays, (MADAs), including a Density-Linked Immunosorbent Assay (DeLISA).

PubMed

Subramaniam, Anand Bala; Gonidec, Mathieu; Shapiro, Nathan D; Kresse, Kayleigh M; Whitesides, George M

2015-02-21

This paper reports the development of Metal-amplified Density Assays, or MADAs - a method of conducting quantitative or multiplexed assays, including immunoassays, by using Magnetic Levitation (MagLev) to measure metal-amplified changes in the density of beads labeled with biomolecules. The binding of target analytes (i.e. proteins, antibodies, antigens) to complementary ligands immobilized on the surface of the beads, followed by a chemical amplification of the binding in a form that results in a change in the density of the beads (achieved by using gold nanoparticle-labeled biomolecules, and electroless deposition of gold or silver), translates analyte binding events into changes in density measureable using MagLev. A minimal model based on diffusion-limited growth of hemispherical nuclei on a surface reproduces the dynamics of the assay. A MADA - when performed with antigens and antibodies - is called a Density-Linked Immunosorbent Assay, or DeLISA. Two immunoassays provided a proof of principle: a competitive quantification of the concentration of neomycin in whole milk, and a multiplexed detection of antibodies against Hepatitis C virus NS3 protein and syphilis T. pallidum p47 protein in serum. MADAs, including DeLISAs, require, besides the requisite biomolecules and amplification reagents, minimal specialized equipment (two permanent magnets, a ruler or a capillary with calibrated length markings) and no electrical power to obtain a quantitative readout of analyte concentration. With further development, the method may be useful in resource-limited or point-of-care settings.

Synthesis and processing of ELISA polymer substitute: The influence of surface chemistry and morphology on detection sensitivity

NASA Astrophysics Data System (ADS)

Hosseini, Samira; Ibrahim, Fatimah; Djordjevic, Ivan; Rothan, Hussin A.; Yusof, Rohana; van der Marel, Cees; Koole, Leo H.

2014-10-01

Despite the known drawbacks of enzyme-linked immunosorbent assay (ELISA), one of the deficiencies that have relatively been ignored is the performance of ELISA substrate itself. Polystyrene (PS), as the cost effective material of choice for mass production of ELISA well-plates, has shown obvious lacks of suitable physical and chemical properties for protein attachment. The general concept of this work was to develop a potential substrate that can be suggested as a material of choice for production of a new generation of ELISA analytical kits. Spin-coated thin films of polymethyl methacrylate-co-methacrylic acid (PMMA-co-MAA) on silicon surfaces were designed and processed for detection of dengue virus. Coated surfaces of different molar ratios have been investigated as carboxyl-functionalized layers for obtaining platform for biomolecule immobilization with high level of protein activity. To improve the sensitivity of detection, we have used amine functional "spacers", hexamethylenediamine (HMDA) and polyethyleneimine (PEI), which were covalently bonded to the surfaces of PMMA-co-MAA coatings. Results demonstrate that the variation of surface concentration of carboxyl groups of PMMA-co-MAA can be used to control the amine surface concentration after carbodiimide coupling with HMDA and PEI spacers. The presence of amine spacers increases hydrophilicity of the coatings and significantly impacts the polymer surface morphology. In particular, protein immobilization via amine-bearing spacers has been achieved in two effective steps: (1) carbodiimide bonding between amine spacer molecules and PMMA-co-MAA polymer coatings; and (2) covalent immobilization of antibody via glutaraldehyde reaction with amine groups from amine-treated surfaces. The application of PEI spacer in comparison to HMDA has shown much higher intensity of detection signal in ELISA experiment, indicating better immobilization efficiency and preservation of antibody activity upon attachment to the polymer surface.

A homolog of Drosophila grainy head is essential for epidermal integrity in mice.

PubMed

Ting, Stephen B; Caddy, Jacinta; Hislop, Nikki; Wilanowski, Tomasz; Auden, Alana; Zhao, Lin-Lin; Ellis, Sarah; Kaur, Pritinder; Uchida, Yoshikazu; Holleran, Walter M; Elias, Peter M; Cunningham, John M; Jane, Stephen M

2005-04-15

The Drosophila cuticle is essential for maintaining the surface barrier defenses of the fly. Integral to cuticle resilience is the transcription factor grainy head, which regulates production of the enzyme required for covalent cross-linking of the cuticular structural components. We report that formation and maintenance of the epidermal barrier in mice are dependent on a mammalian homolog of grainy head, Grainy head-like 3. Mice lacking this factor display defective skin barrier function and deficient wound repair, accompanied by reduced expression of transglutaminase 1, the key enzyme involved in cross-linking the structural components of the superficial epidermis. These findings suggest that the functional mechanisms involving protein cross-linking that maintain the epidermal barrier and induce tissue repair are conserved across 700 million years of evolution.

Bioadhesive control of plasma proteins and blood cells from umbilical cord blood onto the interface grafted with zwitterionic polymer brushes.

PubMed

Chang, Yu; Chang, Yung; Higuchi, Akon; Shih, Yu-Ju; Li, Pei-Tsz; Chen, Wen-Yih; Tsai, Eing-Mei; Hsiue, Ging-Ho

2012-03-06

In this work, bioadhesive behavior of plasma proteins and blood cells from umbilical cord blood (UCB) onto zwitterionic poly(sulfobetaine methacrylate) (polySBMA) polymer brushes was studied. The surface coverage of polySBMA brushes on a hydrophobic polystyrene (PS) well plate with surface grafting weights ranging from 0.02 mg/cm(2) to 0.69 mg/cm(2) can be effectively controlled using the ozone pretreatment and thermal-induced radical graft-polymerization. The chemical composition, grafting structure, surface hydrophilicity, and hydration capability of prepared polySBMA brushes were determined to illustrate the correlations between grafting properties and blood compatibility of zwitterionic-grafted surfaces in contact with human UCB. The protein adsorption of fibrinogen in single-protein solutions and at complex medium of 100% UCB plasma onto different polySBMA brushes with different grafting coverage was measured by enzyme-linked immunosorbent assay (ELISA) with monoclonal antibodies. The grafting density of the zwitterionic brushes greatly affects the PS surface, thus controlling the adsorption of fibrinogen, the adhesion of platelets, and the preservation of hematopoietic stem and progenitor cells (HSPCs) in UCB. The results showed that PS surfaces grafted with polySBMA brushes possess controllable hydration properties through the binding of water molecules, regulating the bioadhesive and bioinert characteristics of plasma proteins and blood platelets in UCB. Interestingly, it was found that the polySBMA brushes with an optimized grafting weight of approximately 0.1 mg/cm(2) at physiologic temperatures show significant hydrated chain flexibility and balanced hydrophilicity to provide the best preservation capacity for HSPCs stored in 100% UCB solution for 2 weeks. This work suggests that, through controlling grafting structures, the hemocompatible nature of grafted zwitterionic polymer brushes makes them well suited to the molecular design of regulated bioadhesive interfaces for use in the preservation of HSPCs from human UCB.

Intracellular targeting of CD44+ cells with self-assembling, protein only nanoparticles.

PubMed

Pesarrodona, Mireia; Ferrer-Miralles, Neus; Unzueta, Ugutz; Gener, Petra; Tatkiewicz, Witold; Abasolo, Ibane; Ratera, Imma; Veciana, Jaume; Schwartz, Simó; Villaverde, Antonio; Vazquez, Esther

2014-10-01

CD44 is a multifunctional cell surface protein involved in proliferation and differentiation, angiogenesis and signaling. The expression of CD44 is up-regulated in several types of human tumors and particularly in cancer stem cells, representing an appealing target for drug delivery in the treatment of cancer. We have explored here several protein ligands of CD44 for the construction of self-assembling modular proteins designed to bind and internalize target cells. Among five tested ligands, two of them (A5G27 and FNI/II/V) drive the formation of protein-only, ring-shaped nanoparticles of about 14 nm that efficiently bind and penetrate CD44(+) cells by an endosomal route. The potential of these newly designed nanoparticles is evaluated regarding the need of biocompatible nanostructured materials for drug delivery in CD44-linked conditions. Copyright © 2014 Elsevier B.V. All rights reserved.

Optically degradable dendrons for temporary adhesion of proteins to DNA.

PubMed

Kostiainen, Mauri A; Kotimaa, Juha; Laukkanen, Marja-Leena; Pavan, Giovanni M

2010-06-18

Experimental studies and molecular dynamics modeling demonstrate that multivalent dendrons can be used to temporarily glue proteins and DNA together with high affinity. We describe N-maleimide-cored polyamine dendrons that can be conjugated with free cysteine residues on protein surfaces through 1,4-conjugate addition to give one-to-one protein-polymer conjugates. We used a genetically engineered cysteine mutant of class II hydrophobin (HFBI) and a single-chain Fragment variable (scFv) antibody as model proteins for the conjugation reactions. The binding affinity of the protein-dendron conjugates towards DNA was experimentally assessed by using the ethidium bromide displacement assay. The binding was found to depend on the generation of the dendron, with the second generation having a stronger affinity than the first generation. Thermodynamic parameters of the binding were obtained from molecular dynamics modeling, which showed that the high binding affinity for each system is almost completely driven by a strong favorable binding enthalpy that is opposed by unfavorable binding entropy. A short exposure to UV (lambda approximately 350 nm) can cleave the photolabile o-nitrobenzyl-linked binding ligands from the surface of the dendron, which results in loss of the multivalent binding interactions and triggers the release of the DNA and protein. The timescale of the release is very rapid and the binding partners can be efficiently released after 3 min of UV exposure.

Endocytosis of GPI-anchored proteins in human lymphocytes: role of glycolipid-based domains, actin cytoskeleton, and protein kinases

PubMed Central

1996-01-01

GPI-anchored surface proteins mediate many important functions, including transport, signal transduction, adhesion, and protection against complement. They cluster into glycolipid-based membrane domains and caveolae, plasmalemmal vesicles involved in the transcytosis and endocytosis of these surface proteins. However, in lymphocytes, neither the characteristic flask shaped caveolae nor caveolin, a transmembrane protein typical of caveolae, have been observed. Here, we show that the GPI-anchored CD59 molecule on Jurkat T cells is internalized after cross-linking, a process inhibited by nystatin, a sterol chelating agent. Clustered CD59 molecules mostly accumulate in non-coated invaginations of the lymphocyte membrane before endocytosis, in marked contrast with the pattern of CD3-TCR internalization. Cytochalasin H blocked CD59 internalization in lymphocytes, but neither CD3 internalization nor transferrin uptake. Confocal microscopy analysis of F-actin distribution within lymphocytes showed that CD59 clusters were associated with patches of polymerized actin. Also, we found that internalization of CD59 was prevented by the protein kinase C inhibitor staurosporine and by the protein kinase A activator forskolin. Thus, in lymphocytes, as in other cell types, glycolipid-based domains provide sites of integration of signaling pathways involved in GPI-anchored protein endocytosis. This process, which is regulated by both protein kinase C and A activity, is tightly controlled by the dynamic organization of actin cytoskeleton, and may be critical for polarized contacts of circulating cells. PMID:8666664

Effects of tunicamycin, mannosamine, and other inhibitors of glycoprotein processing on skeletal alkaline phosphatase in human osteoblast-like cells.

PubMed

Farley, J R; Magnusson, P

2005-01-01

Skeletal alkaline phosphatase (sALP) is a glycoprotein- approximately 20% carbohydrate by weight, with five presumptive sites for N-linked glycosylation, as well as a carboxy-terminal site for attachment of the glycolipid structure (glycosylphosphatidylinositol, GPI), which anchors sALP to the outer surface of osteoblasts. The current studies were intended to characterize the effects of inhibiting glycosylation and glycosyl-processing on the synthesis, plasma membrane attachment, cellular-extracellular distribution, and reaction kinetics of sALP in human osteosarcoma (SaOS-2) cells. sALP synthesis, glycosylation, and GPI-anchor attachment were assessed as total protein synthesis/immunospecific sALP synthesis, sialic acid content (i.e., wheat germ agglutinin precipitation), and insolubility (i.e., temperature-dependent phase-separation), respectively. sALP reaction kinetics were characterized by analysis of dose-dependent initial velocity data, with a phosphoryl substrate. The results of these studies revealed that the inhibition of either N-linked glycosylation or oligosaccharide synthesis for GPI-anchor addition could affect the synthesis and the distribution of sALP, but not the kinetics of the phosphatase reaction. Tunicamycin-which blocks N-linked glycosylation by inhibiting core oligosaccharide synthesis-decreased cell layer protein and the total amount of sALP in the cells, while increasing the relative level of sALP in the cell-conditioned culture medium (CM, i.e., the amount of sALP released). These effects were attributed to dose- and time-dependent decreases in sALP synthesis and N-linked glycosylation, and an increase in apoptotic cell death (P <0.001 for each). In contrast to the effects of tunicamycin on N-linked glycosylation, the effects of mannosamine, which inhibits GPI-anchor glycosylation/formation, included (1) an increase in cell layer protein; (2) decreases in sALP specific activity, in the cells and in the CM; and (3) increases in the percentages of both anchorless and wheat germ agglutinin (WGA)-soluble sALP in the medium, but not in the cells (P <0.005 for each). These effects of mannosamine were, presumably, a consequence of inhibiting the insertion/attachment of sALP to the outside of the plasma membrane surface. Neither mannosammine nor tunicamycin had any effect on the reaction kinetics of sALP or on the apparent affinity (the value of KM) for the phosphoryl substrate.

A green and bio-inspired process to afford durable anti-biofilm properties to stainless steel.

PubMed

Faure, E; Vreuls, C; Falentin-Daudré, C; Zocchi, G; Van de Weerdt, C; Martial, J; Jérôme, C; Duwez, A-S; Detrembleur, C

2012-01-01

A bio-inspired durable anti-biofilm coating was developed for industrial stainless steel (SS) surfaces. Two polymers inspired from the adhesive and cross-linking properties of mussels were designed and assembled from aqueous solutions onto SS surfaces to afford durable coatings. Trypsin, a commercially available broad spectrum serine protease, was grafted as the final active layer of the coating. Its proteolytic activity after long immersion periods was demonstrated against several substrata, viz. a synthetic molecule, N-α-benzoyl-DL-arginine-p-nitroanilide hydrochloride (BAPNA), a protein, FTC-casein, and Gram-positive biofilm forming bacterium Staphylococcus epidermidis.

Weighted gene co-expression network analysis of colorectal cancer liver metastasis genome sequencing data and screening of anti-metastasis drugs.

PubMed

Gao, Bo; Shao, Qin; Choudhry, Hani; Marcus, Victoria; Dong, Kung; Ragoussis, Jiannis; Gao, Zu-Hua

2016-09-01

Approximately 9% of cancer-related deaths are caused by colorectal cancer (CRC). CRC patients are prone to liver metastasis, which is the most important cause for the high CRC mortality rate. Understanding the molecular mechanism of CRC liver metastasis could help us to find novel targets for the effective treatment of this deadly disease. Using weighted gene co-expression network analysis on the sequencing data of CRC with and with metastasis, we identified 5 colorectal cancer liver metastasis related modules which were labeled as brown, blue, grey, yellow and turquoise. In the brown module, which represents the metastatic tumor in the liver, gene ontology (GO) analysis revealed functions including the G-protein coupled receptor protein signaling pathway, epithelial cell differentiation and cell surface receptor linked signal transduction. In the blue module, which represents the primary CRC that has metastasized, GO analysis showed that the genes were mainly enriched in GO terms including G-protein coupled receptor protein signaling pathway, cell surface receptor linked signal transduction, and negative regulation of cell differentiation. In the yellow and turquoise modules, which represent the primary non-metastatic CRC, 13 downregulated CRC liver metastasis-related candidate miRNAs were identified (e.g. hsa-miR-204, hsa-miR-455, etc.). Furthermore, analyzing the DrugBank database and mining the literature identified 25 and 12 candidate drugs that could potentially block the metastatic processes of the primary tumor and inhibit the progression of metastatic tumors in the liver, respectively. Data generated from this study not only furthers our understanding of the genetic alterations that drive the metastatic process, but also guides the development of molecular-targeted therapy of colorectal cancer liver metastasis.

The Missing Link in Epstein-Barr Virus Immune Evasion: the BDLF3 Gene Induces Ubiquitination and Downregulation of Major Histocompatibility Complex Class I (MHC-I) and MHC-II

PubMed Central

Quinn, Laura L.; Williams, Luke R.; White, Claire; Forrest, Calum; Rowe, Martin

2015-01-01

ABSTRACT The ability of Epstein-Barr virus (EBV) to spread and persist in human populations relies on a balance between host immune responses and EBV immune evasion. CD8+ cells specific for EBV late lytic cycle antigens show poor recognition of target cells compared to immediate early and early antigen-specific CD8+ cells. This phenomenon is due in part to the early EBV protein BILF1, whose immunosuppressive activity increases with lytic cycle progression. However, published data suggest the existence of a hitherto unidentified immune evasion protein further enhancing protection against late EBV antigen-specific CD8+ cells. We have now identified the late lytic BDLF3 gene as the missing link accounting for efficient evasion during the late lytic cycle. Interestingly, BDLF3 also contributes to evasion of CD4+ cell responses to EBV. We report that BDLF3 downregulates expression of surface major histocompatibility complex (MHC) class I and class II molecules in the absence of any effect upon other surface molecules screened, including CD54 (ICAM-1) and CD71 (transferrin receptor). BDLF3 both enhanced internalization of surface MHC molecules and reduced the rate of their appearance at the cell surface. The reduced expression of surface MHC molecules correlated with functional protection against CD8+ and CD4+ T cell recognition. The molecular mechanism was identified as BDLF3-induced ubiquitination of MHC molecules and their subsequent downregulation in a proteasome-dependent manner. IMPORTANCE Immune evasion is a necessary feature of viruses that establish lifelong persistent infections in the face of strong immune responses. EBV is an important human pathogen whose immune evasion mechanisms are only partly understood. Of the EBV immune evasion mechanisms identified to date, none could explain why CD8+ T cell responses to late lytic cycle genes are so infrequent and, when present, recognize lytically infected target cells so poorly relative to CD8+ T cells specific for early lytic cycle antigens. The present work identifies an additional immune evasion protein, BDLF3, that is expressed late in the lytic cycle and impairs CD8+ T cell recognition by targeting cell surface MHC class I molecules for ubiquitination and proteasome-dependent downregulation. Interestingly, BDLF3 also targets MHC class II molecules to impair CD4+ T cell recognition. BDLF3 is therefore a rare example of a viral protein that impairs both the MHC class I and class II antigen-presenting pathways. PMID:26468525

The Missing Link in Epstein-Barr Virus Immune Evasion: the BDLF3 Gene Induces Ubiquitination and Downregulation of Major Histocompatibility Complex Class I (MHC-I) and MHC-II.

PubMed

Quinn, Laura L; Williams, Luke R; White, Claire; Forrest, Calum; Zuo, Jianmin; Rowe, Martin

2016-01-01

The ability of Epstein-Barr virus (EBV) to spread and persist in human populations relies on a balance between host immune responses and EBV immune evasion. CD8(+) cells specific for EBV late lytic cycle antigens show poor recognition of target cells compared to immediate early and early antigen-specific CD8(+) cells. This phenomenon is due in part to the early EBV protein BILF1, whose immunosuppressive activity increases with lytic cycle progression. However, published data suggest the existence of a hitherto unidentified immune evasion protein further enhancing protection against late EBV antigen-specific CD8(+) cells. We have now identified the late lytic BDLF3 gene as the missing link accounting for efficient evasion during the late lytic cycle. Interestingly, BDLF3 also contributes to evasion of CD4(+) cell responses to EBV. We report that BDLF3 downregulates expression of surface major histocompatibility complex (MHC) class I and class II molecules in the absence of any effect upon other surface molecules screened, including CD54 (ICAM-1) and CD71 (transferrin receptor). BDLF3 both enhanced internalization of surface MHC molecules and reduced the rate of their appearance at the cell surface. The reduced expression of surface MHC molecules correlated with functional protection against CD8(+) and CD4(+) T cell recognition. The molecular mechanism was identified as BDLF3-induced ubiquitination of MHC molecules and their subsequent downregulation in a proteasome-dependent manner. Immune evasion is a necessary feature of viruses that establish lifelong persistent infections in the face of strong immune responses. EBV is an important human pathogen whose immune evasion mechanisms are only partly understood. Of the EBV immune evasion mechanisms identified to date, none could explain why CD8(+) T cell responses to late lytic cycle genes are so infrequent and, when present, recognize lytically infected target cells so poorly relative to CD8(+) T cells specific for early lytic cycle antigens. The present work identifies an additional immune evasion protein, BDLF3, that is expressed late in the lytic cycle and impairs CD8(+) T cell recognition by targeting cell surface MHC class I molecules for ubiquitination and proteasome-dependent downregulation. Interestingly, BDLF3 also targets MHC class II molecules to impair CD4(+) T cell recognition. BDLF3 is therefore a rare example of a viral protein that impairs both the MHC class I and class II antigen-presenting pathways. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Structural model of the p14/SF3b155 · branch duplex complex.

PubMed

Schellenberg, Matthew J; Dul, Erin L; MacMillan, Andrew M

2011-01-01

Human p14 (SF3b14), a component of the spliceosomal U2 snRNP, interacts directly with the pre-mRNA branch adenosine within the context of the bulged duplex formed between the pre-mRNA branch region and U2 snRNA. This association occurs early in spliceosome assembly and persists within the fully assembled spliceosome. Analysis of the crystal structure of a complex containing p14 and a peptide derived from p14-associated SF3b155 combined with the results of cross-linking studies has suggested that the branch nucleotide interacts with a pocket on a non-canonical RNA binding surface formed by the complex. Here we report a structural model of the p14 · bulged duplex interaction based on a combination of X-ray crystallography of an adenine p14/SF3b155 peptide complex, biochemical comparison of a panel of disulfide cross-linked protein-RNA complexes, and small-angle X-ray scattering (SAXS). These studies reveal specific recognition of the branch adenosine within the p14 pocket and establish the orientation of the bulged duplex RNA bound on the protein surface. The intimate association of one surface of the bulged duplex with the p14/SF3b155 peptide complex described by this model buries the branch nucleotide at the interface and suggests that p14 · duplex interaction must be disrupted before the first step of splicing.

Cross-Linking Studies of Lysozyme Nucleation

NASA Technical Reports Server (NTRS)

Forsythe, Elizabeth; Pusey, Marc

2000-01-01

Tetragonal chicken egg white crystals consist of 4(sub 3) helices running in alternating directions, the helix rows having a two fold symmetry with each other. The unit cell consists of one complete tetrameric turn from each of two adjacent helices (an octamer). PBC analysis indicates that the helix intermolecular bonds are the strongest in the crystal, therefore likely formed first. AFM analysis of the (110) surface shows only complete helices, no half steps or bisected helices being found, while AFM line scans to measure the growth step increments show that they are multiples of the 4(sub 3) helix tetramer dimensions. This supports our thesis that the growth units are in fact multiples of the four molecule 4(sub 3) helix unit, the "average" growth unit size for the (110) face being an octamer (two turns about the helix) and the (101) growth unit averaging about the size of a hexamer. In an effort to better understand the species involved in the crystal nucleation and growth process, we have initiated an experimental program to study the species formed in solution compared to what is found in the crystal through covalent cross-linking studies. These experiments use the heterobifunctional cross-linking agent aminoethyl-4-azidonitroanaline (AEANA). An aliphatic amine at one end is covalently attached to the protein by a carbodiimide-mediated reaction, and a photo reactive group at the other can be used to initiate crosslinking. Modifications to the parent structure can be used to alter the distance between the two reactive groups and thus the cross-linking agents "reach". In practice, the cross-linking agent is first coupled to the asp101 side chain through the amine group. Asp101 lies within the active site cleft, and previous work with fluorescent probes had shown that derivatives at this site still crystallize in the tetragonal space group. This was also found to be the case with the AEANA derivative, which gave red tetragonal crystals. The protein now has a reactive group that can be photoactivated at a specific point in the nucleation or crystal growth process to "capture" protein molecules bound within reach of the crosslinking agent. If those bound protein molecules have a defined geometric relationship with the capturing molecule, such as would be found in a crystal, then the photoreacted cross-linking site should be consistent. Random protein interactions, typical of an amorphous precipitate or interaction, would show a random cross-linking reaction. The results of these and other experiments will be presented.

Protein-Linked Glycan Degradation in Infants Fed Human Milk

PubMed Central

Dallas, David C.; Sela, David; Underwood, Mark A.; German, J. Bruce; Lebrilla, Carlito

2014-01-01

Many human milk proteins are glycosylated. Glycosylation is important in protecting bioactive proteins and peptide fragments from digestion. Protein-linked glycans have a variety of functions; however, there is a paucity of information on protein-linked glycan degradation in either the infant or the adult digestive system. Human digestive enzymes can break down dietary disaccharides and starches, but most of the digestive enzymes required for complex protein-linked glycan degradation are absent from both human digestive secretions and the external brush border membrane of the intestinal lining. Indeed, complex carbohydrates remain intact throughout their transit through the stomach and small intestine, and are undegraded by in vitro incubation with either adult pancreatic secretions or intact intestinal brush border membranes. Human gastrointestinal bacteria, however, produce a wide variety of glycosidases with regio- and anomeric specificities matching those of protein-linked glycan structures. These bacteria degrade a wide array of complex carbohydrates including various protein-linked glycans. That bacteria possess glycan degradation capabilities, whereas the human digestive system, perse, does not, suggests that most dietary protein-linked glycan breakdown will be of bacterial origin. In addition to providing a food source for specific bacteria in the colon, protein-linked glycans from human milk may act as decoys for pathogenic bacteria to prevent invasion and infection of the host. The composition of the intestinal microbiome may be particularly important in the most vulnerable humans-the elderly, the immunocompromised, and infants (particularly premature infants). PMID:24533224

Niosome-loaded cold-set whey protein hydrogels.

PubMed

Abaee, Arash; Madadlou, Ashkan

2016-04-01

The α-tocopherol-carrying niosomes with mean diameter of 5.7 μm were fabricated and charged into a transglutaminase-cross-linked whey protein solution that was subsequently gelled with glucono delta-lactone. Encapsulation efficiency of α-tocopherol within niosomes was ≈80% and encapsulation did not influence the radical scavenging activity of α-tocopherol. Fourier transform infrared (FTIR) spectroscopy suggested formation of ε-(γ-glutamyl) lysine cross-linkages by transglutaminase and that enzymatic cross-linking increased proteins hydrophobicity. FTIR also proposed hydrogen bonding between niosomes and proteins. Dynamic rheometry indicated that transglutaminase cross-linking and niosomes charging of the protein solution enhanced the gelation process. However, charging the cross-linked protein solution with niosomal suspension resulted in lower elastic modulus (G') of the subsequently formed gel compared with both non-cross-linked niosome-loaded and cross-linked niosome-free counterparts. Electron microscopy indicated a discontinuous network for the niosome-loaded cross-linked sample. Niosome loading into the protein gel matrix increased its swelling extent in the enzyme-free simulated gastric fluid. Copyright © 2015 Elsevier Ltd. All rights reserved.

PACSY, a relational database management system for protein structure and chemical shift analysis.

PubMed

Lee, Woonghee; Yu, Wookyung; Kim, Suhkmann; Chang, Iksoo; Lee, Weontae; Markley, John L

2012-10-01

PACSY (Protein structure And Chemical Shift NMR spectroscopY) is a relational database management system that integrates information from the Protein Data Bank, the Biological Magnetic Resonance Data Bank, and the Structural Classification of Proteins database. PACSY provides three-dimensional coordinates and chemical shifts of atoms along with derived information such as torsion angles, solvent accessible surface areas, and hydrophobicity scales. PACSY consists of six relational table types linked to one another for coherence by key identification numbers. Database queries are enabled by advanced search functions supported by an RDBMS server such as MySQL or PostgreSQL. PACSY enables users to search for combinations of information from different database sources in support of their research. Two software packages, PACSY Maker for database creation and PACSY Analyzer for database analysis, are available from http://pacsy.nmrfam.wisc.edu.

Adsorption effectiveness of β-lactoglobulin onto gold surface determined by quartz crystal microbalance.

PubMed

Jachimska, B; Świątek, S; Loch, J I; Lewiński, K; Luxbacher, T

2018-06-01

Bovine β-lactoglobulin (LGB) is a transport protein that can bind to its structure hydrophobic bioactive molecules. Due to the lack of toxicity, high stability and pH-dependent molecular binding mechanism, lactoglobulin can be used as a carrier of sparingly soluble drugs. Dynamic light scattering has confirmed LGB's tendency to create oligomeric forms. The hydrodynamic diameter of LGB molecules varies from 4 nm to 6 nm in the pH range of 2-10 and ionic strength I = 0.001-0.15 M, which corresponds to the presence of mono or dimeric LGB forms. The LGB zeta potential varies from 26.5 mV to -33.3 mV for I = 0.01 M and from 13.3 mV to -16 mV for I = 0.15 M in the pH range of 2-10. The isoelectric point is at pH 4.8. As a result of strong surface charge compensation, the maximum effective ionization degree of the LGB molecule is 35% for ionic strength I = 0.01 M and 22% for I = 0.15 M. The effectiveness of adsorption is linked with the properties of the protein, as well as those of the adsorption surface. The functionalization of gold surfaces with β-lactoglobulin (LGB) was studied using a quartz crystal microbalance with energy dissipation monitoring (QCM-D). The effectiveness of LGB adsorption correlates strongly with a charge of gold surface and the zeta potential of the molecule. The greatest value of the adsorbed mass was observed in the pH range in which LGB has a positive zeta potential values, below pH 4.8. This observation shows that electrostatic interactions play a dominant role in LGB adsorption on gold surfaces. Based on the adsorbed mass, protein orientation on gold surfaces was determined. The preferential side-on orientation of LGB molecules observed in the adsorption layer is consistent with the direction of the molecule dipole momentum determined by molecular dynamics simulations of the protein (MD). The use of the QCM-D method also allowed us to determine the effectiveness of adsorption of LGB on gold surface. Knowing the mechanism of LGB adsorption is significant importance for determining the optimum conditions for immobilizing this protein on solid surfaces. As β-lactoglobulin is a protein that binds various ligands, the binding properties of immobilized β-lactoglobulin can be used to design controlled protein structures for biomedical applications. Copyright © 2018 Elsevier B.V. All rights reserved.

Proteome Analysis and Serological Characterization of Surface-Exposed Proteins of Rickettsia heilongjiangensis

PubMed Central

Qi, Yong; Xiong, Xiaolu; Wang, Xile; Duan, Changsong; Jia, Yinjun; Jiao, Jun; Gong, Wenping; Wen, Bohai

2013-01-01

Background Rickettsia heilongjiangensis, the agent of Far-Eastern spotted fever (FESF), is an obligate intracellular bacterium. The surface-exposed proteins (SEPs) of rickettsiae are involved in rickettsial adherence to and invasion of host cells, intracellular bacterial growth, and/or interaction with immune cells. They are also potential molecular candidates for the development of diagnostic reagents and vaccines against rickettsiosis. Methods R. heilongjiangensis SEPs were identified by biotin-streptavidin affinity purification and 2D electrophoreses coupled with ESI-MS/MS. Recombinant SEPs were probed with various sera to analyze their serological characteristics using a protein microarray and an enzyme-linked immune sorbent assay (ELISA). Results Twenty-five SEPs were identified, most of which were predicted to reside on the surface of R. heilongjiangensis cells. Bioinformatics analysis suggests that these proteins could be involved in bacterial pathogenesis. Eleven of the 25 SEPs were recognized as major seroreactive antigens by sera from R. heilongjiangensis-infected mice and FESF patients. Among the major seroreactive SEPs, microarray assays and/or ELISAs revealed that GroEL, OmpA-2, OmpB-3, PrsA, RplY, RpsB, SurA and YbgF had modest sensitivity and specificity for recognizing R. heilongjiangensis infection and/or spotted fever. Conclusions Many of the SEPs identified herein have potentially important roles in R. heilongjiangensis pathogenicity. Some of them have potential as serodiagnostic antigens or as subunit vaccine antigens against the disease. PMID:23894656

A Library of the Nanoscale Self-Assembly of Amino Acids on Metal Surfaces

NASA Astrophysics Data System (ADS)

Iski, Erin; Yitamben, Esmeralda; Guisinger, Nathan

2012-02-01

The investigation of the hierarchical self-assembly of amino acids on surfaces represents a unique test-bed for the origin of enantio-favoritism in biology and the transmission of chirality from single molecules to complete surface layers. These chiral systems, in particular the assembly of isoleucine and alanine on Cu(111), represent a direct link to the understanding of certain biological processes, specifically the preference for some amino acids to form alpha helices vs. beta-pleated sheets in the secondary structure of proteins. Low temperature, ultra-high vacuum, scanning tunneling microscopy (LT UHV-STM) is used to study the hierarchical self-assembly of different amino acids on a Cu(111) single crystal in an effort to build a library of their two-dimensional structure with molecular-scale resolution for enhanced protein and peptide studies. Both enantiopure and racemic structures are studied in order to elucidate how chirality can affect the self-assembly of the amino acids. In some cases, density functional theory (DFT) models can be used to confirm the experimental structure. The advent of such a library with fully resolved, two-dimensional structures at different molecular coverages would address some of the complex questions surrounding the preferential formation of alpha helices vs. beta-pleated sheets in proteins and lead to a better understanding of the key role played by these amino acids in protein sequencing.

Crystal structure at 2.8 A of the DLLRKN-containing coiled-coil domain of huntingtin-interacting protein 1 (HIP1) reveals a surface suitable for clathrin light chain binding.

PubMed

Ybe, Joel A; Mishra, Sanjay; Helms, Stephen; Nix, Jay

2007-03-16

Huntingtin interacting protein 1 (HIP1) is a member of a family of proteins whose interaction with Huntingtin is critical to prevent cells from initiating apoptosis. HIP1, and related protein HIP12/1R, can also bind to clathrin and membrane phospholipids, and HIP12/1R links the CCV to the actin cytoskeleton. HIP1 and HIP12/1R interact with the clathrin light chain EED regulatory site and stimulate clathrin lattice assembly. Here, we report the X-ray structure of the coiled-coil domain of HIP1 (residues 482-586) that includes residues crucial for binding clathrin light chain. The dimeric HIP1 crystal structure is partially splayed open. The comparison of the HIP1 model with coiled-coil predictions revealed the heptad repeat in the dimeric trunk (S2 path) is offset relative to the register of the heptad repeat from the N-terminal portion (S1 path) of the molecule. Furthermore, surface analysis showed there is a third hydrophobic path (S3) running parallel with S1 and S2. We present structural evidence supporting a role for the S3 path as an interaction surface for clathrin light chain. Finally, comparative analysis suggests the mode of binding between sla2p and clathrin light chain may be different in yeast.

Leishmania cell surface prohibitin: role in host-parasite interaction.

PubMed

Jain, Rohit; Ghoshal, Angana; Mandal, Chitra; Shaha, Chandrima

2010-04-01

Proteins selectively upregulated in infective parasitic forms could be critical for disease pathogenesis. A mammalian prohibitin orthologue is upregulated in infective metacyclic promastigotes of Leishmania donovani, a parasite that causes visceral leishmaniasis. Leishmania donovani prohibitin shares 41% similarity with mammalian prohibitin and 95-100% within the genus. Prohibitin is concentrated at the surface of the flagellar and the aflagellar pole, the aflagellar pole being a region through which host-parasite interactions occur. Prohibitin is attached to the membrane through a GPI anchor. Overexpression of wild-type prohibitin increases protein surface density resulting in parasites with higher infectivity. However, parasites overexpressing a mutant prohibitin with an amino acid substitution at the GPI anchor site to prevent surface expression through GPI-link show lesser surface expression and lower infective abilities. Furthermore, the presence of anti-prohibitin antibodies during macrophage-Leishmania interaction in vitro reduces infection. The cognate binding partner for Leishmania prohibitin on the host cell appears to be macrophage surface HSP70, siRNA mediated downregulation of which abrogates the capability of the macrophage to bind to parasites. Leishmania prohibitin is able to generate a strong humoral response in visceral leishmaniasis patients. The above observations suggest that prohibitin plays an important role in events leading to Leishmania-host interaction.

Structure of the Elastin-Contractile Units in the Thoracic Aorta and How Genes That Cause Thoracic Aortic Aneurysms and Dissections Disrupt This Structure.

PubMed

Karimi, Ashkan; Milewicz, Dianna M

2016-01-01

The medial layer of the aorta confers elasticity and strength to the aortic wall and is composed of alternating layers of smooth muscle cells (SMCs) and elastic fibres. The SMC elastin-contractile unit is a structural unit that links the elastin fibres to the SMCs and is characterized by the following: (1) layers of elastin fibres that are surrounded by microfibrils; (2) microfibrils that bind to the integrin receptors in focal adhesions on the cell surface of the SMCs; and (3) SMC contractile filaments that are linked to the focal adhesions on the inner side of the membrane. The genes that are altered to cause thoracic aortic aneurysms and aortic dissections encode proteins involved in the structure or function of the SMC elastin-contractile unit. Included in this gene list are the genes encoding protein that are structural components of elastin fibres and microfibrils, FBN1, MFAP5, ELN, and FBLN4. Also included are genes that encode structural proteins in the SMC contractile unit, including ACTA2, which encodes SMC-specific α-actin and MYH11, which encodes SMC-specific myosin heavy chain, along with MYLK and PRKG1, which encode kinases that control SMC contraction. Finally, mutations in the gene encoding the protein linking integrin receptors to the contractile filaments, FLNA, also predispose to thoracic aortic disease. Thus, these data suggest that functional SMC elastin-contractile units are important for maintaining the structural integrity of the aorta. Copyright © 2016 Canadian Cardiovascular Society. Published by Elsevier Inc. All rights reserved.

A hydrogel-based versatile screening platform for specific biomolecular recognition in a well plate format.

PubMed

Beer, Meike V; Rech, Claudia; Diederichs, Sylvia; Hahn, Kathrin; Bruellhoff, Kristina; Möller, Martin; Elling, Lothar; Groll, Jürgen

2012-04-01

Precise determination of biomolecular interactions in high throughput crucially depends on a surface coating technique that allows immobilization of a variety of interaction partners in a non-interacting environment. We present a one-step hydrogel coating system based on isocyanate functional six-arm poly(ethylene oxide)-based star polymers for commercially available 96-well microtiter plates that combines a straightforward and robust coating application with versatile bio-functionalization. This system generates resistance to unspecific protein adsorption and cell adhesion, as demonstrated with fluorescently labeled bovine serum albumin and primary human dermal fibroblasts (HDF), and high specificity for the assessment of biomolecular recognition processes when ligands are immobilized on this surface. One particular advantage is the wide range of biomolecules that can be immobilized and convert the per se inert coating into a specifically interacting surface. We here demonstrate the immobilization and quantification of a broad range of biochemically important ligands, such as peptide sequences GRGDS and GRGDSK-biotin, the broadly applicable coupler molecule biocytin, the protein fibronectin, and the carbohydrates N-acetylglucosamine and N-acetyllactosamine. A simplified protocol for an enzyme-linked immunosorbent assay was established for the detection and quantification of ligands on the coating surface. Cell adhesion on the peptide and protein-modified surfaces was assessed using HDF. All coatings were applied using a one-step preparation technique, including bioactivation, which makes the system suitable for high-throughput screening in a format that is compatible with the most routinely used testing systems.

Application of NMR Methods to Identify Detection Reagents for Use in the Development of Robust Nanosensors

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

Cosman, M; Krishnan, V V; Balhorn, R

2004-04-29

Nuclear Magnetic Resonance (NMR) spectroscopy is a powerful technique for studying bi-molecular interactions at the atomic scale. Our NMR lab is involved in the identification of small molecules, or ligands that bind to target protein receptors, such as tetanus (TeNT) and botulinum (BoNT) neurotoxins, anthrax proteins and HLA-DR10 receptors on non-Hodgkin's lymphoma cancer cells. Once low affinity binders are identified, they can be linked together to produce multidentate synthetic high affinity ligands (SHALs) that have very high specificity for their target protein receptors. An important nanotechnology application for SHALs is their use in the development of robust chemical sensors ormore » biochips for the detection of pathogen proteins in environmental samples or body fluids. Here, we describe a recently developed NMR competition assay based on transferred nuclear Overhauser effect spectroscopy (trNOESY) that enables the identification of sets of ligands that bind to the same site, or a different site, on the surface of TeNT fragment C (TetC) than a known ''marker'' ligand, doxorubicin. Using this assay, we can identify the optimal pairs of ligands to be linked together for creating detection reagents, as well as estimate the relative binding constants for ligands competing for the same site.« less

Integrin-mediated signal transduction linked to Ras pathway by GRB2 binding to focal adhesion kinase.

PubMed

Schlaepfer, D D; Hanks, S K; Hunter, T; van der Geer, P

The cytoplasmic focal adhesion protein-tyrosine kinase (FAK) localizes with surface integrin receptors at sites where cells attach to the extracellular matrix. Increased FAK tyrosine phosphorylation occurs upon integrin engagement with fibronectin. Here we show that adhesion of murine NIH3T3 fibroblasts to fibronectin promotes SH2-domain-mediated association of the GRB2 adaptor protein and the c-Src protein-tyrosine kinase (PTK) with FAK in vivo, and also results in activation of mitogen-activated protein kinase (MAPK). In v-Src-transformed NIH3T3, the association of v-Src, GRB2 and Sos with FAK is independent of cell adhesion to fibronectin. The GRB2 SH2 domain binds directly to tyrosine-phosphorylated FAK. Mutation of tyrosine residue 925 of FAK (YENV motif) to phenylalanine blocks GRB2 SH2-domain binding to FAK in vitro. Our results show that fibronectin binding to integrins on NIH3T3 fibroblasts promotes c-Src and FAK association and formation of an integrin-activated signalling complex. Phosphorylation of FAK at Tyr 925 upon fibronectin stimulation creates an SH2-binding site for GRB2 which may link integrin engagement to the activation of the Ras/MAPK signal transduction pathway.

Photoactivable antibody binding protein: site-selective and covalent coupling of antibody.

PubMed

Jung, Yongwon; Lee, Jeong Min; Kim, Jung-won; Yoon, Jeongwon; Cho, Hyunmin; Chung, Bong Hyun

2009-02-01

Here we report new photoactivable antibody binding proteins, which site-selectively capture antibodies and form covalent conjugates with captured antibodies upon irradiation. The proteins allow the site-selective tagging and/or immobilization of antibodies with a highly preferred orientation and omit the need for prior antibody modifications. The minimal Fc-binding domain of protein G, a widely used antibody binding protein, was genetically and chemically engineered to contain a site-specific photo cross-linker, benzophenone. In addition, the domain was further mutated to have an enhanced Fc-targeting ability. This small engineered protein was successfully cross-linked only to the Fc region of the antibody without any nonspecific reactivity. SPR analysis indicated that antibodies can be site-selectively biotinylated through the present photoactivable protein. Furthermore, the system enabled light-induced covalent immobilization of antibodies directly on various solid surfaces, such as those of glass slides, gold chips, and small particles. Antibody coupling via photoactivable antibody binding proteins overcomes several limitations of conventional approaches, such as random chemical reactions or reversible protein binding, and offers a versatile tool for the field of immunosensors.

Monomer–dimer dynamics and distribution of GPI-anchored uPAR are determined by cell surface protein assemblies

PubMed Central

Caiolfa, Valeria R.; Zamai, Moreno; Malengo, Gabriele; Andolfo, Annapaola; Madsen, Chris D.; Sutin, Jason; Digman, Michelle A.; Gratton, Enrico; Blasi, Francesco; Sidenius, Nicolai

2007-01-01

To search for functional links between glycosylphosphatidylinositol (GPI) protein monomer–oligomer exchange and membrane dynamics and confinement, we studied urokinase plasminogen activator (uPA) receptor (uPAR), a GPI receptor involved in the regulation of cell adhesion, migration, and proliferation. Using a functionally active fluorescent protein–uPAR in live cells, we analyzed the effect that extracellular matrix proteins and uPAR ligands have on uPAR dynamics and dimerization at the cell membrane. Vitronectin directs the recruitment of dimers and slows down the diffusion of the receptors at the basal membrane. The commitment to uPA–plasminogen activator inhibitor type 1–mediated endocytosis and recycling modifies uPAR diffusion and induces an exchange between uPAR monomers and dimers. This exchange is fully reversible. The data demonstrate that cell surface protein assemblies are important in regulating the dynamics and localization of uPAR at the cell membrane and the exchange of monomers and dimers. These results also provide a strong rationale for dynamic studies of GPI-anchored molecules in live cells at steady state and in the absence of cross-linker/clustering agents. PMID:18056417

Human Immune Response to Outer Membrane Protein CD of Moraxella catarrhalis in Adults with Chronic Obstructive Pulmonary Disease

PubMed Central

Murphy, Timothy F.; Kirkham, Charmaine; Liu, Dai-Fang; Sethi, Sanjay

2003-01-01

Moraxella catarrhalis is a common cause of lower respiratory tract infection in adults with chronic obstructive pulmonary disease (COPD). The antibody response to outer membrane protein (OMP) CD, a highly conserved surface protein of M. catarrhalis under consideration as a vaccine antigen, was studied in adults with COPD following 40 episodes of infection or colonization. Following infection or colonization, 9 of 40 patients developed new serum immunoglobulin G (IgG) to OMP CD, as measured by enzyme-linked immunosorbent assay. Adsorption assays revealed that a proportion of the serum IgG was directed toward surface-exposed epitopes on OMP CD in six of the nine patients who developed new IgG to OMP CD. Immunoblot assays with fusion peptide constructs indicated that the new antibodies that developed after infection or colonization recognized conformational epitopes, particularly in the carboxy region of the protein. Three of 28 patients developed new mucosal IgA to OMP CD in sputum supernatants. This study establishes that OMP CD is a target of a systemic and mucosal immune response following infection and colonization in some patients with COPD. PMID:12595444

Arrest of Nuclear Division in Plasmodium through Blockage of Erythrocyte Surface Exposed Ribosomal Protein P2

PubMed Central

Das, Sudipta; Basu, Himanish; Korde, Reshma; Tewari, Rita; Sharma, Shobhona

2012-01-01

Malaria parasites reside inside erythrocytes and the disease manifestations are linked to the growth inside infected erythrocytes (IE). The growth of the parasite is mostly confined to the trophozoite stage during which nuclear division occurs followed by the formation of cell bodies (schizogony). The mechanism and regulation of schizogony are poorly understood. Here we show a novel role for a Plasmodium falciparum 60S stalk ribosomal acidic protein P2 (PfP2) (PFC0400w), which gets exported to the IE surface for 6–8 hrs during early schizogony, starting around 26–28 hrs post-merozoite invasion. The surface exposure is demonstrated using multiple PfP2-specific monoclonal antibodies, and is confirmed through transfection using PfP2-GFP. The IE surface-exposed PfP2-protein occurs mainly as SDS-resistant P2-homo-tetramers. Treatment with anti-PfP2 monoclonals causes arrest of IEs at the first nuclear division. Upon removal of the antibodies, about 80–85% of synchronized parasites can be released even after 24 hrs of antibody treatment. It has been reported that a tubovesicular network (TVN) is set up in early trophozoites which is used for nutrient import. Anti-P2 monoclonal antibodies cause a complete fragmentation of TVN by 36 hrs, and impairs lipid import in IEs. These may be downstream causes for the cell-cycle arrest. Upon antibody removal, the TVN is reconstituted, and the cell division progresses. Each of the above properties is observed in the rodent malaria parasite species P. yoelii and P. berghei. The translocation of the P2 protein to the IE surface is therefore likely to be of fundamental importance in Plasmodium cell division. PMID:22912579

Characterisation of surface antigens of Strongylus vulgaris of potential immunodiagnostic importance.

PubMed

Nichol, C; Masterson, W J

1987-08-01

When antigens prepared by detergent washes of Strongylus vulgaris and Parascaris equorum were probed in an enzyme-linked immunosorbent assay test with horse sera from single species infections of S. vulgaris and P. equorum, a high degree of cross-reaction between the species was demonstrated. Western blot analysis of four common horse nematode species showed a large number of common antigens when probed with horse infection sera. Antisera raised in rabbits against the four species, including S. vulgaris, were also found to cross-react considerably. Rabbit anti-S. vulgaris sera were affinity adsorbed over a series of affinity chromatography columns, bound with cross-reactive surface antigens, to obtain S. vulgaris-specific antisera and thereby identify S. vulgaris-specific antigens by Western blotting. These studies revealed potentially specific antigens of apparent molecular weights of 100,000, 52,000, and 36,000. Of these bands, only the 52 kDa and 36 kDa appeared to be found on the surface as judged by 125I-labelling of intact worms by the Iodogen method, although neither protein was immunoprecipitated by horse infection sera. Finally, immunoprecipitation of in vitro translated proteins derived from larval S. vulgaris RNA suggests that two proteins may be parasite-derived. These findings are discussed both with respect to the surface of S. vulgaris and to the use of these species-specific antigens in immunodiagnosis.

Expression of the human hepatitis B virus large surface antigen gene in transgenic tomato plants.

PubMed

Lou, Xiao-Ming; Yao, Quan-Hong; Zhang, Zhen; Peng, Ri-He; Xiong, Ai-Sheng; Wang, Hua-Kun

2007-04-01

The original hepatitis B virus (HBV) large surface antigen gene was synthesized. In order to optimize the expression of this gene in tomato plants, the tobacco pathogenesis-related protein S signal peptide was fused to the 5' end of the modified gene and the sequence encoding amino acids S, E, K, D, E, and L was placed at the 3' end. The gene encoding the modified HBV large surface antigen under the control of a fruit-specific promoter was constructed and expressed in transgenic tomato plants. The expression of the antigen from transgenic plants was confirmed by PCR and reverse transcriptase PCR. Enzyme-linked immunoassays using a monoclonal antibody directed against human serum-derived HBsAg revealed that the maximal level of HBsAg was about 0.02% of the soluble protein in transgenic tomato fruit. The amount of HBsAg in mature fruits was found to be 65- to 171-fold larger than in small or medium fruits and leaf tissues. Examination of transgenic plant samples by transmission electron microscopy proved that HBsAg had been expressed and had accumulated. The HBsAg protein was capable of assembling into capsomers and virus-like particles. To our knowledge, this is the first time the HBV large surface antigen has been expressed in plants. This work suggests the possibility of producing a new alternative vaccine for human HBV.

Fouling behavior of poly(ether)sulfone ultrafiltration membrane during concentration of whey proteins: Effect of hydrophilic modification using atmospheric pressure argon jet plasma.

PubMed

Damar Huner, Irem; Gulec, Haci Ali

2017-12-01

The aim of the study was to investigate the effects of hydrophilic surface modification via atmospheric pressure jet plasma (ApJPls) on the fouling propensity of polyethersulfone (PES) ultrafiltration (UF) membranes during concentration of whey proteins. The distance from nozzle to substrate surface of 30mm and the exposure period of 5 times were determined as the most effective parameters enabling an increase in ΔG iwi value of the plain membrane from (-) 14.92±0.89mJ/m 2 to (+) 17.57±0.67mJ/m 2 . Maximum hydrophilicity and minimum surface roughness achieved by argon plasma action resulted in better antifouling behavior, while the hydraulic permeability and the initial permeate flux were decreased sharply due to the plasma-induced surface cross-linking. A quite steady state flux was obtained throughout the UF with the ApJPls modified PES membrane. The contribution of R frev to R t , which was 94% for the UF through the plain membrane, decreased to 43% after the plasma treatment. The overall results of this study highlighted the ApJPls modification decreased the fouling propensity of PES membrane without affecting the original protein rejection capability and improved the recovery of initial permeate flux after chemical cleaning. Copyright © 2017 Elsevier B.V. All rights reserved.

Structural Insights into the Unusually Strong ATPase Activity of the AAA Domain of the Caenorhabditis elegans Fidgetin-like 1 (FIGL-1) Protein*

PubMed Central

Peng, Wentao; Lin, Zhijie; Li, Weirong; Lu, Jing; Shen, Yuequan; Wang, Chunguang

2013-01-01

The FIGL-1 (fidgetin like-1) protein is a homolog of fidgetin, a protein whose mutation leads to multiple developmental defects. The FIGL-1 protein contains an AAA (ATPase associated with various activities) domain and belongs to the AAA superfamily. However, the biological functions and developmental implications of this protein remain unknown. Here, we show that the AAA domain of the Caenorhabditis elegans FIGL-1 protein (CeFIGL-1-AAA), in clear contrast to homologous AAA domains, has an unusually high ATPase activity and forms a hexamer in solution. By determining the crystal structure of CeFIGL-1-AAA, we found that the loop linking helices α9 and α10 folds into the short helix α9a, which has an acidic surface and interacts with a positively charged surface of the neighboring subunit. Disruption of this charge interaction by mutagenesis diminishes both the ATPase activity and oligomerization capacity of the protein. Interestingly, the acidic residues in helix α9a of CeFIGL-1-AAA are not conserved in other homologous AAA domains that have relatively low ATPase activities. These results demonstrate that the sequence of CeFIGL-1-AAA has adapted to establish an intersubunit charge interaction, which contributes to its strong oligomerization and ATPase activity. These unique properties of CeFIGL-1-AAA distinguish it from other homologous proteins, suggesting that CeFIGL-1 may have a distinct biological function. PMID:23979136

Structural insights into the unusually strong ATPase activity of the AAA domain of the Caenorhabditis elegans fidgetin-like 1 (FIGL-1) protein.

PubMed

Peng, Wentao; Lin, Zhijie; Li, Weirong; Lu, Jing; Shen, Yuequan; Wang, Chunguang

2013-10-11

The FIGL-1 (fidgetin like-1) protein is a homolog of fidgetin, a protein whose mutation leads to multiple developmental defects. The FIGL-1 protein contains an AAA (ATPase associated with various activities) domain and belongs to the AAA superfamily. However, the biological functions and developmental implications of this protein remain unknown. Here, we show that the AAA domain of the Caenorhabditis elegans FIGL-1 protein (CeFIGL-1-AAA), in clear contrast to homologous AAA domains, has an unusually high ATPase activity and forms a hexamer in solution. By determining the crystal structure of CeFIGL-1-AAA, we found that the loop linking helices α9 and α10 folds into the short helix α9a, which has an acidic surface and interacts with a positively charged surface of the neighboring subunit. Disruption of this charge interaction by mutagenesis diminishes both the ATPase activity and oligomerization capacity of the protein. Interestingly, the acidic residues in helix α9a of CeFIGL-1-AAA are not conserved in other homologous AAA domains that have relatively low ATPase activities. These results demonstrate that the sequence of CeFIGL-1-AAA has adapted to establish an intersubunit charge interaction, which contributes to its strong oligomerization and ATPase activity. These unique properties of CeFIGL-1-AAA distinguish it from other homologous proteins, suggesting that CeFIGL-1 may have a distinct biological function.

Development of protein A functionalized microcantilever immunosensors for the analyses of small molecules at parts per trillion levels.

PubMed

Tan, Weiming; Huang, Yuan; Nan, Tiegui; Xue, Changguo; Li, Zhaohu; Zhang, Qingchuan; Wang, Baomin

2010-01-15

Development of microcantilever biosensors for small molecules was exemplified with the beta-adrenergic agonist clenbuterol and the antibiotic chloramphenicol. In this paper, antibody sulfhydrylation and protein A were used to modify the microcantilever Au surface, and the antibody activities on the microcantilever were evaluated with direct competitive enzyme-linked immunosorbent assay (dcELISA). The activity of the antibodies immobilized on the microcantilever via protein A was 1.7-fold of that via the sulfhydrylation reagent 2-iminothiolane hydrochloride. A microcantilever immunosensor method with protein A as the functionalization reagent was established to detect the residues of clenbuterol and chloramphenicol at limits of detection (LOD) of approximately 0.1 and 0.2 ng/mL, respectively. Such LODs were better than that of the corresponding dcELISAs. The concentration of clenbuterol in a fortified feed sample detected with the microcantilever immunosensor after thorough extraction and purification agreed well with that detected with the dcELISA. Protein A showed to be simple and reproducible for functionalization of the antibodies on the Au surface and, thus, has common application values in microcantilever immunosensor development. The results suggest that microcantilever immunosensors be suitable for detection of small molecules, and the assay sensitivity is mainly related to the quality and activities of the antibodies.

Spectroscopy on the wing: naturally inspired SERS substrates for biochemical analysis.

PubMed

Garrett, Natalie L; Vukusic, Peter; Ogrin, Feodor; Sirotkin, Evgeny; Winlove, C Peter; Moger, Julian

2009-03-01

We show that naturally occurring chitinous nanostructures found on the wings of the Graphium butterfly can be used as substrates for surface-enhanced Raman scattering when coated with a thin film of gold or silver. The substrates were found to exhibit excellent biocompatibility and sensitivity, making them ideal for protein assaying. An assay using avidin/biotin binding showed that the substrates could be used to quantify protein binding directly from changes in the surface-enhanced Raman scattering (SERS) spectra and were sensitive over a concentration range comparable with a typical enzyme-linked immunosorbent assays (ELISA) assay. A biomimetic version of the wing nanostructures produced using a highly reproducible, large-scale fabrication process, yielded comparable enhancement factors and biocompatibility. The excellent biocompatibility of the wings and biomimetic substrates is unparalleled by other lithographically produced substrates, and this could pave the way for widespread application of ultrasensitive SERS-based bioassays.

Haem-assisted dityrosine-cross-linking of fibrinogen under non-thermal plasma exposure: one important mechanism of facilitated blood coagulation

PubMed Central

Ke, Zhigang; Huang, Qing

2016-01-01

Although blood coagulation facilitated by non-thermal plasma has been reported several years ago, the insight to the involved mechanisms is still rather limited. In this work, we report our discovery of a new mechanism for the haem-promoted blood-coagulation caused by non-thermal plasma treatment. The reason for the haem role is due to that its oxidized form, namely, hematin, can promote the dityrosine cross-linking of fibrinogen, the most important coagulation protein, to form a membrane-like layer on the surface of the treated blood with plasma exposure. Both haem and non-thermal-plasma generated hydrogen peroxide are requisite for the cross-linking process. We confirmed that fibrinogen can coordinate with the haem iron to form a protein-haem complex which shows pseudo-peroxidase activity, and in the presence of hydrogen peroxide, the complex can induce the dityrosine formation between fibrinogen molecules, leading to the fibrin network necessary for the blood coagulation. Understanding of such an underlying mechanism can be useful to guide more efficient application of non-thermal plasma in the management of hemostasis, thrombosis and etc. PMID:27229173

Tissue factor-dependent vascular endothelial growth factor production by human fibroblasts in response to activated factor VII.

PubMed

Ollivier, V; Bentolila, S; Chabbat, J; Hakim, J; de Prost, D

1998-04-15

The transmembrane protein tissue factor (TF) is the cell surface receptor for coagulation factor VII (FVII) and activated factor VII (FVIIa). Recently, TF has been identified as a regulator of angiogenesis, tumor growth, and metastasis. This study was designed to link the binding of FVII(a) to its receptor, TF, with the subsequent triggering of angiogenesis through vascular endothelial growth factor (VEGF) production by human lung fibroblasts. We report that incubation of fibroblasts, which express constitutive surface TF, with FVII(a) induces VEGF synthesis. FVII(a)-induced VEGF secretion, assessed by a specific enzyme-linked immunosorbent assay, was time- and concentration-dependent. VEGF secretion was maximal after 24 hours of incubation of the cells with 100 nmol/L FVII(a) and represented a threefold induction of the basal VEGF level. Reverse transcriptase-polymerase chain reaction analysis of VEGF detected three mRNA species of 180, 312, and 384 bp corresponding, respectively, to VEGF121, VEGF165, and VEGF189. A 2.5- to 3.5-fold increase was observed for the 180- and 312-bp transcripts at 12 and 24 hours, respectively. FVII(a)-dependent VEGF production was inhibited by a pool of antibodies against TF, pointing to the involvement of this receptor. On specific active-site inhibition with dansyl-glutamyl-glycinyl-arginyl chloromethyl ketone, FVIIa lost 70% of its capacity to elicit VEGF production. Consistent with this, the native form (zymogen) of FVII only had a 1.8-fold stimulating effect. Protein tyrosine kinase and protein kinase C are involved in signal transduction leading to VEGF production, as shown by the inhibitory effects of genistein and GF 109203X. The results of this study indicate that TF is essential for VIIa-induced VEGF production by human fibroblasts and that its role is mainly linked to the proteolytic activity of the TF-VIIa complex.

Weak Long-Range Correlated Motions in a Surface Patch of Ubiquitin Involved in Molecular Recognition

PubMed Central

2011-01-01

Long-range correlated motions in proteins are candidate mechanisms for processes that require information transfer across protein structures, such as allostery and signal transduction. However, the observation of backbone correlations between distant residues has remained elusive, and only local correlations have been revealed using residual dipolar couplings measured by NMR spectroscopy. In this work, we experimentally identified and characterized collective motions spanning four β-strands separated by up to 15 Å in ubiquitin. The observed correlations link molecular recognition sites and result from concerted conformational changes that are in part mediated by the hydrogen-bonding network. PMID:21634390

Protein Viability on Au Nanoparticles during an Electrospray and Electrostatic-Force-Directed Assembly Process

DOE PAGES

Mao, Shun; Lu, Ganhua; Yu, Kehan; ...

2010-01-01

We study the protein viability on Au nanoparticles during an electrospray and electrostatic-force-directed assembly process, through which Au nanoparticle-antibody conjugates are assembled onto the surface of carbon nanotubes (CNTs) to fabricate carbon nanotube field-effect transistor (CNTFET) biosensors. Enzyme-linked immunosorbent assay (ELISA) and field-effect transistor (FET) measurements have been used to investigate the antibody activity after the nanoparticle assembly. Upon the introduction of matching antigens, the colored reaction from the ELISA and the change in the electrical characteristic of the CNTFET device confirm that the antibody activity is preserved during the assembly process.

Exploitation of peptide motif sequences and their use in nanobiotechnology.

PubMed

Shiba, Kiyotaka

2010-08-01

Short amino acid sequences extracted from natural proteins or created using in vitro evolution systems are sometimes associated with particular biological functions. These peptides, called peptide motifs, can serve as functional units for the creation of various tools for nanobiotechnology. In particular, peptide motifs that have the ability to specifically recognize the surfaces of solid materials and to mineralize certain inorganic materials have been linking biological science to material science. Here, I review how these peptide motifs have been isolated from natural proteins or created using in vitro evolution systems, and how they have been used in the nanobiotechnology field. Copyright © 2010 Elsevier Ltd. All rights reserved.

Surface salt bridges modulate DNA wrapping by the type II DNA-binding protein TF1.

PubMed

Grove, Anne

2003-07-29

The histone-like protein HU is involved in compaction of the bacterial genome. Up to 37 bp of DNA may be wrapped about some HU homologues in a process that has been proposed to depend on a linked disruption of surface salt bridges that liberates cationic side chains for interaction with the DNA. Despite significant sequence conservation between HU homologues, binding sites from 9 to 37 bp have been reported. TF1, an HU homologue that is encoded by Bacillus subtilis bacteriophage SPO1, has nM affinity for 37 bp preferred sites in DNA with 5-hydroxymethyluracil (hmU) in place of thymine. On the basis of electrophoretic mobility shift assays, we show that TF1-DNA complex formation is associated with a net release of only approximately 0.5 cations. The structure of TF1 suggests that Asp13 can form a dehydrated surface salt bridge with Lys23; substitution of Asp13 with Ala increases the net release of cations to approximately 1. These data are consistent with complex formation linked to disruption of surface salt bridges. Substitution of Glu90 with Ala, which would expose Lys87 predicted to contact DNA immediately distal to a proline-mediated DNA kink, causes an increase in affinity and an abrogation of the preference for hmU-containing DNA. We propose that hmU preference is due to finely tuned interactions at the sites of kinking that expose a differential flexibility of hmU- and T-containing DNA. Our data further suggest that the difference in binding site size for HU homologues is based on a differential ability to stabilize the DNA kinks.

Expression of Surface Protein LapB by a Wide Spectrum of Listeria monocytogenes Serotypes as Demonstrated with Anti-LapB Monoclonal Antibodies

PubMed Central

Boivin, Teela; Elmgren, Cathie; Brooks, Brian W.; Huang, Hongsheng; Pagotto, Franco

2016-01-01

ABSTRACT Protein antigens expressed on the surface of all strains of Listeria monocytogenes and absent from nonpathogenic Listeria spp. are presumably useful targets for pathogen identification, detection, and isolation using specific antibodies (Abs). To seek such surface proteins expressed in various strains of L. monocytogenes for diagnostic applications, we focused on a set of surface proteins known to be involved or putatively involved in L. monocytogenes virulence and identified Listeria adhesion protein B (LapB) as a candidate based on the bioinformatics analysis of whole-genome sequences showing that the gene coding for LapB was present in L. monocytogenes strains and absent from strains of other Listeria spp. Immunofluorescence microscopy (IFM), performed with rabbit polyclonal antibodies against the recombinant LapB protein (rLapB) of L. monocytogenes serotype 4b strain L10521, confirmed expression of LapB on the surface. A panel of 48 mouse monoclonal antibodies (MAbs) to rLaB was generated, and 7 of them bound strongly to the surface of L. monocytogenes cells as demonstrated using IFM. Further characterization of these 7 anti-LapB MAbs, using an enzyme-linked immunosorbent assay (ELISA), revealed that 6 anti-LapB MAbs (M3484, M3495, M3500, M3509, M3517, and M3519) reacted strongly with 46 (86.8%) of 53 strains representing 10 of the 12 serotypes tested (1/2a, 1/2b, 1/2c, 3a, 3b, 3c, 4ab, 4b, 4d, and 4e). These results indicate that LapB, together with companion anti-LapB MAbs, can be targeted as a biomarker for the detection and isolation of various L. monocytogenes strains from contaminated foods. IMPORTANCE Strains of L. monocytogenes are traditionally grouped into serotypes. Identification of a surface protein expressed in all or the majority of at least 12 serotypes would aid in the development of surface-binding monoclonal antibodies (MAbs) for detection and isolation of L. monocytogenes from foods. Bioinformatics analysis revealed that the gene coding for Listeria adhesion protein B (LapB), a surface protein involved in L. monocytogenes virulence, was present in L. monocytogenes strains and absent from other Listeria spp. Polyclonal antibodies against recombinant LapB (rLapB) detected the exposed epitopes on the surface of L. monocytogenes. Production and extensive assessment of 48 MAbs to rLapB showed that 6 anti-LapB MAbs (M3484, M3495, M3500, M3509, M3517, and M3519) detected the expression of LapB in a wide range of L. monocytogenes isolates representing 10 of 12 serotypes tested, suggesting that LapB, together with specific MAbs, can be targeted as a biomarker for pathogen detection and isolation. PMID:27613687

Expression of Surface Protein LapB by a Wide Spectrum of Listeria monocytogenes Serotypes as Demonstrated with Anti-LapB Monoclonal Antibodies.

PubMed

Boivin, Teela; Elmgren, Cathie; Brooks, Brian W; Huang, Hongsheng; Pagotto, Franco; Lin, Min

2016-11-15

Protein antigens expressed on the surface of all strains of Listeria monocytogenes and absent from nonpathogenic Listeria spp. are presumably useful targets for pathogen identification, detection, and isolation using specific antibodies (Abs). To seek such surface proteins expressed in various strains of L. monocytogenes for diagnostic applications, we focused on a set of surface proteins known to be involved or putatively involved in L. monocytogenes virulence and identified Listeria adhesion protein B (LapB) as a candidate based on the bioinformatics analysis of whole-genome sequences showing that the gene coding for LapB was present in L. monocytogenes strains and absent from strains of other Listeria spp. Immunofluorescence microscopy (IFM), performed with rabbit polyclonal antibodies against the recombinant LapB protein (rLapB) of L. monocytogenes serotype 4b strain L10521, confirmed expression of LapB on the surface. A panel of 48 mouse monoclonal antibodies (MAbs) to rLaB was generated, and 7 of them bound strongly to the surface of L. monocytogenes cells as demonstrated using IFM. Further characterization of these 7 anti-LapB MAbs, using an enzyme-linked immunosorbent assay (ELISA), revealed that 6 anti-LapB MAbs (M3484, M3495, M3500, M3509, M3517, and M3519) reacted strongly with 46 (86.8%) of 53 strains representing 10 of the 12 serotypes tested (1/2a, 1/2b, 1/2c, 3a, 3b, 3c, 4ab, 4b, 4d, and 4e). These results indicate that LapB, together with companion anti-LapB MAbs, can be targeted as a biomarker for the detection and isolation of various L. monocytogenes strains from contaminated foods. Strains of L. monocytogenes are traditionally grouped into serotypes. Identification of a surface protein expressed in all or the majority of at least 12 serotypes would aid in the development of surface-binding monoclonal antibodies (MAbs) for detection and isolation of L. monocytogenes from foods. Bioinformatics analysis revealed that the gene coding for Listeria adhesion protein B (LapB), a surface protein involved in L. monocytogenes virulence, was present in L. monocytogenes strains and absent from other Listeria spp. Polyclonal antibodies against recombinant LapB (rLapB) detected the exposed epitopes on the surface of L. monocytogenes Production and extensive assessment of 48 MAbs to rLapB showed that 6 anti-LapB MAbs (M3484, M3495, M3500, M3509, M3517, and M3519) detected the expression of LapB in a wide range of L. monocytogenes isolates representing 10 of 12 serotypes tested, suggesting that LapB, together with specific MAbs, can be targeted as a biomarker for pathogen detection and isolation. © Crown copyright 2016.

Characterization of rat leydig cell gonadotropin receptor structure by affinity cross-linking

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

Zhang, Q.Y.; Hwang, J.; Menon, K.M.J.

1986-05-01

The gonadotropin receptor from rat leydig cell has been characterized with respect to binding kinetics and physiological regulation. The present study was intended to examine the structure of the receptor. Leydig cell suspension was prepared by either collagenase digestion or by mechanical disruption of the testis. The cells were incubated with /sup 125/I-hCG and the unreacted hCG was removed by centrifugation. The /sup 125/I-hCG was then covalently linked to the cell surface receptor using cleavable (dithiobis (succinimidyl propionate)) and non-cleavable (disuccinimidyl suberate) cross-linking reagents. The extracted cross-linked membrane proteins were resolved on SDS-polyacrylamide gels under reducing and non-reducing conditions andmore » subjected to autoradiographic analysis. Under non-reducing conditions, two labeled species with M/sub r/ = 87,000 and 120,000 were detected. However, only one labeled band was detected under reducing conditions with M/sub r/ = 64,000. The binding of /sup 125/I-hCG to the receptor was inhibited by hCG and LH, but not by a number of peptides and proteins. The data suggest that hCG receptor in leydig cell is an oligomeric complex consisting of four subunits, ..cap alpha cap alpha beta gamma... The ..beta.. and ..gamma.. subunits are each linked to an ..cap alpha.. subunit through disulfide linkage and the hormone binds to each ..cap alpha.. subunit. The two dimers formed (..cap alpha beta cap alpha gamma..) are associated by noncovalent interactions.« less

Spatial segregation of transport and signalling functions between human endothelial caveolae and lipid raft proteomes

PubMed Central

Sprenger, Richard R.; Fontijn, Ruud D.; van Marle, Jan; Pannekoek, Hans; Horrevoets, Anton J. G.

2006-01-01

Lipid rafts and caveolae are biochemically similar, specialized domains of the PM (plasma membrane) that cluster specific proteins. However, they are morphologically distinct, implying different, possibly complementary functions. Two-dimensional gel electrophoresis preceding identification of proteins by MS was used to compare the relative abundance of proteins in DRMs (detergent-resistant membranes) isolated from HUVEC (human umbilical-vein endothelial cells), and caveolae immunopurified from DRM fractions. Various signalling and transport proteins were identified and additional cell-surface biotinylation revealed the majority to be exposed, demonstrating their presence at the PM. In resting endothelial cells, the scaffold of immunoisolated caveolae consists of only few resident proteins, related to structure [CAV1 (caveolin-1), vimentin] and transport (V-ATPase), as well as the GPI (glycosylphosphatidylinositol)-linked, surface-exposed protein CD59. Further quantitative characterization by immunoblotting and confocal microscopy of well-known [eNOS (endothelial nitric oxide synthase) and CAV1], less known [SNAP-23 (23 kDa synaptosome-associated protein) and BASP1 (brain acid soluble protein 1)] and novel [C8ORF2 (chromosome 8 open reading frame 2)] proteins showed different subcellular distributions with none of these proteins being exclusive to either caveolae or DRM. However, the DRM-associated fraction of the novel protein C8ORF2 (∼5% of total protein) associated with immunoseparated caveolae, in contrast with the raft protein SNAP-23. The segregation of caveolae from lipid rafts was visually confirmed in proliferating cells, where CAV1 was spatially separated from eNOS, SNAP-23 and BASP1. These results provide direct evidence for the previously suggested segregation of transport and signalling functions between specialized domains of the endothelial plasma membrane. PMID:16886909

Genetically Targeted Ratiometric and Activated pH Indicator Complexes (TRApHIC) for Receptor Trafficking.

PubMed

Perkins, Lydia A; Yan, Qi; Schmidt, Brigitte F; Kolodieznyi, Dmytro; Saurabh, Saumya; Larsen, Mads Breum; Watkins, Simon C; Kremer, Laura; Bruchez, Marcel P

2018-02-06

Fluorescent protein-based pH sensors are useful tools for measuring protein trafficking through pH changes associated with endo- and exocytosis. However, commonly used pH-sensing probes are ubiquitously expressed with their protein of interest throughout the cell, hindering our ability to focus on specific trafficking pools of proteins. We developed a family of excitation ratiometric, activatable pH responsive tandem dyes, consisting of a pH sensitive Cy3 donor linked to a fluorogenic malachite green acceptor. These cell-excluded dyes are targeted and activated upon binding to a genetically expressed fluorogen-activating protein and are suitable for selective labeling of surface proteins for analysis of endocytosis and recycling in live cells using both confocal and superresolution microscopy. Quantitative profiling of the endocytosis and recycling of tagged β2-adrenergic receptor (B2AR) at a single-vesicle level revealed differences among B2AR agonists, consistent with more detailed pharmacological profiling.

Evolution of an ancient protein function involved in organized multicellularity in animals

PubMed Central

Anderson, Douglas P; Whitney, Dustin S; Hanson-Smith, Victor; Woznica, Arielle; Campodonico-Burnett, William; Volkman, Brian F; King, Nicole; Thornton, Joseph W; Prehoda, Kenneth E

2016-01-01

To form and maintain organized tissues, multicellular organisms orient their mitotic spindles relative to neighboring cells. A molecular complex scaffolded by the GK protein-interaction domain (GKPID) mediates spindle orientation in diverse animal taxa by linking microtubule motor proteins to a marker protein on the cell cortex localized by external cues. Here we illuminate how this complex evolved and commandeered control of spindle orientation from a more ancient mechanism. The complex was assembled through a series of molecular exploitation events, one of which – the evolution of GKPID’s capacity to bind the cortical marker protein – can be recapitulated by reintroducing a single historical substitution into the reconstructed ancestral GKPID. This change revealed and repurposed an ancient molecular surface that previously had a radically different function. We show how the physical simplicity of this binding interface enabled the evolution of a new protein function now essential to the biological complexity of many animals. DOI: http://dx.doi.org/10.7554/eLife.10147.001 PMID:26740169

Comparison of effect of gamma ray irradiation on wild-type and N-terminal mutants of αA-crystallin.

PubMed

Ramkumar, Srinivasagan; Fujii, Noriko; Fujii, Norihiko; Thankappan, Bency; Sakaue, Hiroaki; Ingu, Kim; Natarajaseenivasan, Kalimuthusamy; Anbarasu, Kumarasamy

2014-01-01

To study the comparative structural and functional changes between wild-type (wt) and N-terminal congenital cataract causing αA-crystallin mutants (R12C, R21L, R49C, and R54C) upon exposure to different dosages of gamma rays. Alpha A crystallin N-terminal mutants were created with the site-directed mutagenesis method. The recombinantly overexpressed and purified wt and mutant proteins were used for further studies. A (60)Co source was used to generate gamma rays to irradiate wild and mutant proteins at dosages of 0.5, 1.0, and 2.0 kGy. The biophysical property of the gamma irradiated (GI) and non-gamma irradiated (NGI) αA-crystallin wt and N-terminal mutants were determined. Oligomeric size was determined by size exclusion high-performance liquid chromatography (HPLC), the secondary structure with circular dichroism (CD) spectrometry, conformation of proteins with surface hydrophobicity, and the functional characterization were determined regarding chaperone activity using the alcohol dehydrogenase (ADH) aggregation assay. αA-crystallin N-terminal mutants formed high molecular weight (HMW) cross-linked products as well as aggregates when exposed to GI compared to the NGI wt counterparts. Furthermore, all mutants exhibited changed β-sheet and random coil structure. The GI mutants demonstrated decreased surface hydrophobicity when compared to αA-crystallin wt at 0, 1.0, and 1.5 kGy; however, at 2.0 kGy a drastic increase in hydrophobicity was observed only in the mutant R54C, not the wt. In contrast, chaperone activity toward ADH was gradually elevated at the minimum level in all GI mutants, and significant elevation was observed in the R12C mutant. Our findings suggest that the N-terminal mutants of αA-crystallin are structurally and functionally more sensitive to GI when compared to their NGI counterparts and wt. Protein oxidation as a result of gamma irradiation drives the protein to cross-link and aggregate culminating in cataract formation.

Genetically encoded releasable photo-cross-linking strategies for studying protein-protein interactions in living cells.

PubMed

Yang, Yi; Song, Haiping; He, Dan; Zhang, Shuai; Dai, Shizhong; Xie, Xiao; Lin, Shixian; Hao, Ziyang; Zheng, Huangtao; Chen, Peng R

2017-10-01

Although protein-protein interactions (PPIs) have crucial roles in virtually all cellular processes, the identification of more transient interactions in their biological context remains challenging. Conventional photo-cross-linking strategies can be used to identify transient interactions, but these approaches often suffer from high background due to the cross-linked bait proteins. To solve the problem, we have developed membrane-permeable releasable photo-cross-linkers that allow for prey-bait separation after protein complex isolation and can be installed in proteins of interest (POIs) as unnatural amino acids. Here we describe the procedures for using two releasable photo-cross-linkers, DiZSeK and DiZHSeC, in both living Escherichia coli and mammalian cells. A cleavage after protein photo-cross-linking (CAPP ) strategy based on the photo-cross-linker DiZSeK is described, in which the prey protein pool is released from a POI after affinity purification. Prey proteins are analyzed using mass spectrometry or 2D gel electrophoresis for global comparison of interactomes from different experimental conditions. An in situ cleavage and mass spectrometry (MS)-label transfer after protein photo-cross-linking (IMAPP) strategy based on the photo-cross-linker DiZHSeC is also described. This strategy can be used for the identification of cross-linking sites to allow detailed characterization of PPI interfaces. The procedures for photo-cross-linker incorporation, photo-cross-linking of interaction partners and affinity purification of cross-linked complexes are similar for the two photo-cross-linkers. The final section of the protocol describes prey-bait separation (for CAPP) and MS-label transfer and identification (for IMAPP). After plasmid construction, the CAPP and IMAPP strategies can be completed within 6 and 7 d, respectively.

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.

Electrostatic interaction of positive charges on the surface of Psb31 with photosystem II in the diatom Chaetoceros gracilis.

PubMed

Nagao, Ryo; Suzuki, Takehiro; Okumura, Akinori; Kihira, Tomohiro; Toda, Ayaka; Dohmae, Naoshi; Nakazato, Katsuyoshi; Tomo, Tatsuya

2017-09-01

Psb31, a novel extrinsic protein found in diatom photosystem II (PSII), directly binds to PSII core subunits, independent of the other extrinsic proteins, and functions to maintain optimum oxygen evolution. However, how Psb31 electrostatically interacts with PSII intrinsic proteins remains to be clarified. In this study, we examined electrostatic interaction of Psb31 with PSII complexes isolated from the diatom Chaetoceros gracilis. Positive or negative charges of isolated Psb31 proteins were modified with N-succinimidyl propionate (NSP) or glycine methyl ester (GME), respectively, resulting in formation of uncharged groups. NSP-modified Psb31 did not bind to PSII with a concomitant increase in NSP concentration, whereas GME-modified Psb31 clearly bound to PSII with retention of oxygen-evolving activity, indicating that positive charges of Lys residues and the N-terminus on the surface of Psb31 are involved in electrostatic interactions with PSII intrinsic proteins. Mass spectrometry analysis of NSP-modified Psb31 and sequence comparisons of Psb31 from C. gracilis with other chromophyte algae led to identification of three Lys residues as possible binding sites to PSII. Based on these findings, together with our previous cross-linking study in diatom PSII and a red algal PSII structure, we discuss binding properties of Psb31 with PSII core proteins. Copyright © 2017 Elsevier B.V. All rights reserved.

Induction of DNA-protein cross-links by platinum compounds.

PubMed

Woźniak, K; Walter, Z

2000-01-01

The differences between cis- and trans-diamminedichloroplatinum II (DDP) in forming DNA-protein cross-links in isolated human lymphocytes were investigated. Both cis- and trans-DDP can induce DNA-protein cross-links. We show that cis-DDP forms complexes between DNA and proteins faster than trans-DDP. This results from an increase in the quantity of DNA and platinum together with an increase in drug concentration. Under the same conditions trans-DDP causes a decrease in DNA-forming complexes with proteins. After a 12 h incubation of lymphocytes we observe a similar level of DNA in DNA-protein cross-links induced by DDP isomers, but more platinum appears in complexes induced by trans-DDP. The results obtained demonstrate that the antitumor drug - cis-DDP and the clinically ineffective trans-DDP induce links between DNA and proteins in a different manner. We suggest that the therapeutic activity of cis-DDP can in part arise from rapidly forming DNA-protein complexes which can destroy the most important cellular processes, such as replication and transcription.

One-pot preparation of silica-supported hybrid immobilized metal affinity adsorbent with macroporous surface based on surface imprinting coating technique combined with polysaccharide incorporated sol--gel process.

PubMed

Li, Feng; Li, Xue-Mei; Zhang, Shu-Sheng

2006-10-06

A simple and reliable one-pot approach using surface imprinting coating technique combined with polysaccharide incorporated sol-gel process was established to synthesize a new organic-inorganic hybrid matrix possessing macroporous surface and functional ligand. Using mesoporous silica gel being a support, immobilized metal affinity adsorbent with a macroporous shell/mesoporous core structure was obtained after metal ion loading. In the prepared matrix, covalently bonded coating and morphology manipulation on silica gel was achieved by using one-pot sol-gel process starting from an inorganic precursor, -glycidoxypropyltrimethoxysiloxane (GPTMS), and a functional biopolymer, chitosan (CS) at the atmosphere of imprinting polyethylene glycol (PEG). Self-hydrolysis of GPTMS, self-condensation, and co-condensation of silanol groups (Si-OH) from siloxane and silica gel surface, and in situ covalent cross-linking of CS created an orderly coating on silica gel surface. PEG extraction using hot ammonium hydroxide solution gave a chemically and mechanically stabilized pore structure and deactivated residual epoxy groups. The prepared matrix was characterized by using X-ray energy dispersion spectroscopy (EDX), scanning electron microscopy (SEM) and mercury intrusion porosimetry. The matrix possessed a high capacity for copper ion loading. Protein adsorption performance of the new immobilized metal affinity adsorbent was evaluated by batch adsorption and column chromatographic experiment using bovine serum albumin (BSA) as a simple model protein. Under the optimized coating conditions, the obtained macroporous surface resulted in a fast kinetics and high capability for protein adsorption, while the matrix non-charged with metal ions offered a low non-specific adsorption.

Epilepsy and intellectual disability linked protein Shrm4 interaction with GABABRs shapes inhibitory neurotransmission

PubMed Central

Zapata, Jonathan; Moretto, Edoardo; Hannan, Saad; Murru, Luca; Longatti, Anna; Mazza, Davide; Benedetti, Lorena; Fossati, Matteo; Heise, Christopher; Ponzoni, Luisa; Valnegri, Pamela; Braida, Daniela; Sala, Mariaelvina; Francolini, Maura; Hildebrand, Jeffrey; Kalscheuer, Vera; Fanelli, Francesca; Sala, Carlo; Bettler, Bernhard; Bassani, Silvia; Smart, Trevor G.; Passafaro, Maria

2017-01-01

Shrm4, a protein expressed only in polarized tissues, is encoded by the KIAA1202 gene, whose mutations have been linked to epilepsy and intellectual disability. However, a physiological role for Shrm4 in the brain is yet to be established. Here, we report that Shrm4 is localized to synapses where it regulates dendritic spine morphology and interacts with the C terminus of GABAB receptors (GABABRs) to control their cell surface expression and intracellular trafficking via a dynein-dependent mechanism. Knockdown of Shrm4 in rat severely impairs GABABR activity causing increased anxiety-like behaviour and susceptibility to seizures. Moreover, Shrm4 influences hippocampal excitability by modulating tonic inhibition in dentate gyrus granule cells, in a process involving crosstalk between GABABRs and extrasynaptic δ-subunit-containing GABAARs. Our data highlights a role for Shrm4 in synaptogenesis and in maintaining GABABR-mediated inhibition, perturbation of which may be responsible for the involvement of Shrm4 in cognitive disorders and epilepsy. PMID:28262662

Atypical binding of the Swa2p UBA domain to ubiquitin.

PubMed

Matta-Camacho, Edna; Kozlov, Guennadi; Trempe, Jean-François; Gehring, Kalle

2009-02-20

Swa2p is an auxilin-like yeast protein that is involved in vesicular transport and required for uncoating of clathrin-coated vesicles. Swa2p contains a ubiquitin-associated (UBA) domain, which is present in a variety of proteins involved in ubiquitin (Ub)-mediated processes. We have determined a structural model of the Swa2p UBA domain in complex with Ub using NMR spectroscopy and molecular docking. Ub recognition occurs predominantly through an atypical interaction in which UBA helix alpha1 and the N-terminal part of helix alpha2 bind to Ub. Mutation of Ala148, a key residue in helix alpha1, to polar residues greatly reduced the affinity of the UBA domain for Ub and revealed a second low-affinity Ub-binding site located on the surface formed by helices alpha1 and alpha3. Surface plasmon resonance showed that the Swa2p UBA domain binds K48- and K63-linked di-Ub in a non-linkage-specific manner. These results reveal convergent evolution of a Ub-binding site on helix alpha1 of UBA domains involved in membrane protein trafficking.

Key mediators of intracellular amino acids signaling to mTORC1 activation.

PubMed

Duan, Yehui; Li, Fengna; Tan, Kunrong; Liu, Hongnan; Li, Yinghui; Liu, Yingying; Kong, Xiangfeng; Tang, Yulong; Wu, Guoyao; Yin, Yulong

2015-05-01

Mammalian target of rapamycin complex 1 (mTORC1) is activated by amino acids to promote cell growth via protein synthesis. Specifically, Ras-related guanosine triphosphatases (Rag GTPases) are activated by amino acids, and then translocate mTORC1 to the surface of late endosomes and lysosomes. Ras homolog enriched in brain (Rheb) resides on this surface and directly activates mTORC1. Apart from the presence of intracellular amino acids, Rag GTPases and Rheb, other mediators involved in intracellular amino acid signaling to mTORC1 activation include human vacuolar sorting protein-34 (hVps34) and mitogen-activating protein kinase kinase kinase kinase-3 (MAP4K3). Those molecular links between mTORC1 and its mediators form a complicate signaling network that controls cellular growth, proliferation, and metabolism. Moreover, it is speculated that amino acid signaling to mTORC1 may start from the lysosomal lumen. In this review, we discussed the function of these mediators in mTORC1 pathway and how these mediators are regulated by amino acids in details.

Primary structure and glycosylation of the S-layer protein of Haloferax volcanii.

PubMed Central

Sumper, M; Berg, E; Mengele, R; Strobel, I

1990-01-01

The outer surface of the archaebacterium Haloferax volcanii (formerly named Halobacterium volcanii) is covered with a hexagonally packed surface (S) layer. The gene coding for the S-layer protein was cloned and sequenced. The mature polypeptide is composed of 794 amino acids and is preceded by a typical signal sequence of 34 amino acid residues. A highly hydrophobic stretch of 20 amino acids at the C-terminal end probably serves as a transmembrane domain. Clusters of threonine residues are located adjacent to this membrane anchor. The S-layer protein is a glycoprotein containing both N- and O-glycosidic bonds. Glucosyl-(1----2)-galactose disaccharides are linked to threonine residues. The primary structure and the glycosylation pattern of the S-layer glycoproteins from Haloferax volcanii and from Halobacterium halobium were compared and found to exhibit distinct differences, despite the fact that three-dimensional reconstructions from electron micrographs revealed no structural differences at least to the 2.5-nm level attained so far (M. Kessel, I. Wildhaber, S. Cohe, and W. Baumeister, EMBO J. 7:1549-1554, 1988). Images PMID:2123862

Primary structure and glycosylation of the S-layer protein of Haloferax volcanii.

PubMed

Sumper, M; Berg, E; Mengele, R; Strobel, I

1990-12-01

The outer surface of the archaebacterium Haloferax volcanii (formerly named Halobacterium volcanii) is covered with a hexagonally packed surface (S) layer. The gene coding for the S-layer protein was cloned and sequenced. The mature polypeptide is composed of 794 amino acids and is preceded by a typical signal sequence of 34 amino acid residues. A highly hydrophobic stretch of 20 amino acids at the C-terminal end probably serves as a transmembrane domain. Clusters of threonine residues are located adjacent to this membrane anchor. The S-layer protein is a glycoprotein containing both N- and O-glycosidic bonds. Glucosyl-(1----2)-galactose disaccharides are linked to threonine residues. The primary structure and the glycosylation pattern of the S-layer glycoproteins from Haloferax volcanii and from Halobacterium halobium were compared and found to exhibit distinct differences, despite the fact that three-dimensional reconstructions from electron micrographs revealed no structural differences at least to the 2.5-nm level attained so far (M. Kessel, I. Wildhaber, S. Cohe, and W. Baumeister, EMBO J. 7:1549-1554, 1988).

Mechanisms of Staphylococcus epidermidis adhesion to model biomaterial surfaces: Establising a link between thrombosis and infection

NASA Astrophysics Data System (ADS)

Higashi, Julie Miyo

Infections involving Staphylococcus epidermidis remain a life threatening complication associated with the use of polymer based cardiovascular devices. One of the critical steps in infection pathogenesis is the adhesion of the bacteria to the device surface. Currently, mechanisms of S. epidermidis adhesion are incompletely understood, but are thought to involve interactions between bacteria, device surface, and host blood elements in the form of adsorbed plasma proteins and surface adherent platelets. Our central hypothesis is that elements participating in thrombosis also promote S. epidermidis adhesion by specifically binding to the bacterial surface. The adhesion kinetics of S. epidermidis RP62A to host modified model biomaterial surface octadecyltrichlorosilane (OTS) under hydrodynamic shear conditions were characterized. Steady state adhesion to adsorbed proteins and surface adherent platelets was achieved at 90-120 minutes and 60-90 minutes, respectively. A dose response curve of S. epidermidis adhesion in the concentration range of 10sp7{-}10sp9 bac/mL resembled a multilayer adsorption isotherm. Increasing shear stress was found to LTA, and other LTA blocking agents significantly decreased S. epidermidis adhesion to the fibrin-platelet clots, suggesting that this interaction between S. epidermidis and fibrin-platelet clots is specific. Studies evaluated the adhesion of S. epidermidis to polymer immobilized heparin report conflicting results. Paulsson et al., showed that coagulase negative staphylococci adhered in comparable numbers to both immobilized heparin and nonheparinized surfaces, while exhibiting significantly greater adhesion to both surfaces than S. aureus. Preadsorption of the surfaces with specific heparin binding plasma proteins vitronectin, fibronectin, laminin, and collagen significantly increased adhesion. It was postulated that immobilized heparin contained binding sites for the plasma proteins, exposing bacteria binding domains of the protein. Aggregation of heparin coated (adsorbed) polystyrene beads by the same S. epidermidis strains did not correlate with the adherence assays on the immobilized heparin surfaces and staphylococcal binding was dependent on media ionic strength and pH, suggesting that the conformation of the heparin polysaccharide chains was crucial to the interaction (132). Another study by Arciola et al., showed that heparin modified PMMA intraocular lenses sustained significantly less adherent bacteria than unmodified PMMA lenses, and that the chromatogram of structural fatty acids of only the S. epidermidis adherent to heparin modified PMMA changed (133). Because neither of these investigators thoroughly characterized their heparin modified surfaces, the difference in their results could easily be explained by the notorious heterogeneity of the heparin molecular weight, bioactivity, or surface coverage on these polymers.

Hydrophobic-Interaction-Induced Stiffening of α -Synuclein Fibril Networks

NASA Astrophysics Data System (ADS)

Semerdzhiev, Slav A.; Lindhoud, Saskia; Stefanovic, Anja; Subramaniam, Vinod; van der Schoot, Paul; Claessens, Mireille M. A. E.

2018-05-01

In water, networks of semiflexible fibrils of the protein α -synuclein stiffen significantly with increasing temperature. We make plausible that this reversible stiffening is a result of hydrophobic contacts between the fibrils that become more prominent with increasing temperature. The good agreement of our experimentally observed temperature dependence of the storage modulus of the network with a scaling theory linking network elasticity with reversible cross-linking enables us to quantify the endothermic binding enthalpy and estimate the effective size of hydrophobic patches on the fibril surface. Our findings may not only shed light on the role of amyloid deposits in disease conditions, but can also inspire new approaches for the design of thermoresponsive materials.

Hydrophobic-Interaction-Induced Stiffening of α-Synuclein Fibril Networks.

PubMed

Semerdzhiev, Slav A; Lindhoud, Saskia; Stefanovic, Anja; Subramaniam, Vinod; van der Schoot, Paul; Claessens, Mireille M A E

2018-05-18

In water, networks of semiflexible fibrils of the protein α-synuclein stiffen significantly with increasing temperature. We make plausible that this reversible stiffening is a result of hydrophobic contacts between the fibrils that become more prominent with increasing temperature. The good agreement of our experimentally observed temperature dependence of the storage modulus of the network with a scaling theory linking network elasticity with reversible cross-linking enables us to quantify the endothermic binding enthalpy and estimate the effective size of hydrophobic patches on the fibril surface. Our findings may not only shed light on the role of amyloid deposits in disease conditions, but can also inspire new approaches for the design of thermoresponsive materials.

Vascular effects of advanced glycation endproducts: Clinical effects and molecular mechanisms☆

PubMed Central

Stirban, Alin; Gawlowski, Thomas; Roden, Michael

2013-01-01

The enhanced generation and accumulation of advanced glycation endproducts (AGEs) have been linked to increased risk for macrovascular and microvascular complications associated with diabetes mellitus. AGEs result from the nonenzymatic reaction of reducing sugars with proteins, lipids, and nucleic acids, potentially altering their function by disrupting molecular conformation, promoting cross-linking, altering enzyme activity, reducing their clearance, and impairing receptor recognition. AGEs may also activate specific receptors, like the receptor for AGEs (RAGE), which is present on the surface of all cells relevant to atherosclerotic processes, triggering oxidative stress, inflammation and apoptosis. Understanding the pathogenic mechanisms of AGEs is paramount to develop strategies against diabetic and cardiovascular complications. PMID:24634815

Chemical crosslinking and mass spectrometry studies of the structure and dynamics of membrane proteins and receptors.

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

Haskins, William E.; Leavell, Michael D.; Lane, Pamela

2005-03-01

Membrane proteins make up a diverse and important subset of proteins for which structural information is limited. In this study, chemical cross-linking and mass spectrometry were used to explore the structure of the G-protein-coupled photoreceptor bovine rhodopsin in the dark-state conformation. All experiments were performed in rod outer segment membranes using amino acid 'handles' in the native protein sequence and thus minimizing perturbations to the native protein structure. Cysteine and lysine residues were covalently cross-linked using commercially available reagents with a range of linker arm lengths. Following chemical digestion of cross-linked protein, cross-linked peptides were identified by accurate mass measurementmore » using liquid chromatography-fourier transform mass spectrometry and an automated data analysis pipeline. Assignments were confirmed and, if necessary, resolved, by tandem MS. The relative reactivity of lysine residues participating in cross-links was evaluated by labeling with NHS-esters. A distinct pattern of cross-link formation within the C-terminal domain, and between loop I and the C-terminal domain, emerged. Theoretical distances based on cross-linking were compared to inter-atomic distances determined from the energy-minimized X-ray crystal structure and Monte Carlo conformational search procedures. In general, the observed cross-links can be explained by re-positioning participating side-chains without significantly altering backbone structure. One exception, between C3 16 and K325, requires backbone motion to bring the reactive atoms into sufficient proximity for cross-linking. Evidence from other studies suggests that residues around K325 for a region of high backbone mobility. These findings show that cross-linking studies can provide insight into the structural dynamics of membrane proteins in their native environment.« less

Identification of cross-linked amino acids in the protein pair HmaL23-HmaL29 from the 50S ribosomal subunit of the archaebacterium Haloarcula marismortui.

PubMed

Bergmann, U; Wittmann-Liebold, B

1993-03-23

50S ribosomal subunits from the extreme halophilic archaebacterium Haloarcula marismortui were treated with the homobifunctional protein-protein cross-linking reagents diepoxybutane (4 A) and dithiobis(succinimidyl propionate) (12 A). The dominant product with both cross-linking reagents was identified on the protein level as HmaL23-HmaL29, which is homologous to the protein pair L23-L29 from Escherichia coli [Walleczek, J., Martin, T., Redl, B., Stöffler-Meilicke, M., & Stöffler, G. (1989) Biochemistry 28, 4099-4105] and from Bacillus stearothermophilus [Brockmöller, J., & Kamp, R. M. (1986) Biol. Chem. Hoppe-Seyler 367, 925-935]. To reveal the exact cross-linking site in HmaL23-HmaL29, the cross-linked complex was purified on a preparative scale by conventional and high-performance liquid chromatography. After endoproteolytic fragmentation of the protein pair, the amino acids engaged in cross-link formation were unambiguously identified by N-terminal sequence analysis and mass spectrometry of the cross-linked peptides. The cross-link is formed between lysine-57 in the C-terminal region of HmaL29 and the alpha-amino group of the N-terminal serine in protein HmaL23, irrespective of the cross-linking reagent. This result demonstrates that the N-terminal region of protein HmaL23 and the C-terminal domain of HmaL29 are highly flexible so that the distance between the two polypeptide chains can vary by at least 8 A. Comparison of our cross-linking results with those obtained with B. stearothermophilus revealed that the fine structure within this ribosomal domain is at least partially conserved.

(S)Pot on Mitochondria: Cannabinoids Disrupt Cellular Respiration to Limit Neuronal Activity.

PubMed

Harkany, Tibor; Horvath, Tamas L

2017-01-10

Classical views posit G protein-coupled cannabinoid receptor 1s (CB1Rs) at the cell surface with cytosolic Giα-mediated signal transduction. Hebert-Chatelain et al. (2016) instead place CB 1 Rs at mitochondria limiting neuronal respiration by soluble adenylyl cyclase-dependent modulation of complex I activity. Thus, neuronal bioenergetics link to synaptic plasticity and, globally, learning and memory. Copyright © 2017 Elsevier Inc. All rights reserved.

Biological Nanoplatforms for Self-Assembled Electronics

DTIC Science & Technology

2015-03-24

as M13 , a virus that infects Escherichia coli. Approximately one billion different amino acid sequences are displayed on different viruses in the...sequence when contained within a phage M13 coat protein sequence, not chemically linked to the surface of phage MS2 VLPs. Thus, binding properties may...gallium arsenide in a bacteriophage M13 phage display library, MS2 VLPs modified with the metal binding peptides do not display the same activity

Enzyme-Encapsulated Liposome-Linked Immunosorbent Assay Enabling Sensitive Personal Glucose Meter Readout for Portable Detection of Disease Biomarkers.

PubMed

Lin, Bingqian; Liu, Dan; Yan, Jinmao; Qiao, Zhi; Zhong, Yunxin; Yan, Jiawei; Zhu, Zhi; Ji, Tianhai; Yang, Chaoyong James

2016-03-23

There is considerable demand for sensitive, selective, and portable detection of disease-associated proteins, particularly in clinical practice and diagnostic applications. Portable devices are highly desired for detection of disease biomarkers in daily life due to the advantages of being simple, rapid, user-friendly, and low-cost. Herein we report an enzyme-encapsulated liposome-linked immunosorbent assay for sensitive detection of proteins using personal glucose meters (PGM) for portable quantitative readout. Liposomes encapsulating a large amount of amyloglucosidase or invertase are surface-coated with recognition elements such as aptamers or antibodies for target recognition. By translating molecular recognition signal into a large amount of glucose with the encapsulated enzyme, disease biomarkers such as thrombin or C-reactive protein (CRP) can be quantitatively detected by a PGM with a high detection limit of 1.8 or 0.30 nM, respectively. With the advantages of portability, ease of use, and low-cost, the method reported here has potential for portable and quantitative detection of various targets for different POC testing scenarios, such as rapid diagnosis in clinic offices, health monitoring at the bedside, and chemical/biochemical safety control in the field.

A mitochondria-anchored isoform of the actin-nucleating spire protein regulates mitochondrial division

PubMed Central

Manor, Uri; Bartholomew, Sadie; Golani, Gonen; Christenson, Eric; Kozlov, Michael; Higgs, Henry; Spudich, James; Lippincott-Schwartz, Jennifer

2015-01-01

Mitochondrial division, essential for survival in mammals, is enhanced by an inter-organellar process involving ER tubules encircling and constricting mitochondria. The force for constriction is thought to involve actin polymerization by the ER-anchored isoform of the formin protein inverted formin 2 (INF2). Unknown is the mechanism triggering INF2-mediated actin polymerization at ER-mitochondria intersections. We show that a novel isoform of the formin-binding, actin-nucleating protein Spire, Spire1C, localizes to mitochondria and directly links mitochondria to the actin cytoskeleton and the ER. Spire1C binds INF2 and promotes actin assembly on mitochondrial surfaces. Disrupting either Spire1C actin- or formin-binding activities reduces mitochondrial constriction and division. We propose Spire1C cooperates with INF2 to regulate actin assembly at ER-mitochondrial contacts. Simulations support this model's feasibility and demonstrate polymerizing actin filaments can induce mitochondrial constriction. Thus, Spire1C is optimally positioned to serve as a molecular hub that links mitochondria to actin and the ER for regulation of mitochondrial division. DOI: http://dx.doi.org/10.7554/eLife.08828.001 PMID:26305500

Antimicrobial Peptides in 2014

PubMed Central

Wang, Guangshun; Mishra, Biswajit; Lau, Kyle; Lushnikova, Tamara; Golla, Radha; Wang, Xiuqing

2015-01-01

This article highlights new members, novel mechanisms of action, new functions, and interesting applications of antimicrobial peptides reported in 2014. As of December 2014, over 100 new peptides were registered into the Antimicrobial Peptide Database, increasing the total number of entries to 2493. Unique antimicrobial peptides have been identified from marine bacteria, fungi, and plants. Environmental conditions clearly influence peptide activity or function. Human α-defensin HD-6 is only antimicrobial under reduced conditions. The pH-dependent oligomerization of human cathelicidin LL-37 is linked to double-stranded RNA delivery to endosomes, where the acidic pH triggers the dissociation of the peptide aggregate to release its cargo. Proline-rich peptides, previously known to bind to heat shock proteins, are shown to inhibit protein synthesis. A model antimicrobial peptide is demonstrated to have multiple hits on bacteria, including surface protein delocalization. While cell surface modification to decrease cationic peptide binding is a recognized resistance mechanism for pathogenic bacteria, it is also used as a survival strategy for commensal bacteria. The year 2014 also witnessed continued efforts in exploiting potential applications of antimicrobial peptides. We highlight 3D structure-based design of peptide antimicrobials and vaccines, surface coating, delivery systems, and microbial detection devices involving antimicrobial peptides. The 2014 results also support that combination therapy is preferred over monotherapy in treating biofilms. PMID:25806720

Cell protein cross-linking by erbstatin and related compounds | Center for Cancer Research

Cancer.gov

The scheme depicts a possible mechanism of cross-linking by erbstatin and related analogues. A mechanism of action is proposed which involves initial oxidation to reactive quinone intermediates that subsequently cross-link protein nucleophiles via multiple 1,4-Michael-type additions. Similar alkylation of protein by protein-tyrosine kinase inhibitors, such as herbimycin A, has

Protein bio-corona: critical issue in immune nanotoxicology.

PubMed

Neagu, Monica; Piperigkou, Zoi; Karamanou, Konstantina; Engin, Ayse Basak; Docea, Anca Oana; Constantin, Carolina; Negrei, Carolina; Nikitovic, Dragana; Tsatsakis, Aristidis

2017-03-01

With the expansion of the nanomedicine field, the knowledge focusing on the behavior of nanoparticles in the biological milieu has rapidly escalated. Upon introduction to a complex biological system, nanomaterials dynamically interact with all the encountered biomolecules and form the protein "bio-corona." The decoration with these surface biomolecules endows nanoparticles with new properties. The present review will address updates of the protein bio-corona characteristics as influenced by nanoparticle's physicochemical properties and by the particularities of the encountered biological milieu. Undeniably, bio-corona generation influences the efficacy of the nanodrug and guides the actions of innate and adaptive immunity. Exploiting the dynamic process of protein bio-corona development in combination with the new engineered horizons of drugs linked to nanoparticles could lead to innovative functional nanotherapies. Therefore, bio-medical nanotechnologies should focus on the interactions of nanoparticles with the immune system for both safety and efficacy reasons.

PACSY, a relational database management system for protein structure and chemical shift analysis

PubMed Central

Lee, Woonghee; Yu, Wookyung; Kim, Suhkmann; Chang, Iksoo

2012-01-01

PACSY (Protein structure And Chemical Shift NMR spectroscopY) is a relational database management system that integrates information from the Protein Data Bank, the Biological Magnetic Resonance Data Bank, and the Structural Classification of Proteins database. PACSY provides three-dimensional coordinates and chemical shifts of atoms along with derived information such as torsion angles, solvent accessible surface areas, and hydrophobicity scales. PACSY consists of six relational table types linked to one another for coherence by key identification numbers. Database queries are enabled by advanced search functions supported by an RDBMS server such as MySQL or PostgreSQL. PACSY enables users to search for combinations of information from different database sources in support of their research. Two software packages, PACSY Maker for database creation and PACSY Analyzer for database analysis, are available from http://pacsy.nmrfam.wisc.edu. PMID:22903636

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

Rantalainen, Kimmo I.; Christensen, Peter A.; Hafren, Anders

The viral genome-linked protein (VPg) of Potato virus A (PVA) is a multifunctional protein that belongs to a class of intrinsically disordered proteins. Typically, this type of protein gains a more stable structure upon interactions or posttranslational modifications. In a membrane lipid strip overlay binding assay, PVA VPg was found to bind phosphatidylserine (PS), but not phosphatidylcholine (PC). According to circular dichroism spectroscopy, the secondary structure of PVA VPg was stabilized upon interactions with PS and phosphatidylglycerol (PG), but not with PC vesicles. It is possible that this stabilization favored the formation of alpha-helical structures. Limited tryptic digestion showed thatmore » the interaction with anionic vesicles protected certain, otherwise accessible, trypsin cleavage sites. An electron microscopy study revealed that interaction with VPg substantially increased the vesicle diameter and caused the formation of pore or plaque-like electron dense spots on the vesicle surface, which gradually led to disruption of the vesicles.« less

mRNA 3' of the A site bound codon is located close to protein S3 on the human 80S ribosome.

PubMed

Molotkov, Maxim V; Graifer, Dmitri M; Popugaeva, Elena A; Bulygin, Konstantin N; Meschaninova, Maria I; Ven'yaminova, Aliya G; Karpova, Galina G

2006-07-01

Ribosomal proteins neighboring the mRNA downstream of the codon bound at the decoding site of human 80S ribosomes were identified using three sets of mRNA analogues that contained a UUU triplet at the 5' terminus and a perfluorophenylazide cross-linker at guanosine, adenosine or uridine residues placed at various locations 3' of this triplet. The positions of modified mRNA nucleotides on the ribosome were governed by tRNA(Phe) cognate to the UUU triplet targeted to the P site. Upon mild UV-irradiation, the mRNA analogues cross-linked preferentially to the 40S subunit, to the proteins and to a lesser extent to the 18S rRNA. Cross-linked nucleotides of 18S rRNA were identified previously. In the present study, it is shown that among the proteins the main target for cross-linking with all the mRNA analogues tested was protein S3 (homologous to prokaryotic S3, S3p); minor cross-linking to protein S2 (S5p) was also detected. Both proteins cross-linked to mRNA analogues in the ternary complexes as well as in the binary complexes (without tRNA). In the ternary complexes protein S15 (S19p) also cross-linked, the yield of the cross-link decreased significantly when the modified nucleotide moved from position +5 to position +12 with respect to the first nucleotide of the P site bound codon. In several ternary complexes minor cross-linking to protein S30 was likewise detected. The results of this study indicate that S3 is a key protein at the mRNA binding site neighboring mRNA downstream of the codon at the decoding site in the human ribosome.

Ti4+ to Ti3+ conversion of TiO2 uppermost layer by low-temperature vacuum annealing: interest for titanium biomedical applications.

PubMed

Guillemot, F; Porté, M C; Labrugère, C; Baquey, Ch

2002-11-01

Because of the Ti(3+) defects responsibility for dissociative adsorption of water onto TiO(2) surfaces and due to the hydroxyls influence on the biological behavior of titanium, controlling the Ti(3+) surface defects density by means of low-temperature vacuum annealing is proposed to improve the bone/implant interactions. Experiments have been carried out on Ti-6Al-4V alloys exhibiting a porous surface generated primarily by chemical treatment. XPS investigations have shown that low-temperature vacuum annealing can create a controlled number of Ti(3+) defects (up to 21% Ti(3+)/Ti(4+) at 573 K). High Ti(3+) defect concentration is linked to surface porosity. Such surfaces, exhibiting high hydrophilicity and microporosity, would confer to titanium biomaterials a great ability to interact with surrounding proteins and cells and hence would favor the bone anchorage of as-treated implants.

Alpha-fetoprotein detection by using a localized surface plasmon coupled fluorescence fiber-optic biosensor

NASA Astrophysics Data System (ADS)

Chang, Ying-Feng; Chen, Ran-Chou; Li, Ying-Chang; Yu, Chih-Jen; Hsieh, Bao-Yu; Chou, Chien

2007-11-01

Alpha-fetoprotein (AFP) detection by using a localized surface plasmon coupled fluorescence (LSPCF) fiber-optic biosensor is setup and experimentally demonstrated. It is based on gold nanoparticle (GNP) and coupled with localized surface plasmon wave on the surface of GNP. In this experiment, the fluorophores are labeled on anti-AFP which are bound to protein A conjugated GNP. Thus, LSPCF is excited with high efficiency in the near field of localized surface plasmon wave. Therefore, not only the sensitivity of LSPCF biosensor is enhanced but also the specific selectivity of AFP is improved. Experimentally, the ability of real time measurement in the range of AFP concentration from 0.1ng/ml to 100ng/ml was detected. To compare with conventional methods such as enzyme-linked immunosorbent assay (ELISA) or radioimmunoassay (RIA), the LSPCF fiber-optic biosensor performs higher or comparable detection sensitivity, respectively.

D19S Mutation of the Cationic, Cysteine-Rich Protein PAF: Novel Insights into Its Structural Dynamics, Thermal Unfolding and Antifungal Function

PubMed Central

Burtscher, Laura; Hajdu, Dorottya; Muñoz, Alberto; Gáspári, Zoltán; Read, Nick D.; Batta, Gyula; Marx, Florentine

2017-01-01

The cysteine-rich, cationic, antifungal protein PAF is abundantly secreted into the culture supernatant of the filamentous Ascomycete Penicillium chrysogenum. The five β-strands of PAF form a compact β-barrel that is stabilized by three disulphide bonds. The folding of PAF allows the formation of four surface-exposed loops and distinct charged motifs on the protein surface that might regulate the interaction of PAF with the sensitive target fungus. The growth inhibitory activity of this highly stable protein against opportunistic fungal pathogens provides great potential in antifungal drug research. To understand its mode of action, we started to investigate the surface-exposed loops of PAF and replaced one aspartic acid at position 19 in loop 2 that is potentially involved in PAF active or binding site, with a serine (Asp19 to Ser19). We analysed the overall effects, such as unfolding, electrostatic changes, sporadic conformers and antifungal activity when substituting this specific amino acid to the fairly indifferent amino acid serine. Structural analyses revealed that the overall 3D solution structure is virtually identical with that of PAF. However, PAFD19S showed slightly increased dynamics and significant differences in the surface charge distribution. Thermal unfolding identified PAFD19S to be rather a two-state folder in contrast to the three-state folder PAF. Functional comparison of PAFD19S and PAF revealed that the exchange at residue 19 caused a dramatic loss of antifungal activity: the binding and internalization of PAFD19S by target cells was reduced and the protein failed to trigger an intracellular Ca2+ response, all of which are closely linked to the antifungal toxicity of PAF. We conclude that the negatively charged residue Asp19 in loop 2 is essential for full function of the cationic protein PAF. PMID:28072824

Clinical roundtable monograph: Paroxysmal nocturnal hemoglobinuria: a case-based discussion.

PubMed

Szer, Jeff; Hill, Anita; Weitz, Ilene Ceil

2012-11-01

Paroxysmal nocturnal hemoglobinuria (PNH) is a rare, acquired disorder characterized by chronic intravascular hemolysis as the primary clinical manifestation and morbidities that include anemia, thrombosis, renal impairment, pulmonary hypertension, and bone marrow failure. The prevalence of the PNH clone (from <1-100% PNH granulocytes) is approximately 16 per million, and careful monitoring is required. The average age of onset of the clinical disease is the early 30s, although it can present at all ages. PNH is caused by the acquisition of a somatic mutation of the gene phosphatidylinositol glycan anchor (PIG-A) in a multipotent hematopoietic stem cell (HSC), with clonal expansion of the mutated HSC. The mutation causes a deficiency in the synthesis of glycosylphosphatidylinositol (GPI). In cells derived from normal HSCs, the complement regulatory proteins CD55 and CD59 are anchored to the hematopoietic cell membrane surface via GPI, protecting the cells from complement-mediated lysis. However, in patients with PNH, these 2 proteins, along with numerous other GPI-linked proteins, are absent from the cell surface of red cells, granulocytes, monocytes, and platelets, resulting in complement-mediated intravascular hemolysis and other complications. Lysis of red blood cells is the most obvious manifestation, but as other cell lineages are also affected, this complement-mediated attack contributes to additional complications, such as thrombosis. Eculizumab, a humanized monoclonal antibody against the C5 complement protein, is the only effective drug therapy for PNH patients. The antibody prevents cleavage of the C5 protein by C5 convertase, in turn preventing generation of C5b-9 and release of C5a, thereby protecting from hemolysis of cells lacking the CD59 surface protein and other complications associated with complement activation. Drs. Ilene C. Weitz, Anita Hill, and Jeff Szer discuss 3 recent cases of patients with PNH.

Detecting protein-protein interactions in the intact cell of Bacillus subtilis (ATCC 6633).

PubMed

Winters, Michael S; Day, R A

2003-07-01

The salt bridge, paired group-specific reagent cyanogen (ethanedinitrile; C(2)N(2)) converts naturally occurring pairs of functional groups into covalently linked products. Cyanogen readily permeates cell walls and membranes. When the paired groups are shared between associated proteins, isolation of the covalently linked proteins allows their identity to be assigned. Examination of organisms of known genome sequence permits identification of the linked proteins by mass spectrometric techniques applied to peptides derived from them. The cyanogen-linked proteins were isolated by polyacrylamide gel electrophoresis. Digestion of the isolated proteins with proteases of known specificity afforded sets of peptides that could be analyzed by mass spectrometry. These data were compared with those derived theoretically from the Swiss Protein Database by computer-based comparisons (Protein Prospector; http://prospector.ucsf.edu). Identification of associated proteins in the ribosome of Bacillus subtilis strain ATCC 6633 showed that there is an association homology with the association patterns of the ribosomal proteins of Haloarcula marismortui and Thermus thermophilus. In addition, other proteins involved in protein biosynthesis were shown to be associated with ribosomal proteins.

Detecting Protein-Protein Interactions in the Intact Cell of Bacillus subtilis (ATCC 6633)

PubMed Central

Winters, Michael S.; Day, R. A.

2003-01-01

The salt bridge, paired group-specific reagent cyanogen (ethanedinitrile; C2N2) converts naturally occurring pairs of functional groups into covalently linked products. Cyanogen readily permeates cell walls and membranes. When the paired groups are shared between associated proteins, isolation of the covalently linked proteins allows their identity to be assigned. Examination of organisms of known genome sequence permits identification of the linked proteins by mass spectrometric techniques applied to peptides derived from them. The cyanogen-linked proteins were isolated by polyacrylamide gel electrophoresis. Digestion of the isolated proteins with proteases of known specificity afforded sets of peptides that could be analyzed by mass spectrometry. These data were compared with those derived theoretically from the Swiss Protein Database by computer-based comparisons (Protein Prospector; http://prospector.ucsf.edu). Identification of associated proteins in the ribosome of Bacillus subtilis strain ATCC 6633 showed that there is an association homology with the association patterns of the ribosomal proteins of Haloarcula marismortui and Thermus thermophilus. In addition, other proteins involved in protein biosynthesis were shown to be associated with ribosomal proteins. PMID:12837803

Preparation of Sulfobetaine-Grafted PVDF Hollow Fiber Membranes with a Stably Anti-Protein-Fouling Performance

PubMed Central

Li, Qian; Lin, Han-Han; Wang, Xiao-Lin

2014-01-01

Based on a two-step polymerization method, two sulfobetaine-based zwitterionic monomers, including 3-(methacryloylamino) propyl-dimethyl-(3-sulfopropyl) ammonium hydroxide (MPDSAH) and 2-(methacryloyloxyethyl) ethyl-dimethyl-(3-sulfopropyl) ammonium (MEDSA), were successfully grafted from poly(vinylidene fluoride) (PVDF) hollow fiber membrane surfaces in the presence of N,N′-methylene bisacrylamide (MBAA) as a cross-linking agent. The mechanical properties of the PVDF membrane were improved by the zwitterionic surface layers. The surface hydrophilicity of PVDF membranes was significantly enhanced and the polyMPDSAH-g-PVDF membrane showed a higher hydrophilicity due to the higher grafting amount. Compared to the polyMEDSA-g-PVDF membrane, the polyMPDSAH-g-PVDF membrane showed excellent significantly better anti-protein-fouling performance with a flux recovery ratio (RFR) higher than 90% during the cyclic filtration of a bovine serum albumin (BSA) solution. The polyMPDSAH-g-PVDF membrane showed an obvious electrolyte-responsive behavior and its protein-fouling-resistance performance was improved further during the filtration of the protein solution with 100 mmol/L of NaCl. After cleaned with a membrane cleaning solution for 16 days, the grafted MPDSAH layer on the PVDF membrane could be maintain without any chang; however, the polyMEDSA-g-PVDF membrane lost the grafted MEDSA layer after this treatment. Therefore, the amide group of sulfobetaine, which contributed significantly to the higher hydrophilicity and stability, was shown to be imperative in modifying the PVDF membrane for a stable anti-protein-fouling performance via the two-step polymerization method. PMID:24957171

Vibrio parahaemolyticus enolase is an adhesion-related factor that binds plasminogen and functions as a protective antigen.

PubMed

Jiang, Wei; Han, Xiangan; Wang, Quan; Li, Xintong; Yi, Li; Liu, Yongjie; Ding, Chan

2014-06-01

Vibrio parahaemolyticus, an emerging food and waterborne pathogen, is a leading cause of seafood poisoning worldwide. Surface proteins can directly participate in microbial virulence by facilitating pathogen dissemination via interactions with host factors. Screening and identification of protective antigens is important for developing therapies against V. parahaemolyticus infections. Here, we systematically characterized a novel immunogenic enolase of V. parahaemolyticus. The enolase gene of V. parahaemolyticus ATCC33847 was cloned, sequenced, and expressed in Escherichia coli BL21. Enzymatic assays revealed that the purified recombinant V. parahaemolyticus enolase protein catalyzes the dehydration of 2-phospho-D-glycerate to phosphoenolpyruvate. Western blot analysis showed that V. parahaemolyticus enolase was detectable in the extracellular, outer membrane (OM) and cytoplasmic protein fractions using antibodies against the recombinant enolase. Surface expression of enolase was further confirmed by immunogold staining and mass spectrometry (liquid chromatography-tandem mass spectrometry) analysis of OM protein profiles. Notably, V. parahaemolyticus enolase was identified as a human plasminogen-binding protein with the enzyme-linked immunosorbent assay. The values obtained for adherence and inhibition suggest a role of surface-exposed enolase in epithelial adherence of V. parahaemolyticus. We further showed that enolase confers efficient immunity against challenge with a lethal dose of V. parahaemolyticus in a mouse model. To our knowledge, this is the first study to demonstrate the plasminogen-binding activity of enolase that is an adhesion-related factor of V. parahaemolyticus. Our findings collectively imply that enolase plays important roles in pathogenicity, supporting its utility as a novel vaccine candidate against V. parahaemolyticus infection.

Sexual orientation, fraternal birth order, and the maternal immune hypothesis: a review.

PubMed

Bogaert, Anthony F; Skorska, Malvina

2011-04-01

In 1996, psychologists Ray Blanchard and Anthony Bogaert found evidence that gay men have a greater number of older brothers than do heterosexual men. This "fraternal birth order" (FBO) effect has been replicated numerous times, including in non-Western samples. More recently, strong evidence has been found that the FBO effect is of prenatal origin. Although there is no direct support for the exact prenatal mechanism, the most plausible explanation may be immunological in origin, i.e., a mother develops an immune reaction against a substance important in male fetal development during pregnancy, and that this immune effect becomes increasingly likely with each male gestation. This immune effect is hypothesized to cause an alteration in (some) later born males' prenatal brain development. The target of the immune response may be molecules (i.e., Y-linked proteins) on the surface of male fetal brain cells, including in sites of the anterior hypothalamus, which has been linked to sexual orientation in other research. Antibodies might bind to these molecules and thus alter their role in typical sexual differentiation, leading some later born males to be attracted to men as opposed to women. Here we review evidence in favor of this hypothesis, including recent research showing that mothers of boys develop an immune response to one Y-linked protein (i.e., H-Y antigen; SMCY) important in male fetal development, and that this immune effect becomes increasingly likely with each additional boy to which a mother gives birth. We also discuss other Y-linked proteins that may be relevant if this hypothesis is correct. Finally, we discuss issues in testing the maternal immune hypothesis of FBO. Copyright © 2011 Elsevier Inc. All rights reserved.

Quantitative evaluation of protein conformation in pharmaceuticals using cross-linking reactions coupled with LC-MS/MS analysis.

PubMed

Yamaguchi, Hideto; Hirakura, Yutaka; Shirai, Hiroki; Mimura, Hisashi; Toyo'oka, Toshimasa

2011-06-01

The need for a simple and high-throughput method for identifying the tertiary structure of protein pharmaceuticals has increased. In this study, a simple method for mapping the protein fold is proposed for use as a complementary quality test. This method is based on cross-linking a protein using a [bis(sulfosuccinimidyl)suberate (BS(3))], followed by peptide mapping by LC-MS. Consensus interferon (CIFN) was used as the model protein. The tryptic map obtained via liquid chromatography tandem mass spectroscopy (LC-MS/MS) and the mass mapping obtained via matrix-assisted laser desorption/ionization time-of-flight mass spectroscopy were used to identify cross-linked peptides. While LC-MS/MS analyses found that BS(3) formed cross-links in the loop region of the protein, which was regarded as the biologically active site, sodium dodecyl-sulfate polyacrylamide gel electrophoresis demonstrated that cross-linking occurred within a protein molecule, but not between protein molecules. The occurrence of cross-links at the active site depends greatly on the conformation of the protein, which is determined by the denaturing conditions. Quantitative evaluation of the tertiary structure of CIFN was thus possible by monitoring the amounts of cross-linked peptides generated. Assuming that background information is available at the development stage, this method may be applicable to process development as a complementary test for quality control. Copyright © 2011 Elsevier B.V. All rights reserved.

The structure of human 4F2hc ectodomain provides a model for homodimerization and electrostatic interaction with plasma membrane.

PubMed

Fort, Joana; de la Ballina, Laura R; Burghardt, Hans E; Ferrer-Costa, Carles; Turnay, Javier; Ferrer-Orta, Cristina; Usón, Isabel; Zorzano, Antonio; Fernández-Recio, Juan; Orozco, Modesto; Lizarbe, María Antonia; Fita, Ignacio; Palacín, Manuel

2007-10-26

4F2hc (CD98hc) is a multifunctional type II membrane glycoprotein involved in amino acid transport and cell fusion, adhesion, and transformation. The structure of the ectodomain of human 4F2hc has been solved using monoclinic (Protein Data Bank code 2DH2) and orthorhombic (Protein Data Bank code 2DH3) crystal forms at 2.1 and 2.8 A, respectively. It is composed of a (betaalpha)(8) barrel and an antiparallel beta(8) sandwich related to bacterial alpha-glycosidases, although lacking key catalytic residues and consequently catalytic activity. 2DH3 is a dimer with Zn(2+) coordination at the interface. Human 4F2hc expressed in several cell types resulted in cell surface and Cys(109) disulfide bridge-linked homodimers with major architectural features of the crystal dimer, as demonstrated by cross-linking experiments. 4F2hc has no significant hydrophobic patches at the surface. Monomer and homodimer have a polarized charged surface. The N terminus of the solved structure, including the position of Cys(109) residue located four residues apart from the transmembrane domain, is adjacent to the positive face of the ectodomain. This location of the N terminus and the Cys(109)-intervening disulfide bridge imposes space restrictions sufficient to support a model for electrostatic interaction of the 4F2hc ectodomain with membrane phospholipids. These results provide the first crystal structure of heteromeric amino acid transporters and suggest a dynamic interaction of the 4F2hc ectodomain with the plasma membrane.

Cross-linking measurements of the Potato leafroll virus reveal protein interaction topologies required for virion stability, aphid transmission, and virus-plant interactions.

PubMed

Chavez, Juan D; Cilia, Michelle; Weisbrod, Chad R; Ju, Ho-Jong; Eng, Jimmy K; Gray, Stewart M; Bruce, James E

2012-05-04

Protein interactions are critical determinants of insect transmission for viruses in the family Luteoviridae. Two luteovirid structural proteins, the capsid protein (CP) and the readthrough protein (RTP), contain multiple functional domains that regulate virus transmission. There is no structural information available for these economically important viruses. We used Protein Interaction Reporter (PIR) technology, a strategy that uses chemical cross-linking and high resolution mass spectrometry, to discover topological features of the Potato leafroll virus (PLRV) CP and RTP that are required for the diverse biological functions of PLRV virions. Four cross-linked sites were repeatedly detected, one linking CP monomers, two within the RTP, and one linking the RTP and CP. Virus mutants with triple amino acid deletions immediately adjacent to or encompassing the cross-linked sites were defective in virion stability, RTP incorporation into the capsid, and aphid transmission. Plants infected with a new, infectious PLRV mutant lacking 26 amino acids encompassing a cross-linked site in the RTP exhibited a delay in the appearance of systemic infection symptoms. PIR technology provided the first structural insights into luteoviruses which are crucially lacking and are involved in vector-virus and plant-virus interactions. These are the first cross-linking measurements on any infectious, insect-transmitted virus.

Cross-linking measurements of the Potato leafroll virus reveal protein interaction topologies required for virion stability, aphid transmission, and virus-plant interactions

PubMed Central

Chavez, Juan D.; Cilia, Michelle; Weisbrod, Chad R.; Ju, Ho-Jong; Eng, Jimmy K.; Gray, Stewart M.; Bruce, James E.

2012-01-01

Protein interactions are critical determinants of insect-transmission for viruses in the family Luteoviridae. Two luteovirid structural proteins, the capsid protein (CP) and the readthrough protein (RTP), contain multiple functional domains that regulate virus transmission. There is no structural information available for these economically important viruses. We used Protein Interaction Reporter (PIR) technology, a strategy that uses chemical cross-linking and high resolution mass spectrometry, to discover topological features of the Potato leafroll virus (PLRV) CP and RTP that are required for the diverse biological functions of PLRV virions. Four cross-linked sites were repeatedly detected, one linking CP monomers, two within the RTP, and one linking the RTP and CP. Virus mutants with triple amino acid deletions immediately adjacent to or encompassing the cross-linked sites were defective in virion stability, RTP incorporation into the capsid, and aphid transmission. Plants infected with a new, infectious PLRV mutant lacking 26 amino acids encompassing a cross-linked site in the RTP exhibited a delay in the appearance of systemic infection symptoms. PIR technology provided the first structural insights into luteoviruses which are crucially lacking and that are involved in vector-virus and plant-virus interactions. These are the first cross-linking measurements on any infectious, insect-transmitted virus. PMID:22390342

Genetic ablation of zyxin causes Mena/VASP mislocalization, increased motility, and deficits in actin remodeling

PubMed Central

Hoffman, Laura M.; Jensen, Christopher C.; Kloeker, Susanne; Wang, C.-L. Albert; Yoshigi, Masaaki; Beckerle, Mary C.

2006-01-01

Focal adhesions are specialized regions of the cell surface where integrin receptors and associated proteins link the extracellular matrix to the actin cytoskeleton. To define the cellular role of the focal adhesion protein zyxin, we characterized the phenotype of fibroblasts in which the zyxin gene was deleted by homologous recombination. Zyxin-null fibroblasts display enhanced integrin-dependent adhesion and are more migratory than wild-type fibroblasts, displaying reduced dependence on extracellular matrix cues. We identified differences in the profiles of 75- and 80-kD tyrosine-phosphorylated proteins in the zyxin-null cells. Tandem array mass spectrometry identified both modified proteins as isoforms of the actomyosin regulator caldesmon, a protein known to influence contractility, stress fiber formation, and motility. Zyxin-null fibroblasts also show deficits in actin stress fiber remodeling and exhibit changes in the molecular composition of focal adhesions, most notably by severely reduced accumulation of Ena/VASP proteins. We postulate that zyxin cooperates with Ena/VASP proteins and caldesmon to influence integrin-dependent cell motility and actin stress fiber remodeling. PMID:16505170

Cross-Linking/Mass Spectrometry for Studying Protein Structures and Protein-Protein Interactions: Where Are We Now and Where Should We Go from Here?

PubMed

Sinz, Andrea

2018-05-28

Structural mass spectrometry (MS) is gaining increasing importance for deriving valuable three-dimensional structural information on proteins and protein complexes, and it complements existing techniques, such as NMR spectroscopy and X-ray crystallography. Structural MS unites different MS-based techniques, such as hydrogen/deuterium exchange, native MS, ion-mobility MS, protein footprinting, and chemical cross-linking/MS, and it allows fundamental questions in structural biology to be addressed. In this Minireview, I will focus on the cross-linking/MS strategy. This method not only delivers tertiary structural information on proteins, but is also increasingly being used to decipher protein interaction networks, both in vitro and in vivo. Cross-linking/MS is currently one of the most promising MS-based approaches to derive structural information on very large and transient protein assemblies and intrinsically disordered proteins. © 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Rapid endocytosis of the low density lipoprotein receptor-related protein modulates cell surface distribution and processing of the beta-amyloid precursor protein.

PubMed

Cam, Judy A; Zerbinatti, Celina V; Li, Yonghe; Bu, Guojun

2005-04-15

The low density lipoprotein receptor-related protein (LRP) is a approximately 600-kDa multifunctional endocytic receptor that is highly expressed in the brain. LRP and its ligands apolipoprotein E, alpha2-macroglobulin, and beta-amyloid precursor protein (APP), are genetically linked to Alzheimer disease and are found in characteristic plaque deposits in brains of patients with Alzheimer disease. To identify which extracellular domains of LRP interact with APP, we used minireceptors of each of the individual LRP ligand binding domains and assessed their ability to bind and degrade a soluble APP fragment. LRP minireceptors containing ligand binding domains II and IV, but not I or III, interacted with APP. To test whether APP trafficking is directly related to the rapid endocytosis of LRP, we generated stable Chinese hamster ovary cell lines expressing either a wild-type LRP minireceptor or its endocytosis mutants. Chinese hamster ovary cells stably expressing wild-type LRP minireceptor had less cell surface APP than pcDNA3 vector-transfected cells, whereas those stably expressing endocytosis-defective LRP minireceptors accumulated APP at the cell surface. We also found that the steady-state levels of the amyloid beta-peptides (Abeta) is dictated by the relative expression levels of APP and LRP, probably reflecting the dual roles of LRP in both Abeta production and clearance. Together, these data establish a relationship between LRP rapid endocytosis and APP trafficking and proteolytic processing to generate Abeta.

Crystal structure at 2.8 Å of the DLLRKN-containing coiled-coil domain of Huntingtin-interacting protein 1 (HIP1) reveals a surface suitable for clathrin light chain binding

PubMed Central

Ybe, Joel A.; Mishra, Sanjay; Helms, Stephen; Nix, Jay

2007-01-01

Summary Huntingtin interacting protein 1 (HIP1) is a member of a family of proteins whose interaction with Huntingtin is critical to prevent cells from initiating apoptosis. HIP1, and related protein HIP12/1R, can also bind to clathrin and membrane phospholipids and HIP12/1R links the CCV to the actin cytoskeleton. HIP1 and HIP12/1R interact with the clathrin light chain EED regulatory site and stimulate clathrin lattice assembly. Here we report the X-ray structure of the coiled-coil domain of HIP1 from 482–586 that includes residues crucial for binding clathrin light chain. The dimeric HIP1 crystal structure is partially splayed open. The comparison of the HIP1 model with coiled-coil predictions revealed the heptad repeat in the dimeric trunk (S2 path) is offset relative to the register of the heptad repeat from the N-terminal portion (S1 path) of the molecule. Furthermore, surface analysis showed there is a third hydrophobic path (S3) running parallel to S1 and S2. We present structural evidence supporting a role for S3 path as an interaction surface for clathrin light chain. Finally, comparative analysis suggests the mode of binding between sla2p and clathrin light chain may be different in yeast. PMID:17257618

Aggregated silver nanoparticles based surface-enhanced Raman scattering enzyme-linked immunosorbent assay for ultrasensitive detection of protein biomarkers and small molecules.

PubMed

Liang, Jiajie; Liu, Hongwu; Huang, Caihong; Yao, Cuize; Fu, Qiangqiang; Li, Xiuqing; Cao, Donglin; Luo, Zhi; Tang, Yong

2015-06-02

Lowering the detection limit is critical to the design of bioassays required for medical diagnostics, environmental monitoring, and food safety regulations. The current sensitivity of standard color-based analyte detection limits the further use of enzyme-linked immunosorbent assays (ELISAs) in research and clinical diagnoses. Here, we demonstrate a novel method that uses the Raman signal as the signal-generating system of an ELISA and combines surface-enhanced Raman scattering (SERS) with silver nanoparticles aggregation for ultrasensitive analyte detection. The enzyme label of the ELISA controls the dissolution of Raman reporter-labeled silver nanoparticles through hydrogen peroxide and generates a strong Raman signal when the analyte is present. Using this assay, prostate-specific antigen (PSA) and the adrenal stimulant ractopamine (Rac) were detected in whole serum and urine at the ultralow concentrations of 10(-9) and 10(-6) ng/mL, respectively. The methodology proposed here could potentially be applied to other molecules detection as well as PSA and Rac.

Studies of the viral binding proteins of shrimp BP53, a receptor of white spot syndrome virus.

PubMed

Li, Chen; Gao, Xiao-Xiao; Huang, Jie; Liang, Yan

2016-02-01

The specific binding between viral attachment proteins (VAPs) of a virus and its cellular receptors on host cells mediates virus entry into host cells, which triggers subsequent viral infections. Previous studies indicate that F1 ATP synthase β subunit (named BP53), is found on the surface of shrimp cells and involved in white spot syndrome virus (WSSV) infection by functioning as a potential viral receptor. Herein, in a far-western blotting assay, three WSSV proteins with molecular weights of 28 kDa, 37 kDa, and >50 kDa were found to interact with BP53. The 28 kDa and 37 kDa proteins were identified as the envelope protein VP28 and VP37 of WSSV respectively, which could be recognized by the polyclonal antibodies. Enzyme-linked immunosorbent binding assays revealed that VP37 contributed to almost 80% of the binding capability for BP53 compared with the same amount of total WSSV protein. The relationship between BP53 and its complementary interacting protein, VP37, was visualized using a co-localization assay. Bound VP37 on the cell surface co-localized with BP53 and shared a similar subcellular location on the outer surface of shrimp cells. Pearson's correlation coefficients reached to 0.67 ± 0.05 and the Mander's overlap coefficients reached 0.70 ± 0.05, which indicated a strong relationship between the localization of BP53 and bound rVP37. This provides evidence for an interaction between BP53 and VP37 obtained at the molecular and cellular levels, supporting the hypothesis that BP53 serves as a receptor for WSSV by binding to VP37. The identification of the viral binding proteins of shrimp BP53 is helpful for better understanding the pathogenic mechanisms of WSSV to infect shrimp at the cellular level. Copyright © 2016 Elsevier Inc. All rights reserved.

Interaction of Myosin Phosphatase Target Subunit (MYPT1) with Myosin Phosphatase-RhoA Interacting Protein (MRIP): A Role of Glutamic Acids in the Interaction.

PubMed

Lee, Eunhee; Stafford, Walter F

2015-01-01

Scaffold proteins bind to and functionally link protein members of signaling pathways. Interaction of the scaffold proteins, myosin phosphatase target subunit (MYPT1) and myosin phosphatase-RhoA interacting protein (MRIP), causes co-localization of myosin phosphatase and RhoA to actomyosin. To examine biophysical properties of interaction of MYPT1 with MRIP, we employed analytical ultracentrifugation and surface plasmon resonance. In regard to MRIP, its residues 724-837 are sufficient for the MYPT1/MRIP interaction. Moreover, MRIP binds to MYPT1 as either a monomer or a dimer. With respect to MYPT1, its leucine repeat region, LR (residues 991-1030) is sufficient to account for the MYPT1/MRIP interaction. Furthermore, point mutations that replace glutamic acids 998-1000 within LR reduced the binding affinity toward MRIP. This suggests that the glutamic acids of MYPT1 play an important role in the interaction.

Covalent modification of soy protein isolate by (-)-epigallocatechin-3-gallate: effects on structural and emulsifying properties.

PubMed

Tao, Fei; Jiang, He; Chen, Wenwei; Zhang, Yongyong; Pan, Jiarong; Jiang, Jiaxin; Jia, Zhenbao

2018-05-07

Soy protein isolate (SPI) has promising applications in various food products because of its excellent functional properties and nutritional quality. The structural and emulsifying properties of covalently modified SPI by (-)-epigallocatechin-3-gallate (EGCG) were investigated. SPI was covalently modified by EGCG under alkaline conditions. SDS-PAGE analysis revealed that EGCG modification caused cross-linking of SPI proteins. Circular dichroism spectra demonstrated that the secondary structure of SPI proteins was changed by EGCG modification. In addition, the modifications resulted in the perturbation of the tertiary structure of SPI as evidenced by intrinsic fluorescence spectra and surface hydrophobicity measurements. Oil-in-water emulsions of modified SPI had smaller droplet sizes and better creaming stability compared to those from unmodified SPI. The covalent modification by EGCG improved the emulsifying property of SPI. This study provided an innovative approach for improving the emulsifying properties of proteins. This article is protected by copyright. All rights reserved.

The synaptic vesicle-associated protein amphiphysin is the 128-kD autoantigen of Stiff-Man syndrome with breast cancer

PubMed Central

1993-01-01

Stiff-Man syndrome (SMS) is a rare disease of the central nervous system (CNS) characterized by progressive rigidity of the body musculature with superimposed painful spasms. An autoimmune origin of the disease has been proposed. In a caseload of more than 100 SMS patients, 60% were found positive for autoantibodies directed against the GABA-synthesizing enzyme glutamic acid decarboxylase (GAD). Few patients, all women affected by breast cancer, were negative for GAD autoantibodies but positive for autoantibodies directed against a 128- kD synaptic protein. We report here that this antigen is amphiphysin. GAD and amphiphysin are nonintrinsic membrane proteins that are concentrated in nerve terminals, where a pool of both proteins is associated with the cytoplasmic surface of synaptic vesicles. GAD and amphiphysin are the only two known targets of CNS autoimmunity with this distribution. This finding suggests a possible link between autoimmunity directed against cytoplasmic proteins associated with synaptic vesicles and SMS. PMID:8245793

Guar foaming albumin: a low molecular mass protein with high foaming activity and foam stability isolated from guar meal.

PubMed

Shimoyama, Ami; Kido, Shoko; Kinekawa, Yoh-ichi; Doi, Yukio

2008-10-08

The water extract of guar meal ( Cyamopsis tetragonolobus) was examined for its foamability. Compared with egg white, the extract showed an extraordinary foam stability: no drainage after 3 h of standing in contrast to 65% drainage for egg white at the same protein concentration. The acid-precipitated protein from the extract was responsible for the high foamability and designated guar foaming albumin (GFA). The foaming activity of GFA was 20 times higher than that of egg white. GFA consisted of two subunits with molecular masses of 6 and 11 kDa linked to each other through disulfide bonds. The cleavage of disulfide bonds in GFA affected the foamability only slightly. GFA remarkably decreased the surface tension of water at low protein concentrations. Immunoblotting analysis demonstrated that GFA did not react to the antisera from allergic patients against plant food. These results suggest that GFA serves as an effective food additive in developing protein-stabilized foam.

Intrinsic Pleckstrin Homology (PH) Domain Motion in Phospholipase C-β Exposes a Gβγ Protein Binding Site.

PubMed

Kadamur, Ganesh; Ross, Elliott M

2016-05-20

Mammalian phospholipase C-β (PLC-β) isoforms are stimulated by heterotrimeric G protein subunits and members of the Rho GTPase family of small G proteins. Although recent structural studies showed how Gαq and Rac1 bind PLC-β, there is a lack of consensus regarding the Gβγ binding site in PLC-β. Using FRET between cerulean fluorescent protein-labeled Gβγ and the Alexa Fluor 594-labeled PLC-β pleckstrin homology (PH) domain, we demonstrate that the PH domain is the minimal Gβγ binding region in PLC-β3. We show that the isolated PH domain can compete with full-length PLC-β3 for binding Gβγ but not Gαq, Using sequence conservation, structural analyses, and mutagenesis, we identify a hydrophobic face of the PLC-β PH domain as the Gβγ binding interface. This PH domain surface is not solvent-exposed in crystal structures of PLC-β, necessitating conformational rearrangement to allow Gβγ binding. Blocking PH domain motion in PLC-β by cross-linking it to the EF hand domain inhibits stimulation by Gβγ without altering basal activity or Gαq response. The fraction of PLC-β cross-linked is proportional to the fractional loss of Gβγ response. Cross-linked PLC-β does not bind Gβγ in a FRET-based Gβγ-PLC-β binding assay. We propose that unliganded PLC-β exists in equilibrium between a closed conformation observed in crystal structures and an open conformation where the PH domain moves away from the EF hands. Therefore, intrinsic movement of the PH domain in PLC-β modulates Gβγ access to its binding site. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Intrinsic Pleckstrin Homology (PH) Domain Motion in Phospholipase C-β Exposes a Gβγ Protein Binding Site*

PubMed Central

Kadamur, Ganesh

2016-01-01

Mammalian phospholipase C-β (PLC-β) isoforms are stimulated by heterotrimeric G protein subunits and members of the Rho GTPase family of small G proteins. Although recent structural studies showed how Gαq and Rac1 bind PLC-β, there is a lack of consensus regarding the Gβγ binding site in PLC-β. Using FRET between cerulean fluorescent protein-labeled Gβγ and the Alexa Fluor 594-labeled PLC-β pleckstrin homology (PH) domain, we demonstrate that the PH domain is the minimal Gβγ binding region in PLC-β3. We show that the isolated PH domain can compete with full-length PLC-β3 for binding Gβγ but not Gαq, Using sequence conservation, structural analyses, and mutagenesis, we identify a hydrophobic face of the PLC-β PH domain as the Gβγ binding interface. This PH domain surface is not solvent-exposed in crystal structures of PLC-β, necessitating conformational rearrangement to allow Gβγ binding. Blocking PH domain motion in PLC-β by cross-linking it to the EF hand domain inhibits stimulation by Gβγ without altering basal activity or Gαq response. The fraction of PLC-β cross-linked is proportional to the fractional loss of Gβγ response. Cross-linked PLC-β does not bind Gβγ in a FRET-based Gβγ-PLC-β binding assay. We propose that unliganded PLC-β exists in equilibrium between a closed conformation observed in crystal structures and an open conformation where the PH domain moves away from the EF hands. Therefore, intrinsic movement of the PH domain in PLC-β modulates Gβγ access to its binding site. PMID:27002154

Enrichment of Cross-Linked Peptides Using Charge-Based Fractional Diagonal Chromatography (ChaFRADIC).

PubMed

Tinnefeld, Verena; Venne, A Saskia; Sickmann, Albert; Zahedi, René P

2017-02-03

Chemical cross-linking of proteins is an emerging field with huge potential for the structural investigation of proteins and protein complexes. Owing to the often relatively low yield of cross-linking products, their identification in complex samples benefits from enrichment procedures prior to mass spectrometry analysis. So far, this is mainly accomplished by using biotin moieties in specific cross-linkers or by applying strong cation exchange chromatography (SCX) for a relatively crude enrichment. We present a novel workflow to enrich cross-linked peptides by utilizing charge-based fractional diagonal chromatography (ChaFRADIC). On the basis of two-dimensional diagonal SCX separation, we could increase the number of identified cross-linked peptides for samples of different complexity: pure cross-linked BSA, cross-linked BSA spiked into a simple protein mixture, and cross-linked BSA spiked into a HeLa lysate. We also compared XL-ChaFRADIC with size exclusion chromatography-based enrichment of cross-linked peptides. The XL-ChaFRADIC approach is straightforward, reproducible, and independent of the cross-linking chemistry and cross-linker properties.

A functionalized poly(ethylene glycol)-based bioassay surface chemistry that facilitates bio-immobilization and inhibits non-specific protein, bacterial, and mammalian cell adhesion

PubMed Central

Harbers, Gregory M.; Emoto, Kazunori; Greef, Charles; Metzger, Steven W.; Woodward, Heather N.; Mascali, James J.; Grainger, David W.; Lochhead, Michael J.

2008-01-01

This paper describes a new bioassay surface chemistry that effectively inhibits non-specific biomolecular and cell binding interactions, while providing a capacity for specific immobilization of desired biomolecules. Poly(ethylene glycol) (PEG) as the primary component in nonfouling film chemistry is well-established, but the multicomponent formulation described here is unique in that it (1) is applied in a single, reproducible, solution-based coating step; (2) can be applied to diverse substrate materials without the use of special primers; and (3) is readily functionalized to provide specific attachment chemistries. Surface analysis data are presented, detailing surface roughness, polymer film thickness, and film chemistry. Protein non-specific binding assays demonstrate significant inhibition of serum, fibrinogen, and lysozyme adsorption to coated glass, indium tin oxide, and tissue culture polystyrene dishes. Inhibition of S. aureus and K. pneumoniae microbial adhesion in a microfluidic flow cell, and inhibition of fibroblast cell adhesion from serum-based cell culture is shown. Effective functionalization of the coating is demonstrated by directing fibroblast adhesion to polymer surfaces activated with an RGD peptide. Batch-to-batch reproducibility data are included. The in situ cross-linked PEG-based coating chemistry is unique in its formulation, and its surface properties are attractive for a broad range of in vitro bioassay applications. PMID:18815622

Functional analysis of the Helicobacter pullorum N-linked protein glycosylation system.

PubMed

Jervis, Adrian J; Wood, Alison G; Cain, Joel A; Butler, Jonathan A; Frost, Helen; Lord, Elizabeth; Langdon, Rebecca; Cordwell, Stuart J; Wren, Brendan W; Linton, Dennis

2018-04-01

N-linked protein glycosylation systems operate in species from all three domains of life. The model bacterial N-linked glycosylation system from Campylobacter jejuni is encoded by pgl genes present at a single chromosomal locus. This gene cluster includes the pglB oligosaccharyltransferase responsible for transfer of glycan from lipid carrier to protein. Although all genomes from species of the Campylobacter genus contain a pgl locus, among the related Helicobacter genus only three evolutionarily related species (H. pullorum, H. canadensis and H. winghamensis) potentially encode N-linked protein glycosylation systems. Helicobacter putative pgl genes are scattered in five chromosomal loci and include two putative oligosaccharyltransferase-encoding pglB genes per genome. We have previously demonstrated the in vitro N-linked glycosylation activity of H. pullorum resulting in transfer of a pentasaccharide to a peptide at asparagine within the sequon (D/E)XNXS/T. In this study, we identified the first H. pullorum N-linked glycoprotein, termed HgpA. Production of histidine-tagged HgpA in the background of insertional knockout mutants of H. pullorum pgl/wbp genes followed by analysis of HgpA glycan structures demonstrated the role of individual gene products in the PglB1-dependent N-linked protein glycosylation pathway. Glycopeptide purification by zwitterionic-hydrophilic interaction liquid chromatography coupled with tandem mass spectrometry identified six glycosites from five H. pullorum proteins, which was consistent with proteins reactive with a polyclonal antiserum generated against glycosylated HgpA. This study demonstrates functioning of a H. pullorum N-linked general protein glycosylation system.

Developing a Multiplexed Quantitative Cross-Linking Mass Spectrometry Platform for Comparative Structural Analysis of Protein Complexes.

PubMed

Yu, Clinton; Huszagh, Alexander; Viner, Rosa; Novitsky, Eric J; Rychnovsky, Scott D; Huang, Lan

2016-10-18

Cross-linking mass spectrometry (XL-MS) represents a recently popularized hybrid methodology for defining protein-protein interactions (PPIs) and analyzing structures of large protein assemblies. In particular, XL-MS strategies have been demonstrated to be effective in elucidating molecular details of PPIs at the peptide resolution, providing a complementary set of structural data that can be utilized to refine existing complex structures or direct de novo modeling of unknown protein structures. To study structural and interaction dynamics of protein complexes, quantitative cross-linking mass spectrometry (QXL-MS) strategies based on isotope-labeled cross-linkers have been developed. Although successful, these approaches are mostly limited to pairwise comparisons. In order to establish a robust workflow enabling comparative analysis of multiple cross-linked samples simultaneously, we have developed a multiplexed QXL-MS strategy, namely, QMIX (Quantitation of Multiplexed, Isobaric-labeled cross (X)-linked peptides) by integrating MS-cleavable cross-linkers with isobaric labeling reagents. This study has established a new analytical platform for quantitative analysis of cross-linked peptides, which can be directly applied for multiplexed comparisons of the conformational dynamics of protein complexes and PPIs at the proteome scale in future studies.

The advancement of chemical cross-linking and mass spectrometry for structural proteomics: from single proteins to protein interaction networks.

PubMed

Sinz, Andrea

2014-12-01

During the last 15 years, chemical cross-linking combined with mass spectrometry (MS) and computational modeling has advanced from investigating 3D-structures of isolated proteins to deciphering protein interaction networks. In this article, the author discusses the advent, the development and the current status of the chemical cross-linking/MS strategy in the context of recent technological developments. A direct way to probe in vivo protein-protein interactions is by site-specific incorporation of genetically encoded photo-reactive amino acids or by non-directed incorporation of photo-reactive amino acids. As the chemical cross-linking/MS approach allows the capture of transient and weak interactions, it has the potential to become a routine technique for unraveling protein interaction networks in their natural cellular environment.

The antigenic determinants on HIV p24 for CD4+ T cell inhibiting antibodies as determined by limited proteolysis, chemical modification, and mass spectrometry.

PubMed

Williams, Jason G; Tomer, Kenneth B; Hioe, Catarina E; Zolla-Pazner, Susan; Norris, Philip J

2006-11-01

In the last decade, mass spectrometry has been employed by more and more researchers for identifying the proteins in a macromolecular complex as well as for defining the surfaces of their binding interfaces. This characterization of protein-protein interfaces usually involves at least one of several different methodologies in addition to the actual mass spectrometry. For example, limited proteolysis is often used as a first step in defining regions of a protein that are protected from proteolysis when the protein of interest is part of a macromolecular complex. Other techniques used in conjunction with mass spectrometry for determining regions of a protein involved in protein-protein interactions include chemical modification, such as covalent cross-linking, acetylation of lysines, hydrogen-deuterium exchange, or other forms of modification. In this report, both limited proteolysis and chemical modification were combined with several mass spectrometric techniques in efforts to define the protein surface on the HIV core protein, p24, recognized by two different monoclonal human antibodies that were isolated from HIV+ patients. One of these antibodies, 1571, strongly inhibits the CD4+ T cell proliferative response to a known epitope (PEVIPMFSALSEGATP), while the other antibody, 241-D, does not inhibit as strongly. The epitopes for both of these antibodies were determined to be discontinuous and localized to the N-terminus of p24. Interestingly, the epitope recognized by the strongly inhibiting antibody, 1571, completely overlaps the T cell epitope PEVIPMFSALSEGATP, while the antibody 241-D binds to a region adjacent to the region of p24 recognized by the antibody 1571. These results suggest that, possibly due to epitope competition, antibodies produced during HIV infection can negatively affect CD4+ T cell-mediated immunity against the virus.

Evolutionarily Conserved Linkage between Enzyme Fold, Flexibility, and Catalysis

PubMed Central

Ramanathan, Arvind; Agarwal, Pratul K.

2011-01-01

Proteins are intrinsically flexible molecules. The role of internal motions in a protein's designated function is widely debated. The role of protein structure in enzyme catalysis is well established, and conservation of structural features provides vital clues to their role in function. Recently, it has been proposed that the protein function may involve multiple conformations: the observed deviations are not random thermodynamic fluctuations; rather, flexibility may be closely linked to protein function, including enzyme catalysis. We hypothesize that the argument of conservation of important structural features can also be extended to identification of protein flexibility in interconnection with enzyme function. Three classes of enzymes (prolyl-peptidyl isomerase, oxidoreductase, and nuclease) that catalyze diverse chemical reactions have been examined using detailed computational modeling. For each class, the identification and characterization of the internal protein motions coupled to the chemical step in enzyme mechanisms in multiple species show identical enzyme conformational fluctuations. In addition to the active-site residues, motions of protein surface loop regions (>10 Å away) are observed to be identical across species, and networks of conserved interactions/residues connect these highly flexible surface regions to the active-site residues that make direct contact with substrates. More interestingly, examination of reaction-coupled motions in non-homologous enzyme systems (with no structural or sequence similarity) that catalyze the same biochemical reaction shows motions that induce remarkably similar changes in the enzyme–substrate interactions during catalysis. The results indicate that the reaction-coupled flexibility is a conserved aspect of the enzyme molecular architecture. Protein motions in distal areas of homologous and non-homologous enzyme systems mediate similar changes in the active-site enzyme–substrate interactions, thereby impacting the mechanism of catalyzed chemistry. These results have implications for understanding the mechanism of allostery, and for protein engineering and drug design. PMID:22087074

Evolutionarily conserved linkage between enzyme fold, flexibility, and catalysis.

PubMed

Ramanathan, Arvind; Agarwal, Pratul K

2011-11-01

Proteins are intrinsically flexible molecules. The role of internal motions in a protein's designated function is widely debated. The role of protein structure in enzyme catalysis is well established, and conservation of structural features provides vital clues to their role in function. Recently, it has been proposed that the protein function may involve multiple conformations: the observed deviations are not random thermodynamic fluctuations; rather, flexibility may be closely linked to protein function, including enzyme catalysis. We hypothesize that the argument of conservation of important structural features can also be extended to identification of protein flexibility in interconnection with enzyme function. Three classes of enzymes (prolyl-peptidyl isomerase, oxidoreductase, and nuclease) that catalyze diverse chemical reactions have been examined using detailed computational modeling. For each class, the identification and characterization of the internal protein motions coupled to the chemical step in enzyme mechanisms in multiple species show identical enzyme conformational fluctuations. In addition to the active-site residues, motions of protein surface loop regions (>10 Å away) are observed to be identical across species, and networks of conserved interactions/residues connect these highly flexible surface regions to the active-site residues that make direct contact with substrates. More interestingly, examination of reaction-coupled motions in non-homologous enzyme systems (with no structural or sequence similarity) that catalyze the same biochemical reaction shows motions that induce remarkably similar changes in the enzyme-substrate interactions during catalysis. The results indicate that the reaction-coupled flexibility is a conserved aspect of the enzyme molecular architecture. Protein motions in distal areas of homologous and non-homologous enzyme systems mediate similar changes in the active-site enzyme-substrate interactions, thereby impacting the mechanism of catalyzed chemistry. These results have implications for understanding the mechanism of allostery, and for protein engineering and drug design.

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

Ramanathan, Arvind; Agarwal, Pratul K

Proteins are intrinsically flexible molecules. The role of internal motions in a protein's designated function is widely debated. The role of protein structure in enzyme catalysis is well established, and conservation of structural features provides vital clues to their role in function. Recently, it has been proposed that the protein function may involve multiple conformations: the observed deviations are not random thermodynamic fluctuations; rather, flexibility may be closely linked to protein function, including enzyme catalysis. We hypothesize that the argument of conservation of important structural features can also be extended to identification of protein flexibility in interconnection with enzyme function.more » Three classes of enzymes (prolyl-peptidyl isomerase, oxidoreductase, and nuclease) that catalyze diverse chemical reactions have been examined using detailed computational modeling. For each class, the identification and characterization of the internal protein motions coupled to the chemical step in enzyme mechanisms in multiple species show identical enzyme conformational fluctuations. In addition to the active-site residues, motions of protein surface loop regions (>10 away) are observed to be identical across species, and networks of conserved interactions/residues connect these highly flexible surface regions to the active-site residues that make direct contact with substrates. More interestingly, examination of reaction-coupled motions in non-homologous enzyme systems (with no structural or sequence similarity) that catalyze the same biochemical reaction shows motions that induce remarkably similar changes in the enzyme substrate interactions during catalysis. The results indicate that the reaction-coupled flexibility is a conserved aspect of the enzyme molecular architecture. Protein motions in distal areas of homologous and non-homologous enzyme systems mediate similar changes in the active-site enzyme substrate interactions, thereby impacting the mechanism of catalyzed chemistry. These results have implications for understanding the mechanism of allostery, and for protein engineering and drug design.« less

Sensitive and simultaneous surface plasmon resonance detection of free and p53-bound MDM2 proteins from human sarcomas.

PubMed

Wu, Ling; Tang, Hailin; Hu, Shengqiang; Xia, Yonghong; Lu, Zhixuan; Fan, Yujuan; Wang, Zixiao; Yi, Xinyao; Zhou, Feimeng; Wang, Jianxiu

2018-04-30

Murine double minute 2 (MDM2) is an oncoprotein mediating the degradation of the tumor suppressor p53 protein. The physiological levels of MDM2 protein are closely related to malignant transformation and tumor growth. In this work, the simultaneous and label-free determination of free and p53-bound MDM2 proteins from sarcoma tissue extracts was conducted using a dual-channel surface plasmon resonance (SPR) instrument. Free MDM2 protein was measured in one fluidic channel covered with the consensus double-stranded (ds)-DNA/p53 conjugate, while MDM2 bound to p53 was captured by the consensus ds-DNA immobilized onto the other channel. To achieve higher sensitivity and to confirm specificity, an MDM2-specific monoclonal antibody (2A10) was used to recognize both the free and p53-bound MDM2 proteins. The resultant method afforded a detection limit of 0.55 pM of MDM2. The amenability of the method to the analysis of free and p53-bound MDM2 proteins was demonstrated for normal and sarcoma tissue extracts from three patients. Our data reveal that both free and total MDM2 (free and bound forms combined) proteins from sarcoma tissue extracts are of much higher concentrations than those from normal tissue extracts and the p53-bound MDM2 protein only constitutes a small fraction of the total MDM2 concentration. In comparison with enzyme-linked immunosorbent assay (ELISA), the proposed method possesses higher sensitivity, is more cost-effective, and is capable of determining free and p53-bound MDM2 proteins in clinical samples.

Application of a fast sorting algorithm to the assignment of mass spectrometric cross-linking data.

PubMed

Petrotchenko, Evgeniy V; Borchers, Christoph H

2014-09-01

Cross-linking combined with MS involves enzymatic digestion of cross-linked proteins and identifying cross-linked peptides. Assignment of cross-linked peptide masses requires a search of all possible binary combinations of peptides from the cross-linked proteins' sequences, which becomes impractical with increasing complexity of the protein system and/or if digestion enzyme specificity is relaxed. Here, we describe the application of a fast sorting algorithm to search large sequence databases for cross-linked peptide assignments based on mass. This same algorithm has been used previously for assigning disulfide-bridged peptides (Choi et al., ), but has not previously been applied to cross-linking studies. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

A novel nanoemulsion-based method to produce ultrasmall, water-dispersible nanoparticles from chitosan, surface modified with cell-penetrating peptide for oral delivery of proteins and peptides

PubMed Central

Barbari, Ghullam Reza; Dorkoosh, Farid Abedin; Amini, Mohsen; Sharifzadeh, Mohammad; Atyabi, Fateme; Balalaie, Saeed; Rafiee Tehrani, Niyousha; Rafiee Tehrani, Morteza

2017-01-01

A simple and reproducible water-in-oil (W/O) nanoemulsion technique for making ultrasmall (<15 nm), monodispersed and water-dispersible nanoparticles (NPs) from chitosan (CS) is reported. The nano-sized (50 nm) water pools of the W/O nanoemulsion serve as “nano-containers and nano-reactors”. The entrapped polymer chains of CS inside these “nano-reactors” are covalently cross-linked with the chains of polyethylene glycol (PEG), leading to rigidification and formation of NPs. These NPs possess excessive swelling properties in aqueous medium and preserve integrity in all pH ranges due to chemical cross-linking with PEG. A potent and newly developed cell-penetrating peptide (CPP) is further chemically conjugated to the surface of the NPs, leading to development of a novel peptide-conjugated derivative of CS with profound tight-junction opening properties. The CPP-conjugated NPs can easily be loaded with almost all kinds of proteins, peptides and nucleotides for oral delivery applications. Feasibility of this nanoparticulate system for oral delivery of a model peptide (insulin) is investigated in Caco-2 cell line. The cell culture results for translocation of insulin across the cell monolayer are very promising (15%–19% increase), and animal studies are actively under progress and will be published separately. PMID:28496323

Nanoswitch-linked immunosorbent assay (NLISA) for fast, sensitive, and specific protein detection.

PubMed

Hansen, Clinton H; Yang, Darren; Koussa, Mounir A; Wong, Wesley P

2017-09-26

Protein detection and quantification play critical roles in both basic research and clinical practice. Current detection platforms range from the widely used ELISA to more sophisticated, and more expensive, approaches such as digital ELISA. Despite advances, there remains a need for a method that combines the simplicity and cost-effectiveness of ELISA with the sensitivity and speed of modern approaches in a format suitable for both laboratory and rapid, point-of-care applications. Building on recent developments in DNA structural nanotechnology, we introduce the nanoswitch-linked immunosorbent assay (NLISA), a detection platform based on easily constructed DNA nanodevices that change conformation upon binding to a target protein with the results read out by gel electrophoresis. NLISA is surface-free and includes a kinetic-proofreading step for purification, enabling both enhanced sensitivity and reduced cross-reactivity. We demonstrate femtomolar-level detection of prostate-specific antigen in biological fluids, as well as reduced cross-reactivity between different serotypes of dengue and also between a single-mutation and wild-type protein. NLISA is less expensive, uses less sample volume, is more rapid, and, with no washes, includes fewer hands-on steps than ELISA, while also achieving superior sensitivity. Our approach also has the potential to enable rapid point-of-care assays, as we demonstrate by performing NLISA with an iPad/iPhone camera for imaging.

Highly efficient antibody immobilization with multimeric protein Gs coupled magnetic silica nanoparticles

NASA Astrophysics Data System (ADS)

Lee, J. H.; Choi, H. K.; Chang, J. H.

2011-10-01

This work reports the immobilization of monomeric, dimeric and trimer protein Gs onto silica magnetic nanoparticles for self-oriented antibody immobilization. To achieve this, we initially prepared the silica-coated magnetic nanoparticle having about 170 nm diameters. The surface of the silica coated magnetic nanoparticles was modified with 3- aminopropyl-trimethoxysilane (APTMS) to chemically link to multimeric protein Gs. The conjugation of amino groups on the SiO2-MNPs to cysteine tagged in multimeric protein Gs was performed using a sulfo-SMCC coupling procedure. The binding efficiencies of monomer, dimer and trimer were 77 %, 67 % and 55 % respectively. However, the efficiencies of antibody immobilization were 70 %, 83 % and 95 % for monomeric, dimeric and trimeric protein G, respectively. To prove the enhancement of accessibility by using multimeric protein G, FITC labeled goat-anti-mouse IgG was treated to mouse IgG immobilized magnetic silica nanoparticles through multimeric protein G. FITC labeled goat anti-mouse IgGs were more easily bound to mouse IgG immobilized by trimeric protein G than others. Finally protein G bound silica magnetic nanoparticles were utilized to develop highly sensitive immunoassay to detect hepatitis B antigen.

Probing structures of large protein complexes using zero-length cross-linking.

PubMed

Rivera-Santiago, Roland F; Sriswasdi, Sira; Harper, Sandra L; Speicher, David W

2015-11-01

Structural mass spectrometry (MS) is a field with growing applicability for addressing complex biophysical questions regarding proteins and protein complexes. One of the major structural MS approaches involves the use of chemical cross-linking coupled with MS analysis (CX-MS) to identify proximal sites within macromolecules. Identified cross-linked sites can be used to probe novel protein-protein interactions or the derived distance constraints can be used to verify and refine molecular models. This review focuses on recent advances of "zero-length" cross-linking. Zero-length cross-linking reagents do not add any atoms to the cross-linked species due to the lack of a spacer arm. This provides a major advantage in the form of providing more precise distance constraints as the cross-linkable groups must be within salt bridge distances in order to react. However, identification of cross-linked peptides using these reagents presents unique challenges. We discuss recent efforts by our group to minimize these challenges by using multiple cycles of LC-MS/MS analysis and software specifically developed and optimized for identification of zero-length cross-linked peptides. Representative data utilizing our current protocol are presented and discussed. Copyright © 2015 Elsevier Inc. All rights reserved.

Surface Display of the Receptor-Binding Region of the Lactobacillus brevis S-Layer Protein in Lactococcus lactis Provides Nonadhesive Lactococci with the Ability To Adhere to Intestinal Epithelial Cells

PubMed Central

Åvall-Jääskeläinen, Silja; Lindholm, Agneta; Palva, Airi

2003-01-01

Lactobacillus brevis is a promising lactic acid bacterium for use as a probiotic dietary adjunct and a vaccine vector. The N-terminal region of the S-layer protein (SlpA) of L. brevis ATCC 8287 was recently shown to mediate adhesion to various human cell lines in vitro. In this study, a surface display cassette was constructed on the basis of this SlpA receptor-binding domain, a proteinase spacer, and an autolysin anchor. The cassette was expressed under control of the nisA promoter in Lactococcus lactis NZ9000. Western blot assay of lactococcal cell wall extracts with anti-SlpA antibodies confirmed that the SlpA adhesion domain of the fusion protein was expressed and located within the cell wall layer. Whole-cell enzyme-linked immunosorbent assay and immunofluorescence microscopy verified that the SlpA adhesion-mediating region was accessible on the lactococcal cell surface. In vitro adhesion assays with the human intestinal epithelial cell line Intestine 407 indicated that the recombinant lactococcal cells had gained an ability to adhere to Intestine 407 cells significantly greater than that of wild-type L. lactis NZ9000. Serum inhibition assay further confirmed that adhesion of recombinant lactococci to Intestine 407 cells was indeed mediated by the N terminus-encoding part of the slpA gene. The ability of the receptor-binding region of SlpA to adhere to fibronectin was also confirmed with this lactococcal surface display system. These results show that, with the aid of the receptor-binding region of the L. brevis SlpA protein, the ability to adhere to gut epithelial cells can indeed be transferred to another, nonadhesive, lactic acid bacterium. PMID:12676705

Surface display of the receptor-binding region of the Lactobacillus brevis S-layer protein in Lactococcus lactis provides nonadhesive lactococci with the ability to adhere to intestinal epithelial cells.

PubMed

Avall-Jääskeläinen, Silja; Lindholm, Agneta; Palva, Airi

2003-04-01

Lactobacillus brevis is a promising lactic acid bacterium for use as a probiotic dietary adjunct and a vaccine vector. The N-terminal region of the S-layer protein (SlpA) of L. brevis ATCC 8287 was recently shown to mediate adhesion to various human cell lines in vitro. In this study, a surface display cassette was constructed on the basis of this SlpA receptor-binding domain, a proteinase spacer, and an autolysin anchor. The cassette was expressed under control of the nisA promoter in Lactococcus lactis NZ9000. Western blot assay of lactococcal cell wall extracts with anti-SlpA antibodies confirmed that the SlpA adhesion domain of the fusion protein was expressed and located within the cell wall layer. Whole-cell enzyme-linked immunosorbent assay and immunofluorescence microscopy verified that the SlpA adhesion-mediating region was accessible on the lactococcal cell surface. In vitro adhesion assays with the human intestinal epithelial cell line Intestine 407 indicated that the recombinant lactococcal cells had gained an ability to adhere to Intestine 407 cells significantly greater than that of wild-type L. lactis NZ9000. Serum inhibition assay further confirmed that adhesion of recombinant lactococci to Intestine 407 cells was indeed mediated by the N terminus-encoding part of the slpA gene. The ability of the receptor-binding region of SlpA to adhere to fibronectin was also confirmed with this lactococcal surface display system. These results show that, with the aid of the receptor-binding region of the L. brevis SlpA protein, the ability to adhere to gut epithelial cells can indeed be transferred to another, nonadhesive, lactic acid bacterium.

G-quadruplex aptamer targeting Protein A and its capability to detect Staphylococcus aureus demonstrated by ELONA.

PubMed

Stoltenburg, Regina; Krafčiková, Petra; Víglaský, Viktor; Strehlitz, Beate

2016-09-21

Aptamers for whole cell detection are selected mostly by the Cell-SELEX procedure. Alternatively, the use of specific cell surface epitopes as target during aptamer selections allows the development of aptamers with ability to bind whole cells. In this study, we integrated a formerly selected Protein A-binding aptamer PA#2/8 in an assay format called ELONA (Enzyme-Linked OligoNucleotide Assay) and evaluated the ability of the aptamer to recognise and bind to Staphylococcus aureus presenting Protein A on the cell surface. The full-length aptamer and one of its truncated variants could be demonstrated to specifically bind to Protein A-expressing intact cells of S. aureus, and thus have the potential to expand the portfolio of aptamers that can act as an analytical agent for the specific recognition and rapid detection of the bacterial pathogen. The functionality of the aptamer was found to be based on a very complex, but also highly variable structure. Two structural key elements were identified. The aptamer sequence contains several G-clusters allowing folding into a G-quadruplex structure with the potential of dimeric and multimeric assembly. An inverted repeat able to form an imperfect stem-loop at the 5'-end also contributes essentially to the aptameric function.

G-quadruplex aptamer targeting Protein A and its capability to detect Staphylococcus aureus demonstrated by ELONA

PubMed Central

Stoltenburg, Regina; Krafčiková, Petra; Víglaský, Viktor; Strehlitz, Beate

2016-01-01

Aptamers for whole cell detection are selected mostly by the Cell-SELEX procedure. Alternatively, the use of specific cell surface epitopes as target during aptamer selections allows the development of aptamers with ability to bind whole cells. In this study, we integrated a formerly selected Protein A-binding aptamer PA#2/8 in an assay format called ELONA (Enzyme-Linked OligoNucleotide Assay) and evaluated the ability of the aptamer to recognise and bind to Staphylococcus aureus presenting Protein A on the cell surface. The full-length aptamer and one of its truncated variants could be demonstrated to specifically bind to Protein A-expressing intact cells of S. aureus, and thus have the potential to expand the portfolio of aptamers that can act as an analytical agent for the specific recognition and rapid detection of the bacterial pathogen. The functionality of the aptamer was found to be based on a very complex, but also highly variable structure. Two structural key elements were identified. The aptamer sequence contains several G-clusters allowing folding into a G-quadruplex structure with the potential of dimeric and multimeric assembly. An inverted repeat able to form an imperfect stem-loop at the 5′-end also contributes essentially to the aptameric function. PMID:27650576

Imaging and three-dimensional reconstruction of chemical groups inside a protein complex using atomic force microscopy

NASA Astrophysics Data System (ADS)

Kim, Duckhoe; Sahin, Ozgur

2015-03-01

Scanning probe microscopes can be used to image and chemically characterize surfaces down to the atomic scale. However, the localized tip-sample interactions in scanning probe microscopes limit high-resolution images to the topmost atomic layer of surfaces, and characterizing the inner structures of materials and biomolecules is a challenge for such instruments. Here, we show that an atomic force microscope can be used to image and three-dimensionally reconstruct chemical groups inside a protein complex. We use short single-stranded DNAs as imaging labels that are linked to target regions inside a protein complex, and T-shaped atomic force microscope cantilevers functionalized with complementary probe DNAs allow the labels to be located with sequence specificity and subnanometre resolution. After measuring pairwise distances between labels, we reconstruct the three-dimensional structure formed by the target chemical groups within the protein complex using simple geometric calculations. Experiments with the biotin-streptavidin complex show that the predicted three-dimensional loci of the carboxylic acid groups of biotins are within 2 Å of their respective loci in the corresponding crystal structure, suggesting that scanning probe microscopes could complement existing structural biological techniques in solving structures that are difficult to study due to their size and complexity.

Surface dynamics of aerolysin on the plasma membrane of living cells.

PubMed

Abrami, L; Fivaz, M; van der Goot, F G

2000-10-01

Aerolysin secreted by the human pathogen Aeromonas hydrophila belongs to a group of bacterial toxins that are hemolytic and form channels in biological membranes. The toxin is secreted as an inactive precursor proaerolysin that must be proteolytically processed at its C-terminus to become active. The toxin then polymerizes into a heptameric ring that is amphipathic and can insert into a lipid bilayer and form a pore. We have examined these various steps at the surface of target cells. The toxin binds to specific receptors. Various receptors have been identified, all of which are anchored to the plasma membrane via a glycosylphosphatidyl inositol (GPI)-anchored moiety. The GPI anchor confers to the protein that is linked to it two usual properties: (i) the protein has a higher lateral mobility in a phospholipid bilayer than its transmembrane counterpart, (ii) the protein has the capacity to transiently associate with cholesterol-glycosphingolipid-rich microdomains. We have shown that both these properties of GPI-anchored proteins are exploited by proaerolysin bound to its receptor. The high lateral mobility within the phosphoglyceride region of the plasma membrane favors the encounter of the protoxin with its converting enzyme furin. The ability to associate with microdomains on the other hand favors the oligomerization process presumably by concentrating the toxin locally.

Protein adsorption and cell adhesion on nanoscale bioactive coatings formed from poly(ethylene glycol) and albumin microgels

PubMed Central

Scott, Evan A.; Nichols, Michael D.; Cordova, Lee H.; George, Brandon J.; Jun, Young-Shin; Elbert, Donald L.

2008-01-01

Late-term thrombosis on drug-eluting stents is an emerging problem that might be addressed using extremely thin, biologically-active hydrogel coatings. We report a dip-coating strategy to covalently link poly(ethylene glycol) (PEG) to substrates, producing coatings with <≈100 nm thickness. Gelation of PEG-octavinylsulfone with amines in either bovine serum albumin (BSA) or PEG-octaamine was monitored by dynamic light scattering (DLS), revealing the presence of microgels before macrogelation. NMR also revealed extremely high end group conversions prior to macrogelation, consistent with the formation of highly crosslinked microgels and deviation from Flory-Stockmayer theory. Before macrogelation, the reacting solutions were diluted and incubated with nucleophile-functionalized surfaces. Using optical waveguide lightmode spectroscopy (OWLS) and quartz crystal microbalance with dissipation (QCM-D), we identified a highly hydrated, protein-resistant layer with a thickness of approximately 75 nm. Atomic force microscopy in buffered water revealed the presence of coalesced spheres of various sizes but with diameters less than about 100 nm. Microgel-coated glass or poly(ethylene terephthalate) exhibited reduced protein adsorption and cell adhesion. Cellular interactions with the surface could be controlled by using different proteins to cap unreacted vinylsulfone groups within the coating. PMID:18771802

Extending the cross-linking/mass spectrometry strategy: Facile incorporation of photo-activatable amino acids into the model protein calmodulin in Escherichia coli cells.

PubMed

Piotrowski, Christine; Ihling, Christian H; Sinz, Andrea

2015-11-01

Photo-induced cross-linking is a highly promising technique to investigate protein conformations and protein-protein interactions in their natural cellular environment. One strategy relies on the non-directed incorporation of diazirine-containing photo-activatable amino acids into proteins and a subsequent cross-link formation induced by UV-A irradiation. The advantage of this photo-cross-linking strategy is that it is not restricted to lysine residues and that hydrophobic regions in proteins can also be targeted, which is advantageous for investigating membrane proteins. Here, we present a simplified protocol that relies on the use of mineral salts medium without any special requirements for the incorporation of photo-methionines into proteins in Escherichia coli cells. The possibility to perform these experiments in E. coli is especially valuable as it is the major system for recombinant protein production. The method is exemplified for the Ca(2+) regulating protein calmodulin containing nine methionines, which were found to be replaced by their photo-activatable analogues. Our protocol allows the facile and stochastic incorporation of photo-methionines as the basis for conducting photo-cross-linking experiments in E. coli in an efficient manner. Copyright © 2015 Elsevier Inc. All rights reserved.

Characterization of hyaluronate binding proteins isolated from 3T3 and murine sarcoma virus transformed 3T3 cells

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

Turley, E.A.; Moore, D.; Hayden, L.J.

1987-06-02

A hyaluronic acid binding fraction was purified from the supernatant media of both 3T3 and murine sarcoma virus (MSV) transformed 3T3 cultures by hyaluronate and immunoaffinity chromatography. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis resolved the hyaluronate affinity-purified fraction into three major protein bands of estimated molecular weight (M/sub r,e/) 70K, 66K, and 56K which contained hyaluronate binding activity and which were termed hyaluronate binding proteins (HABP). Hyaluronate affinity chromatography combined with immunoaffinity chromatography, using antibody directed against the larger HABP, allowed a 20-fold purification of HABP. Fractions isolated from 3T3 supernatant medium also contained additional binding molecules in the molecular weightmore » range of 20K. This material was present in vanishingly small amounts and was not detected with a silver stain or with (/sup 35/S)methionine label. The three protein species isolated by hyaluronate affinity chromatography (M/sub r,e/ 70K, 66K, and 56K) were related to one another since they shared antigenic determinants and exhibited similar pI values. In isocratic conditions, HABP occurred as aggregates of up to 580 kilodaltons. Their glycoprotein nature was indicated by their incorporation of /sup 3/H-sugars. Enzyme-linked immunoadsorbent assay showed they were antigenically distinct from other hyaluronate binding proteins such as fibronectin, cartilage link protein, and the hyaluronate binding region of chondroitin sulfate proteoglycan. The results are discussed with regard both to the functional significance of hyaluronate-cell surface interactions in transformed as well as normal cells and to the relationship of HABP to other reported hyaluronate binding proteins.« less

Top surface blade residues and the central channel water molecules are conserved in every repeat of the integrin-like β-propeller structures.

PubMed

Denesyuk, Alexander; Denessiouk, Konstantin; Johnson, Mark S

2018-02-01

An integrin-like β-propeller domain contains seven repeats of a four-stranded antiparallel β-sheet motif (blades). Previously we described a 3D structural motif within each blade of the integrin-type β-propeller. Here, we show unique structural links that join different blades of the β-propeller structure, which together with the structural motif for a single blade are repeated in a β-propeller to provide the functional top face of the barrel, found to be involved in protein-protein interactions and substrate recognition. We compare functional top face diagrams of the integrin-type β-propeller domain and two non-integrin type β-propeller domains of virginiamycin B lyase and WD Repeat-Containing Protein 5. Copyright © 2017 Elsevier Inc. All rights reserved.

RBscore&NBench: a high-level web server for nucleic acid binding residues prediction with a large-scale benchmarking database.

PubMed

Miao, Zhichao; Westhof, Eric

2016-07-08

RBscore&NBench combines a web server, RBscore and a database, NBench. RBscore predicts RNA-/DNA-binding residues in proteins and visualizes the prediction scores and features on protein structures. The scoring scheme of RBscore directly links feature values to nucleic acid binding probabilities and illustrates the nucleic acid binding energy funnel on the protein surface. To avoid dataset, binding site definition and assessment metric biases, we compared RBscore with 18 web servers and 3 stand-alone programs on 41 datasets, which demonstrated the high and stable accuracy of RBscore. A comprehensive comparison led us to develop a benchmark database named NBench. The web server is available on: http://ahsoka.u-strasbg.fr/rbscorenbench/. © The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

A multicomponent complex is required for the AAUAAA-dependent cross-linking of a 64-kilodalton protein to polyadenylation substrates.

PubMed Central

Wilusz, J; Shenk, T; Takagaki, Y; Manley, J L

1990-01-01

A 64-kilodalton (kDa) polypeptide that is cross-linked by UV light specifically to polyadenylation substrate RNAs containing a functional AAUAAA element has been identified previously. Fractionated HeLa nuclear components that can be combined to regenerate efficient and accurate polyadenylation in vitro have now been screened for the presence of the 64-kDa protein. None of the individual components contained an activity which could generate the 64-kDa species upon UV cross-linking in the presence of substrate RNA. It was necessary to mix two components, cleavage stimulation factor and specificity factor, to reconstitute 64-kDa protein-RNA cross-linking. The addition of cleavage factors to this mixture very efficiently reconstituted the AAUAAA-specific 64-kDa protein-RNA interaction. The 64-kDa protein, therefore, is present in highly purified, reconstituted polyadenylation reactions. However, it is necessary to form a multicomponent complex to efficiently cross-link the protein to a substrate RNA. Images PMID:2304466

γ-PGA and MTGase improve the formation of ε-(γ-glutamyl) lysine cross-links within hairtail (Trichiurus haumela) surimi protein.

PubMed

Hu, Yaqin; Shao, Ying; Wu, Chunhua; Yuan, Chunhong; Ishimura, Gakushi; Liu, Wenjuan; Chen, Shiguo

2018-03-01

The present study investigated the mechanism of ε-(γ-glutamyl) lysine cross-links within hairtail (Trichiurus haumela) surimi protein via γ-polyglutamic acid (γ-PGA) and MTGase. The results indicated that the addition of MTGase and γ-PGA markedly improved the gelation properties of hairtail surimi protein, including its maximum breaking force and deformation, water holding capacity and gel strength. The maximum improvements were achieved by adding 0.5units MTGase/g meat paste in combination with 0.06% γ-PGA. SDS-PAGE showed that the band intensity of cross-linked proteins increased, whereas that of myosin heavy chain decreased after treatments. Further scanning electron microscopy (SEM) analysis showed the formation of a denser gel matrix, which was caused by much stronger and more inter- and intra-molecular cross-linking of proteins, via MTGase catalysing ε-(γ-glutamyl) lysine cross-links formed between lysine residues in the gel protein and glutamic residues in the hydrolytic γ-PGA. The results provide reliable guidance for the improvement of hairtail surimi protein gelation properties. Copyright © 2017 Elsevier Ltd. All rights reserved.

Network organization of the human autophagy system.

PubMed

Behrends, Christian; Sowa, Mathew E; Gygi, Steven P; Harper, J Wade

2010-07-01

Autophagy, the process by which proteins and organelles are sequestered in autophagosomal vesicles and delivered to the lysosome/vacuole for degradation, provides a primary route for turnover of stable and defective cellular proteins. Defects in this system are linked with numerous human diseases. Although conserved protein kinase, lipid kinase and ubiquitin-like protein conjugation subnetworks controlling autophagosome formation and cargo recruitment have been defined, our understanding of the global organization of this system is limited. Here we report a proteomic analysis of the autophagy interaction network in human cells under conditions of ongoing (basal) autophagy, revealing a network of 751 interactions among 409 candidate interacting proteins with extensive connectivity among subnetworks. Many new autophagy interaction network components have roles in vesicle trafficking, protein or lipid phosphorylation and protein ubiquitination, and affect autophagosome number or flux when depleted by RNA interference. The six ATG8 orthologues in humans (MAP1LC3/GABARAP proteins) interact with a cohort of 67 proteins, with extensive binding partner overlap between family members, and frequent involvement of a conserved surface on ATG8 proteins known to interact with LC3-interacting regions in partner proteins. These studies provide a global view of the mammalian autophagy interaction landscape and a resource for mechanistic analysis of this critical protein homeostasis pathway.

The role of the C-domain of bacteriophage T4 gene 32 protein in ssDNA binding and dsDNA helix-destabilization: Kinetic, single-molecule, and cross-linking studies

PubMed Central

Pant, Kiran; Anderson, Brian; Perdana, Hendrik; Malinowski, Matthew A.; Win, Aye T.; Williams, Mark C.

2018-01-01

The model single-stranded DNA binding protein of bacteriophage T4, gene 32 protein (gp32) has well-established roles in DNA replication, recombination, and repair. gp32 is a single-chain polypeptide consisting of three domains. Based on thermodynamics and kinetics measurements, we have proposed that gp32 can undergo a conformational change where the acidic C-terminal domain binds internally to or near the single-stranded (ss) DNA binding surface in the core (central) domain, blocking ssDNA interaction. To test this model, we have employed a variety of experimental approaches and gp32 variants to characterize this conformational change. Utilizing stopped-flow methods, the association kinetics of wild type and truncated forms of gp32 with ssDNA were measured. When the C-domain is present, the log-log plot of k vs. [NaCl] shows a positive slope, whereas when it is absent (*I protein), there is little rate change with salt concentration, as expected for this model.A gp32 variant lacking residues 292–296 within the C-domain, ΔPR201, displays kinetic properties intermediate between gp32 and *I. The single molecule force-induced DNA helix-destabilizing activitiesas well as the single- and double-stranded DNA affinities of ΔPR201 and gp32 truncated at residue 295 also fall between full-length protein and *I. Finally, chemical cross-linking of recombinant C-domain and gp32 lacking both N- and C-terminal domains is inhibited by increasing concentrations of a short single-stranded oligonucleotide, and the salt dependence of cross-linking mirrors that expected for the model. Taken together, these results provide the first evidence in support of this model that have been obtained through structural probes. PMID:29634784

Analysis of glycation induced protein cross-linking inhibitory effects of some antidiabetic plants and spices.

PubMed

Perera, Handunge Kumudu Irani; Handuwalage, Charith Sandaruwan

2015-06-09

Protein cross-linking which occurs towards the latter part of protein glycation is implicated in the development of chronic diabetic complications. Glycation induced protein cross-linking inhibitory effects of nine antidiabetic plants and three spices were evaluated in this study using a novel, simple, electrophoresis based method. Methanol extracts of thirteen plants including nine antidiabetic plants and three spices were used. Lysozyme and fructose were incubated at 37 °C in the presence or absence of different concentrations of plant extracts up to 31 days. Standard glycation inhibitor aminoguanidine and other appropriate controls were included. A recently established sodium dodecyl polyacrylamide gel electrophoresis (SDS-PAGE) method was used to detect the products of protein cross-linking in the incubation mixtures. High molecular weight protein products representing the dimer, trimer and tetramer of lysozyme were detected in the presence of fructose. Among the nine antidiabetic plants, seven showed glycation induced protein cross-linking inhibitory effects namely Ficus racemosa (FR) stem bark, Gymnema sylvestre (GS) leaves, Musa paradisiaca (MP) yam, Phyllanthus debilis (PD) whole plant, Phyllanthus emblica (PE) fruit, Pterocarpus marsupium (PM) latex and Tinospora cordifolia (TC) leaves. Inhibition observed with Coccinia grandis (CG) leaves and Strychnos potatorum (SP) seeds were much low. Leaves of Gymnema lactiferum (GL), the plant without known antidiabetic effects showed the lowest inhibition. All three spices namely Coriandrum sativum (CS) seeds, Cinnamomum zeylanicum (CZ) bark and Syzygium aromaticum (SA) flower buds showed cross-link inhibitory effects with higher effects in CS and SA. PD, PE, PM, CS and SA showed almost complete inhibition on the formation of cross-linking with 25 μg/ml extracts. Methanol extracts of PD, PE, PM, CS and SA have shown promising inhibitory effects on glycation induced protein cross-linking.

Structural interactions between retroviral Gag proteins examined by cysteine cross-linking.

PubMed Central

Hansen, M S; Barklis, E

1995-01-01

We have examined structural interactions between Gag proteins within Moloney murine leukemia virus (M-MuLV) particles by making use of the cysteine-specific cross-linking agents iodine and bis-maleimido hexane. Virion-associated wild-type M-MuLV Pr65Gag proteins in immature particles were intermolecularly cross-linked at cysteines to form Pr65Gag oligomers, from dimers to pentamers or hexamers. Following a systematic approach of cysteine-to-serine mutagenesis, we have shown that cross-linking of Pr65Gag occurred at cysteines of the nucleocapsid (NC) Cys-His motif, suggesting that the Cys-His motifs within virus particles are packed in close proximity. The M-MuLV Pr65Gag protein did not cross-link to the human immunodeficiency virus Pr55Gag protein when the two molecules were coexpressed, indicating either that they did not coassemble or that heterologous Gag proteins were not in close enough proximity to be cross-linked. Using an assembly-competent, protease-minus, cysteine-minus Pr65Gag protein as a template, novel cysteine residues were generated in the M-MuLV capsid domain major homology region (MHR). Cross-linking of proteins containing MHR cysteines showed above-background levels of Gag-Gag dimers but also identified a novel cellular factor, present in virions, that cross-linked to MHR residues. Although the NC cysteine mutation was compatible with M-MuLV particle assembly, deletions of the NC domain were not tolerated. These results suggest that the Cys-His motif is held in close proximity within immature M-MuLV particles by interactions between CA domains and/or non-Cys-His motif domains of the NC. PMID:7815493

Hekate: Software Suite for the Mass Spectrometric Analysis and Three-Dimensional Visualization of Cross-Linked Protein Samples

PubMed Central

2013-01-01

Chemical cross-linking of proteins combined with mass spectrometry provides an attractive and novel method for the analysis of native protein structures and protein complexes. Analysis of the data however is complex. Only a small number of cross-linked peptides are produced during sample preparation and must be identified against a background of more abundant native peptides. To facilitate the search and identification of cross-linked peptides, we have developed a novel software suite, named Hekate. Hekate is a suite of tools that address the challenges involved in analyzing protein cross-linking experiments when combined with mass spectrometry. The software is an integrated pipeline for the automation of the data analysis workflow and provides a novel scoring system based on principles of linear peptide analysis. In addition, it provides a tool for the visualization of identified cross-links using three-dimensional models, which is particularly useful when combining chemical cross-linking with other structural techniques. Hekate was validated by the comparative analysis of cytochrome c (bovine heart) against previously reported data.1 Further validation was carried out on known structural elements of DNA polymerase III, the catalytic α-subunit of the Escherichia coli DNA replisome along with new insight into the previously uncharacterized C-terminal domain of the protein. PMID:24010795

Bactericidal activity of partially oxidized nanodiamonds.

PubMed

Wehling, Julia; Dringen, Ralf; Zare, Richard N; Maas, Michael; Rezwan, Kurosch

2014-06-24

Nanodiamonds are a class of carbon-based nanoparticles that are rapidly gaining attention, particularly for biomedical applications, i.e., as drug carriers, for bioimaging, or as implant coatings. Nanodiamonds have generally been considered biocompatible with a broad variety of eukaryotic cells. We show that, depending on their surface composition, nanodiamonds kill Gram-positive and -negative bacteria rapidly and efficiently. We investigated six different types of nanodiamonds exhibiting diverse oxygen-containing surface groups that were created using standard pretreatment methods for forming nanodiamond dispersions. Our experiments suggest that the antibacterial activity of nanodiamond is linked to the presence of partially oxidized and negatively charged surfaces, specifically those containing acid anhydride groups. Furthermore, proteins were found to control the bactericidal properties of nanodiamonds by covering these surface groups, which explains the previously reported biocompatibility of nanodiamonds. Our findings describe the discovery of an exciting property of partially oxidized nanodiamonds as a potent antibacterial agent.

Binary bacterial toxins: biochemistry, biology, and applications of common Clostridium and Bacillus proteins.

PubMed

Barth, Holger; Aktories, Klaus; Popoff, Michel R; Stiles, Bradley G

2004-09-01

Certain pathogenic species of Bacillus and Clostridium have developed unique methods for intoxicating cells that employ the classic enzymatic "A-B" paradigm for protein toxins. The binary toxins produced by B. anthracis, B. cereus, C. botulinum, C. difficile, C. perfringens, and C. spiroforme consist of components not physically associated in solution that are linked to various diseases in humans, animals, or insects. The "B" components are synthesized as precursors that are subsequently activated by serine-type proteases on the targeted cell surface and/or in solution. Following release of a 20-kDa N-terminal peptide, the activated "B" components form homoheptameric rings that subsequently dock with an "A" component(s) on the cell surface. By following an acidified endosomal route and translocation into the cytosol, "A" molecules disable a cell (and host organism) via disruption of the actin cytoskeleton, increasing intracellular levels of cyclic AMP, or inactivation of signaling pathways linked to mitogen-activated protein kinase kinases. Recently, B. anthracis has gleaned much notoriety as a biowarfare/bioterrorism agent, and of primary interest has been the edema and lethal toxins, their role in anthrax, as well as the development of efficacious vaccines and therapeutics targeting these virulence factors and ultimately B. anthracis. This review comprehensively surveys the literature and discusses the similarities, as well as distinct differences, between each Clostridium and Bacillus binary toxin in terms of their biochemistry, biology, genetics, structure, and applications in science and medicine. The information may foster future studies that aid novel vaccine and drug development, as well as a better understanding of a conserved intoxication process utilized by various gram-positive, spore-forming bacteria.

Binary Bacterial Toxins: Biochemistry, Biology, and Applications of Common Clostridium and Bacillus Proteins

PubMed Central

Barth, Holger; Aktories, Klaus; Popoff, Michel R.; Stiles, Bradley G.

2004-01-01

Certain pathogenic species of Bacillus and Clostridium have developed unique methods for intoxicating cells that employ the classic enzymatic “A-B” paradigm for protein toxins. The binary toxins produced by B. anthracis, B. cereus, C. botulinum, C. difficile, C. perfringens, and C. spiroforme consist of components not physically associated in solution that are linked to various diseases in humans, animals, or insects. The “B” components are synthesized as precursors that are subsequently activated by serine-type proteases on the targeted cell surface and/or in solution. Following release of a 20-kDa N-terminal peptide, the activated “B” components form homoheptameric rings that subsequently dock with an “A” component(s) on the cell surface. By following an acidified endosomal route and translocation into the cytosol, “A” molecules disable a cell (and host organism) via disruption of the actin cytoskeleton, increasing intracellular levels of cyclic AMP, or inactivation of signaling pathways linked to mitogen-activated protein kinase kinases. Recently, B. anthracis has gleaned much notoriety as a biowarfare/bioterrorism agent, and of primary interest has been the edema and lethal toxins, their role in anthrax, as well as the development of efficacious vaccines and therapeutics targeting these virulence factors and ultimately B. anthracis. This review comprehensively surveys the literature and discusses the similarities, as well as distinct differences, between each Clostridium and Bacillus binary toxin in terms of their biochemistry, biology, genetics, structure, and applications in science and medicine. The information may foster future studies that aid novel vaccine and drug development, as well as a better understanding of a conserved intoxication process utilized by various gram-positive, spore-forming bacteria. PMID:15353562

Cross-linking proteins with bimetallic tetracarboxylate compounds of transition metals

DOEpatents

Kostic, Nenad M.; Chen, Jian

1991-03-05

Stable cross-linked complexes of transition-metal tetracarboxylates and proteins are formed. The preferred transition-metal is rhodium. The protein may be collagen or an enzyme such as a proteolytic enzyme.

Insights in understanding aggregate formation and dissociation in cation exchange chromatography for a structurally unstable Fc-fusion protein.

PubMed

Chen, Zhiqiang; Huang, Chao; Chennamsetty, Naresh; Xu, Xuankuo; Li, Zheng Jian

2016-08-19

Cation-exchange chromatography (CEX) of a structurally unstable Fc-fusion protein exhibited multi-peak elution profile upon a salt-step elution due to protein aggregation during intra-column buffer transition where low pH and high salt coexisted. The protein exhibited a single-peak elution behavior during a pH-step elution; nevertheless, the levels of soluble aggregates (i.e. high molecular weight species, HMW) in the CEX eluate were still found up to 12-fold higher than that for the load material. The amount of the aggregates formed upon the pH-step elution was dependent on column loading with maximum HMW achieved at intermediate loading levels, supporting the hypothesis that the aggregation was the result of both the conformational changes of the bound protein and the solution concentration of the aggregation-susceptible proteins during elution. Factors such as high load pH, short protein/resin contact time, hydrophilic resin surface, and weak ionizable ligand were effective, to some extent, to reduce aggregate formation by improving the structural integrity of the bound protein. An orthogonal technique, differential scanning fluorimetry (DSF) using Sypro Orange dye confirmed that the bound protein exposed more hydrophobic area than the native molecule in free solution, especially in the pH 4-5 range. The Sypro Orange dye study of resin surface property also demonstrated that the poly[styrene-divinylbenzene]-based Poros XS with polyhydroxyl surface coating is more hydrophobic compared to the agarose-based CM Sepharose FF and SP Sepharose FF. The hydrophobic property of Poros XS contributed to stronger interactions with the partially unfolded bound protein and consequently to the higher aggregate levels seen in Poros XS eluate. This work also investigates the aggregation reversibility in CEX eluate where up to 66% of the aggregates were observed to dissociate into native monomers over a period of 120h, and links the aggregate stability to such conditions as resin surface properties and charged ligand type. Experimental data was correlated semi-quantitatively with theoretical protein charge and hydrophobicity calculations using homology modeling within the BIOVIA Discovery Studio software. Finally, an arginine-sulphopropyl (Arg-SP) agarose resin immobilized with multi-functional ligands was prepared to verify the proposed hypothesis and to eliminate the aggregate formation. The findings of this work provide general insights in understanding aggregate formation and dissociation for structurally unstable proteins in the CEX step. Copyright © 2016 Elsevier B.V. All rights reserved.

Characterization of a midgut mucin-like glycoconjugate of Lutzomyia longipalpis with a potential role in Leishmania attachment.

PubMed

Myšková, Jitka; Dostálová, Anna; Pěničková, Lucie; Halada, Petr; Bates, Paul A; Volf, Petr

2016-07-25

Leishmania parasites are transmitted by phlebotomine sand flies and a crucial step in their life-cycle is the binding to the sand fly midgut. Laboratory studies on sand fly competence to Leishmania parasites suggest that the sand flies fall into two groups: several species are termed "specific/restricted" vectors that support the development of one Leishmania species only, while the others belong to so-called "permissive" vectors susceptible to a wide range of Leishmania species. In a previous study we revealed a correlation between specificity vs permissivity of the vector and glycosylation of its midgut proteins. Lutzomyia longipalpis and other four permissive species tested possessed O-linked glycoproteins whereas none were detected in three specific vectors examined. We used a combination of biochemical, molecular and parasitological approaches to characterize biochemical and biological properties of O-linked glycoprotein of Lu. longipalpis. Lectin blotting and mass spectrometry revealed that this molecule with an apparent molecular weight about 45-50 kDa corresponds to a putative 19 kDa protein with unknown function detected in a midgut cDNA library of Lu. longipalpis. We produced a recombinant glycoprotein rLuloG with molecular weight around 45 kDa. Anti-rLuloG antibodies localize the native glycoprotein on epithelial midgut surface of Lu. longipalpis. Although we could not prove involvement of LuloG in Leishmania attachment by blocking the native protein with anti-rLuloG during sand fly infections, we demonstrated strong binding of rLuloG to whole surface of Leishmania promastigotes. We characterized a novel O-glycoprotein from sand fly Lutzomyia longipalpis. It has mucin-like properties and is localized on the luminal side of the midgut epithelium. Recombinant form of the protein binds to Leishmania parasites in vitro. We propose a role of this molecule in Leishmania attachment to sand fly midgut.

Characterization of N-Glycan Structures on the Surface of Mature Dengue 2 Virus Derived from Insect Cells.

PubMed

Lei, Y; Yu, H; Dong, Y; Yang, J; Ye, W; Wang, Y; Chen, W; Jia, Z; Xu, Z; Li, Z; Zhang, F

2015-01-01

DENV envelope glycoprotein (E) is responsible for interacting with host cell receptors and is the main target for the development of a dengue vaccine based on an induction of neutralizing antibodies. It is well known that DENV E glycoprotein has two potential N-linked glycosylation sites at Asn67 and Asn153. The N-glycans of E glycoprotein have been shown to influence the proper folding of the protein, its cellular localization, its interactions with receptors and its immunogenicity. However, the precise structures of the N-glycans that are attached to E glycoprotein remain elusive, although the crystal structure of DENV E has been determined. This study characterized the structures of envelope protein N-linked glycans on mature DENV-2 particles derived from insect cells via an integrated method that used both lectin microarray and MALDI-TOF-MS. By combining these methods, a high heterogeneity of DENV N-glycans was found. Five types of N-glycan were identified on DENV-2, including mannose, GalNAc, GlcNAc, fucose and sialic acid; high mannose-type N-linked oligosaccharides and the galactosylation of N-glycans were the major structures that were found. Furthermore, a complex between a glycan on DENV and the carbohydrate recognition domain (CRD) of DC-SIGN was mimicked with computational docking experiments. For the first time, this study provides a comprehensive understanding of the N-linked glycan profile of whole DENV-2 particles derived from insect cells.

FAST TRACK COMMUNICATION: Deposition of amino-rich thin films by RF magnetron sputtering of nylon

NASA Astrophysics Data System (ADS)

Kylián, O.; Hanuš, J.; Choukourov, A.; Kousal, J.; Slavínská, D.; Biederman, H.

2009-07-01

RF magnetron sputtering of a nylon target in different gas mixtures was studied in order to evaluate the capability of this process to deposit amino-rich coatings needed in a wide range of biomedical applications. It has been demonstrated that both the deposition rate of the coatings and the surface density of primary amino groups are strongly linked with working gas mixture composition. From this point of view, a sufficiently high deposition rate as well as the highest amine efficiency reaching a NH2/C value of 18% was observed in the N2/H2 discharge, which leads to the surface exhibiting a high rate of protein adsorption.

Biosynthesis and processing of the Autographa californica nuclear polyhedrosis virus gp64 protein.

PubMed

Jarvis, D L; Garcia, A

1994-11-15

gp64 is a major virion envelope glycoprotein of the baculovirus Autographa californica multicapsid nuclear polyhedrosis virus (AcMNPV). gp64 plays an important role in AcMNPV infection, probably mediating penetration of one form of the virus into host cells through the endocytic pathway. gp64 also represents an excellent probe for studying the membrane glycoprotein processing capabilities of baculovirus-infected insect cells, which are used widely as a eucaryotic expression system. The goals of this study were to characterize gp64 biosynthesis and processing and determine how N-glycosylation and N-linked oligosaccharide processing influence the fate and function of gp64 in AcMNPV-infected insect cells. We found that gp64 was synthesized in a biphasic fashion, with peaks at 8 and 24 hr postinfection in both the intracellular and extracellular fractions. Interestingly, the first peak preceded detectable budded virus (BV) production, suggesting that gp64 is shed from infected cells early in infection. Transcriptional regulation accounted for the biphasic mode of gp64 protein synthesis, as transcription initiated at a consensus early motif during early times of infection, at a late motif during late times of infection, and there was a lag between the peak of early and the onset of late transcription. In vitro transcription-translation assays showed that the second ATG in the AcMNPV gp64 long open reading frame is used as the translational initiation codon and that downstream sequences encode a functional signal peptide. Pulse-chase analyses, endoglycosidases, and various inhibitors were used to show that some N-linked oligosaccharides on gp64 are processed by glucosidases and alpha-mannosidases in AcMNPV-infected insect cells. These experiments also revealed that at least two differentially processed gp64 glycoforms are produced in these cells and that both can reach the cell surface and assemble into progeny BV. However, N-linked oligosaccharide processing was not required for gp64 cell surface expression, its assembly into infectious BV, or its fusogenic activity. This suggested that any gp64 glycoform produced during infection, regardless of its N-linked carbohydrate structure, can have essentially normal biological properties. By contrast, transport of gp64 to the cell surface, production of infectious BV, and fusogenic activity were reduced in the absence of N-glycosylation, indicating that this modification is necessary for optimal gp64 function.

Dysregulated expression of cell surface glycoprotein CDCP1 in prostate cancer

PubMed Central

Yang, Lifang; Dutta, Sucharita M.; Troyer, Dean A.; Lin, Jefferson B.; Lance, Raymond A.; Nyalwidhe, Julius O.; Drake, Richard R; Semmes, O. John

2015-01-01

CUB-domain-containing protein 1 (CDCP1) is a trans-membrane protein regulator of cell adhesion with a potent pro-migratory function in tumors. Given that proteolytic cleavage of the ectodomain correlates with outside-in oncogenic signaling, we characterized glycosylation in the context of cellular processing and expression of CDCP1 in prostate cancer. We detected 135 kDa full-length and proteolytic processed 70 kDa species in a panel of PCa cell models. The relative expression of full-length CDCP1 correlated with the metastatic potential of syngeneic cell models and an increase in surface membrane expression of CDCP1 was observed in tumor compared to adjacent normal prostate tissues. We demonstrated that glycosylation of CDCP1 is a prerequisite for protein stability and plasma membrane localization, and that the expression level and extent of N-glycosylation of CDCP1 correlated with metastatic status. Interestingly, complex N-linked glycans with sialic acid chains were restricted to the N-terminal half of the ectodomain and absent in the truncated species. Characterization of the extracellular expression of CDCP1 identified novel circulating forms and revealed that extracellular vesicles provide additional processing pathways. Employing immunoaffinity mass spectrometry, we detected elevated levels of circulating CDCP1 in patient urine with high-risk disease. Our results establish that differential glycosylation, cell surface presentation and extracellular expression of CDCP1 are hallmarks of PCa progression. PMID:26497208

Teaching Genetics in Secondary Classrooms: a Linguistic Analysis of Teachers' Talk About Proteins

NASA Astrophysics Data System (ADS)

Thörne, Karin; Gericke, Niklas

2014-02-01

This study investigates Swedish biology teachers' inclusion of proteins when teaching genetics in grade nine (students 15-16 years old). For some years, there has been a call to give attention to proteins when teaching genetics as a means of linking the concepts `gene' and `trait'. Students are known to have problems with this relation because the concepts belong to different organizational levels. However, we know little about how the topic is taught and therefore this case study focuses on how teachers talk about proteins while teaching genetics and if they use proteins as a link between the micro and macro level. Four teachers were recorded during entire genetics teaching sequences, 45 lessons in total. The teachers' verbal communication was then analyzed using thematic pattern analysis, which is based in systemic functional linguistics. The linguistic analysis of teachers' talk in action revealed great variations in both the extent to which they used proteins in explanations of genetics and the ways they included proteins in linking genes and traits. Two of the teachers used protein as a link between gene and trait, while two did not. Three of the four teachers included instruction about protein synthesis. The common message from all teachers was that proteins are built, but none of the teachers talked about genes as exclusively encoding proteins. Our results suggest that students' common lack of understanding of proteins as an intermediate link between gene and trait could be explained by limitations in the way the subject is taught.

Patterns of the adsorption of bovine serum albumin on carboxymethyl dextran and carboxymethyl cellulose films

NASA Astrophysics Data System (ADS)

Paribok, I. V.; Solomyanskii, A. E.; Zhavnerko, G. K.

2016-02-01

Patterns of the adsorption of bovine serum albumin on carboxymethyl dextran and carboxymethyl cellulose films are studied by means of microcontact printing, atomic force microscopy, and quartz crystal microbalance. It is shown that both the charge of polysaccharide macromolecules and the technique for deposition of their films onto the surface (via adsorption from a solution or covalent cross-linking) are factors that determine the degree of nonspecific adsorption of the protein on such films.

Cross-linking proteins with bimetallic tetracarboxylate compounds of transition metals

DOEpatents

Kostic, N.M.; Chen, J.

1991-03-05

Stable cross-linked complexes of transition-metal tetracarboxylates and proteins are formed. The preferred transition-metal is rhodium. The protein may be collagen or an enzyme such as a proteolytic enzyme. No Drawings

Rho proteins of plants--functional cycle and regulation of cytoskeletal dynamics.

PubMed

Mucha, Elena; Fricke, Inka; Schaefer, Antje; Wittinghofer, Alfred; Berken, Antje

2011-11-01

Rho-related ROP proteins are molecular switches that essentially regulate a wide variety of processes. Of central interest is their influence on the plant cytoskeleton by which they affect vital processes like cell division, growth, morphogenesis, and pathogen defense. ROPs switch between GTP- and GDP-bound conformations by strictly regulated nucleotide exchange and GTP-hydrolysis, and only the active GTP-form interacts with downstream effectors to ultimately provoke a biological response. However, the mode of action of the engaged regulators and effectors as well as their upstream and downstream interaction partners have long been largely unknown. As opposed to analogous systems in animals and fungi, plants use specific GTPase activating proteins (RopGAPs) with a unique domain composition and novel guanine nucleotide exchange factors (RopGEFs) with a probable link to cell surface receptors. Moreover, plants comprise novel effector molecules and adapters connecting ROPs to mostly unknown downstream targets on the route to the cytoskeleton. This review aims to summarize recent knowledge on the molecular mechanisms and reaction cascades involved in ROP dependent cytoskeletal rearrangements, addressing the structure and function of the unusual RopGAPs, RopGEFs and effectors, and the upstream and downstream pathways linking ROPs to cell receptor-like kinases, actin filaments, and microtubules. Copyright © 2010 Elsevier GmbH. All rights reserved.

Optimization of tetanus toxoid ammonium sulfate precipitation process using response surface methodology.

PubMed

Brgles, Marija; Prebeg, Pero; Kurtović, Tihana; Ranić, Jelena; Marchetti-Deschmann, Martina; Allmaier, Günter; Halassy, Beata

2016-10-02

Tetanus toxoid (TTd) is a highly immunogenic, detoxified form of tetanus toxin, a causative agent of tetanus disease, produced by Clostridium tetani. Since tetanus disease cannot be eradicated but is easily prevented by vaccination, the need for the tetanus vaccine is permanent. The aim of this work was to investigate the possibility of optimizing TTd purification, i.e., ammonium sulfate precipitation process. The influence of the percentage of ammonium sulfate, starting amount of TTd, buffer type, pH, temperature, and starting purity of TTd on the purification process were investigated using optimal design for response surface models. Responses measured for evaluation of the ammonium sulfate precipitation process were TTd amount (Lf/mL) and total protein content. These two parameters were used to calculate purity (Lf/mgPN) and the yield of the process. Results indicate that citrate buffer, lower temperature, and lower starting amount of TTd result in higher purities of precipitates. Gel electrophoresis combined with matrix-assisted laser desorption ionization-mass spectrometric analysis of precipitates revealed that there are no inter-protein cross-links and that all contaminating proteins have pIs similar to TTd, so this is most probably the reason for the limited success of purification by precipitation.

WAIF1 Is a Cell-Surface CTHRC1 Binding Protein Coupling Bone Resorption and Formation.

PubMed

Matsuoka, Kazuhiko; Kohara, Yukihiro; Naoe, Yoshinori; Watanabe, Atsushi; Ito, Masako; Ikeda, Kyoji; Takeshita, Sunao

2018-04-06

The osteoclast-derived collagen triple helix repeat containing 1 (CTHRC1) protein stimulates osteoblast differentiation, but the underlying mechanism remains unclear. Here, we identified Wnt-activated inhibitory factor 1 (WAIF1)/5T4 as a cell-surface protein binding CTHRC1. The WAIF1-encoding Trophoblast glycoprotein (Tpbg) gene, which is abundantly expressed in the brain and bone but not in other tissues, showed the same expression pattern as Cthrc1. Tpbg downregulation in marrow stromal cells reduced CTHRC1 binding and CTHRC1-stimulated alkaline phosphatase activity through PKCδ activation of MEK/ERK, suggesting a novel WAIF1/PKCδ/ERK pathway triggered by CTHRC1. Unexpectedly, osteoblast lineage-specific deletion of Tpbg downregulated Rankl expression in mouse bones and reduced both bone formation and resorption; importantly, it impaired bone mass recovery following RANKL-induced resorption, reproducing the phenotype of osteoclast-specific Cthrc1 deficiency. Thus, the binding of osteoclast-derived CTHRC1 to WAIF1 in stromal cells activates PKCδ-ERK osteoblastogenic signaling and serves as a key molecular link between bone resorption and formation during bone remodeling. © 2018 American Society for Bone and Mineral Research. © 2018 American Society for Bone and Mineral Research.

Plasma treatment of paper for protein immobilization on paper-based chemiluminescence immunodevice.

PubMed

Zhao, Mei; Li, Huifang; Liu, Wei; Guo, Yumei; Chu, Weiru

2016-05-15

A novel protein immobilization method based on plasma treatment of paper on the low-cost paper-based immunodevice was established in this work. By using a benchtop plasma cleaner, the paper microzone was treated by oxygen plasma treatment for 4 min and then the antibody can be directly immobilized on the paper surface. Aldehyde group was produced after the plasma treatment, which can be verified from the fourier transform infrared spectroscopy (FT-IR) spectra and x-ray photoelectron spectroscopy (XPS) spectra. By linked to aldehyde group, the antibody can be immobilized on the paper surface without any other pretreatment. A paper-based immunodevice was introduced here through this antibody immobilization method. With sandwich chemiluminescence (CL) immunoassay method, the paper-based immunodevice was successfully performed for carcinoembryonic antigen (CEA) detection in human serum with a linear range of 0.1-80.0 ng/mL. The detection limit was 0.03 ng/mL, which was 30 times lower than the clinical CEA level. Comparing to the other protein immobilization methods on paper-based device, this strategy was faster and simpler and had potential applications in point-of-care testing, public health and environmental monitoring. Copyright © 2015 Elsevier B.V. All rights reserved.

Three-Dimensional Hierarchical Plasmonic Nano-Architecture Enhanced Surface-Enhanced Raman Scattering Immuno-Sensor for Cancer Biomarker Detection in Blood Plasma

PubMed Central

Li, Ming; Cushing, Scott K.; Zhang, Jianming; Suri, Savan; Evans, Rebecca; Petros, William P.; Gibson, Laura F.; Ma, Dongling; Liu, Yuxin; Wu, Nianqiang

2013-01-01

A three-dimensional (3D) hierarchical plasmonic nano-architecture has been designed for a sensitive surface-enhanced Raman scattering (SERS) immuno-sensor for protein biomarker detection. The capture antibody molecules are immobilized on a plasmonic gold triangle nano-array pattern. On the other hand, the detection antibody molecules are linked to the gold nano-star@Raman-reporter@silica sandwich nanoparticles. When protein biomarkers are present, the sandwich nanoparticles are captured over the gold triangle nano-array, forming a confined 3D plasmonic field, leading to the enhanced electromagnetic field in intensity and in 3D space. As a result, the Raman reporter molecules are exposed to a high density of “hot spots”, which amplifies the Raman signal remarkably, improving the sensitivity of the SERS immuno-sensor. This SERS immuno-sensor exhibits a wide linear range (0.1 pg/mL to 10 ng/mL), and a low limit of detection (7 fg/mL) toward human immunoglobulin G (IgG) protein in the buffer solution. This biosensor has been successfully used for detection of the vascular endothelial growth factor (VEGF) in the human blood plasma from clinical breast cancer patient samples. PMID:23659430

CLMSVault: A Software Suite for Protein Cross-Linking Mass-Spectrometry Data Analysis and Visualization.

PubMed

Courcelles, Mathieu; Coulombe-Huntington, Jasmin; Cossette, Émilie; Gingras, Anne-Claude; Thibault, Pierre; Tyers, Mike

2017-07-07

Protein cross-linking mass spectrometry (CL-MS) enables the sensitive detection of protein interactions and the inference of protein complex topology. The detection of chemical cross-links between protein residues can identify intra- and interprotein contact sites or provide physical constraints for molecular modeling of protein structure. Recent innovations in cross-linker design, sample preparation, mass spectrometry, and software tools have significantly improved CL-MS approaches. Although a number of algorithms now exist for the identification of cross-linked peptides from mass spectral data, a dearth of user-friendly analysis tools represent a practical bottleneck to the broad adoption of the approach. To facilitate the analysis of CL-MS data, we developed CLMSVault, a software suite designed to leverage existing CL-MS algorithms and provide intuitive and flexible tools for cross-platform data interpretation. CLMSVault stores and combines complementary information obtained from different cross-linkers and search algorithms. CLMSVault provides filtering, comparison, and visualization tools to support CL-MS analyses and includes a workflow for label-free quantification of cross-linked peptides. An embedded 3D viewer enables the visualization of quantitative data and the mapping of cross-linked sites onto PDB structural models. We demonstrate the application of CLMSVault for the analysis of a noncovalent Cdc34-ubiquitin protein complex cross-linked under different conditions. CLMSVault is open-source software (available at https://gitlab.com/courcelm/clmsvault.git ), and a live demo is available at http://democlmsvault.tyerslab.com/ .

Utilizing Mechanistic Cross-Linking Technology to Study Protein-Protein Interactions: An Experiment Designed for an Undergraduate Biochemistry Lab

ERIC Educational Resources Information Center

Finzel, Kara; Beld, Joris; Burkart, Michael D.; Charkoudian, Louise K.

2017-01-01

Over the past decade, mechanistic cross-linking probes have been used to study protein-protein interactions in natural product biosynthetic pathways. This approach is highly interdisciplinary, combining elements of protein biochemistry, organic chemistry, and computational docking. Herein, we described the development of an experiment to engage…

Optimization of Formaldehyde Cross-Linking for Protein Interaction Analysis of Non-Tagged Integrin β1

PubMed Central

Klockenbusch, Cordula; Kast, Juergen

2010-01-01

Formaldehyde cross-linking of protein complexes combined with immunoprecipitation and mass spectrometry analysis is a promising technique for analysing protein-protein interactions, including those of transient nature. Here we used integrin β1 as a model to describe the application of formaldehyde cross-linking in detail, particularly focusing on the optimal parameters for cross-linking, the detection of formaldehyde cross-linked complexes, the utility of antibodies, and the identification of binding partners. Integrin β1 was found in a high molecular weight complex after formaldehyde cross-linking. Eight different anti-integrin β1 antibodies were used for pull-down experiments and no loss in precipitation efficiency after cross-linking was observed. However, two of the antibodies could not precipitate the complex, probably due to hidden epitopes. Formaldehyde cross-linked complexes, precipitated from Jurkat cells or human platelets and analyzed by mass spectrometry, were found to be composed of integrin β1, α4 and α6 or β1, α6, α2, and α5, respectively. PMID:20634879

SALSA-A dance on a slippery floor with changing partners.

PubMed

Reichhardt, M P; Holmskov, U; Meri, S

2017-09-01

It is becoming increasingly clear that the connections between our immune system and the microbiota colonizing us have a tremendous impact on human health. A number of innate molecular defence mechanisms cooperate to selectively target unwanted microorganisms at the mucosal surfaces. Amongst others these include the complement system, IgA and the SALSA molecule. The salivary scavenger and agglutinin (SALSA), also known as deleted in malignant brain tumors 1 (DMBT1), salivary agglutinin (SAG) or gp340 is a multifunctional molecule with important functions in innate immunity, inflammation and epithelial homeostasis. The SALSA protein is expressed at most mucosal surfaces, where it is one of the most abundant proteins. In the fetal meconium and infant intestine it may constitute even up to 10% of the total protein amount. SALSA is found either directly associated with the epithelial surface or secreted into the lining fluids. In the fluid-phase SALSA interacts with a number of bacterial and viral organisms, as well as with endogenous ligands, including IgA, lactoferrin, surfactant proteins and complement components. While complement has been shown to impact the mucosal environment, this remains an area of limited research. The multiple interactions of the SALSA molecule provide a scaffold, where this potent defence system may engage in cooperative microbial clearance together with corresponding mucosal host ligands. With its high abundance, and multiple effects on both host and microbes, the SALSA molecule is a key player in maintaining the immunological balance at the mucosal surfaces. This is further supported by observations linking the expression of different SALSA isoforms to the development of chronic inflammatory conditions, such as Crohn's disease and ulcerative colitis. This review describes the latest advances in understanding functions of SALSA and its different isoforms. Recently recognized functions are related to complement activation and regulation, endothelial development and epithelial homeostasis. In addition, we suggest mechanisms how SALSA regulates inflammation at the mucosal surfaces. Copyright © 2017 Elsevier Ltd. All rights reserved.

Modification of silicone elastomer with zwitterionic silane for durable antifouling properties.

PubMed

Yeh, Shiou-Bang; Chen, Chien-Sheng; Chen, Wen-Yih; Huang, Chun-Jen

2014-09-30

Biofouling on medical devices generally causes adverse complications, such as thrombosis, infection, and pathogenic calcification. Silicone is a widely used material for medical applications. Its surface modification typically encounters undesirable "hydrophobic recovery", leading to deterioration of surface engineering. In this study, we developed a stable superhydrophilic zwitterionic interface on polydimethylsiloxane (PDMS) elastomer by covalent silanization of sulfobetaine silane (SBSi) to resist nonspecific adsorption of bacteria, proteins, and lipids. SBSi is a zwitterionic organosilane assembly, enabling resisting surface reconstruction by forming a cross-linked network and polar segregation. Surface elemental composition was confirmed by X-ray photoelectron spectroscopy (XPS), and the long-term stability of modification was accessed using a contact angle goniometer. The biofouling tests were carried out by exposing substrates to bacterial, protein, and lipid solutions, revealing the excellent bioinertness of SBSi-tailored PDMS, even after 30 day storage in ambient. For the real-world application, we modified commercially available silicone hydrogel contact lenses with developed zwitterionic silane, presenting its antibacterial adhesion property. Moreover, the cytotoxicity of SBSi was accessed with NIH-3T3 fibroblast by the MTT assay, showing negligible cytotoxicity up to a concentration of 5 mM. Consequently, the strategy of surface engineering in this work can effectively retard the "hydrophobic recovery" occurrence and can be applied to other silicone-based medical devices in a facile way.

Biophysical characterization of a de novo elastin

NASA Astrophysics Data System (ADS)

Greenland, Kelly Nicole

Natural human elastin is found in tissue such as the lungs, arteries, and skin. This protein is formed at birth with no mechanism present to repair or supplement the initial quantity formed. As a result, the functionality and durability of elastin's elasticity is critically important. To date, the mechanics of this ability to stretch and recoil is not fully understood. This study utilizes de novo protein design to create a small library of simplistic versions of elastin-like proteins, demonstrate the elastin-like proteins, maintain elastin's functionality, and inquire into its structure using solution nuclear magnetic resonance (NMR). Elastin is formed from cross-linked tropoelastin. Therefore, the first generation of designed proteins consisted of one protein that utilized homogony of interspecies tropoelastin by using three common domains, two hydrophobic and one cross-linking domains. Basic modifications were made to open the hydrophobic region and also to make the protein easier to purify and characterize. The designed protein maintained its functionality, self-aggregating as the temperature increased. Uniquely, the protein remained self-aggregated as the temperature returned below the critical transition temperature. Self-aggregation was additionally induced by increasing salt concentrations and by modifying the pH. The protein appeared to have little secondary structure when studied with solution NMR. These results fueled a second generation of designed elastin-like proteins. This generation contained variations designed to study the cross-linking domain, one specific hydrophobic domain, and the effect of the length of the elastin-like protein. The cross-linking domain in one variation has been significantly modified while the flanking hydrophobic domains have remained unchanged. This characterization of this protein will answer questions regarding the specificity of the homologous nature of the cross-linking domain of tropoelastin across species. A second protein has additional hydrophobic domains flanking the originally designed elastin-like protein. The characterization of this protein will answer questions regarding the functionality of longer or more hydrophobic elastin-like proteins. The final variation designed is one hydrophobic domain and the new cross-linking domain repeating several times. The characterization of this protein will answer questions regarding the specific hydrophobic domain and its functionality.

Intra-molecular cross-linking of acidic residues for protein structure studies.

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

Kruppa, Gary Hermann; Young, Malin M.; Novak, Petr

2005-03-01

Intra-molecular cross-linking has been suggested as a method of obtaining distance constraints that would be useful in developing structural models of proteins. Recent work published on intra-molecular cross-linking for protein structural studies has employed commercially available primary amine selective reagents that can cross-link lysine residues to other lysine residues or the amino terminus. Previous work using these cross-linkers has shown that for several proteins of known structure, the number of cross-links that can be obtained experimentally may be small compared to what would be expected from the known structure, due to the relative reactivity, distribution, and solvent accessibility of themore » lysines in the protein sequence. To overcome these limitations we have investigated the use of cross-linking reagents that can react with other reactive sidechains in proteins. We used 1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) to activate the carboxylic acid containing residues, aspartic acid (D), glutamic acid (E), and the carboxy terminus (O), for cross-linking reactions. Once activated, the DEO sidechains can react to form 'zero-length' cross-links with nearby primary amine containing resides, lysines (K) and the amino terminus (X), via the formation of a new amide bond. We also show that the EDC-activated DEO sidechains can be cross-linked to each other using dihydrazides, two hydrazide moieties connected by an alkyl cross-linker ann of variable length. Using these reagents, we have found three new 'zero-length' cross-links in ubiquitin consistent with its known structure (M1-E16, M1-E18, and K63-E64). Using the dihydrazide cross-linkers, we have identified 2 new cross-links (D21-D32 and E24-D32) unambiguously. Using a library of dihydrazide cross-linkers with varying arm length, we have shown that there is a minimum arm length required for the DEO-DEO cross-links of 5.8 angstroms. These results show that additional structural information can be obtained by exploiting new cross-linker chemistry, increasing the probability that the protein target of choice will yield sufficient distance constraints to develop a structural model.« less

Actin Cross-link Assembly and Disassembly Mechanics for α-Actinin and Fascin*

PubMed Central

Courson, David S.; Rock, Ronald S.

2010-01-01

Self-assembly of complex structures is commonplace in biology but often poorly understood. In the case of the actin cytoskeleton, a great deal is known about the components that include higher order structures, such as lamellar meshes, filopodial bundles, and stress fibers. Each of these cytoskeletal structures contains actin filaments and cross-linking proteins, but the role of cross-linking proteins in the initial steps of structure formation has not been clearly elucidated. We employ an optical trapping assay to investigate the behaviors of two actin cross-linking proteins, fascin and α-actinin, during the first steps of structure assembly. Here, we show that these proteins have distinct binding characteristics that cause them to recognize and cross-link filaments that are arranged with specific geometries. α-Actinin is a promiscuous cross-linker, linking filaments over all angles. It retains this flexibility after cross-links are formed, maintaining a connection even when the link is rotated. Conversely, fascin is extremely selective, only cross-linking filaments in a parallel orientation. Surprisingly, bundles formed by either protein are extremely stable, persisting for over 0.5 h in a continuous wash. However, using fluorescence recovery after photobleaching and fluorescence decay experiments, we find that the stable fascin population can be rapidly competed away by free fascin. We present a simple avidity model for this cross-link dissociation behavior. Together, these results place constraints on how cytoskeletal structures assemble, organize, and disassemble in vivo. PMID:20551315

Incomplete synthesis of N-glycans in congenital dyserythropoietic anemia type II caused by a defect in the gene encoding. alpha. -mannosidase II

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

Fukuda, M.N.; Masri, K.A.; Dell, A.

1990-10-01

Congenital dyserythropoietic anemia type II, or hereditary erythroblastic multinuclearity with a positive acidified-serum-lysis test (HEMPAS), is a genetic anemia in humans inherited by an autosomally recessive mode. The enzyme defect in most HEMPAS patients has previously been proposed as a lowered activity of N-acetylglucosaminyltransferase II, resulting in a lack of polylactosamine on proteins and leading to the accumulation of polylactosaminyl lipids. A recent HEMPAS case, G.C., has now been analyzed by cell-surface labeling, fast-atom-bombardment mass spectrometry of glycopeptides, and activity assay of glycosylation enzymes. Significantly decreased glycosylation of polylactosaminoglycan proteins and incompletely processed asparagine-linked oligosaccharides were detected in the erythrocytemore » membranes of G.C. These results suggest that G.C. cells contain a mutation in {alpha}-ManII-encoding gene that results in inefficient expression of {alpha}-ManII mRNA, either through reduced transcription or message instability. This report demonstrates that HEMPAS is caused by a defective gene encoding an enzyme necessary for the synthesis of asparagine-linked oligosaccharides.« less

A highly sensitive and selective diagnostic assay based on virus nanoparticles

NASA Astrophysics Data System (ADS)

Park, Jin-Seung; Cho, Moon Kyu; Lee, Eun Jung; Ahn, Keum-Young; Lee, Kyung Eun; Jung, Jae Hun; Cho, Yunjung; Han, Sung-Sik; Kim, Young Keun; Lee, Jeewon

2009-04-01

Early detection of the protein marker troponin I in patients with a higher risk of acute myocardial infarction can reduce the risk of death from heart attacks. Most troponin assays are currently based on the conventional enzyme linked immunosorbent assay and have detection limits in the nano- and picomolar range. Here, we show that by combining viral nanoparticles, which are engineered to have dual affinity for troponin antibodies and nickel, with three-dimensional nanostructures including nickel nanohairs, we can detect troponin levels in human serum samples that are six to seven orders of magnitude lower than those detectable using conventional enzyme linked immunosorbent assays. The viral nanoparticle helps to orient the antibodies for maximum capture of the troponin markers. High densities of antibodies on the surfaces of the nanoparticles and nanohairs lead to greater binding of the troponin markers, which significantly enhances detection sensitivities. The nickel nanohairs are re-useable and can reproducibly differentiate healthy serum from unhealthy ones. We expect other viral nanoparticles to form similar highly sensitive diagnostic assays for a variety of other protein markers.

Cross-linking mass spectrometry and mutagenesis confirm the functional importance of surface interactions between CYP3A4 and holo/apo cytochrome b(5).

PubMed

Zhao, Chunsheng; Gao, Qiuxia; Roberts, Arthur G; Shaffer, Scott A; Doneanu, Catalin E; Xue, Song; Goodlett, David R; Nelson, Sidney D; Atkins, William M

2012-11-27

Cytochrome b(5) (cyt b(5)) is one of the key components in the microsomal cytochrome P450 monooxygenase system. Consensus has not been reached about the underlying mechanism of cyt b(5) modulation of CYP catalysis. Both cyt b(5) and apo b(5) are reported to stimulate the activity of several P450 isoforms. In this study, the surface interactions of both holo and apo b(5) with CYP3A4 were investigated and compared for the first time. Chemical cross-linking coupled with mass spectrometric analysis was used to identify the potential electrostatic interactions between the protein surfaces. Subsequently, the models of interaction of holo/apo b(5) with CYP3A4 were built using the identified interacting sites as constraints. Both cyt b(5) and apo b(5) were predicted to bind to the same groove on CYP3A4 with close contacts to the B-B' loop of CYP3A4, a substrate recognition site. Mutagenesis studies further confirmed that the interacting sites on CYP3A4 (Lys96, Lys127, and Lys421) are functionally important. Mutation of these residues reduced or abolished cyt b(5) binding affinity. The critical role of Arg446 on CYP3A4 in binding to cyt b(5) and/or cytochrome P450 reductase was also discovered. The results indicated that electrostatic interactions on the interface of the two proteins are functionally important. The results indicate that apo b(5) can dock with CYP3A4 in a manner analogous to that of holo b(5), so electron transfer from cyt b(5) is not required for its effects.

Enzyme-linked immunosorbent assay detection and bioactivity of Cry1Ab protein fragments

USDA-ARS?s Scientific Manuscript database

Enzyme-linked immunosorbent assay (ELISA) has emerged as the preferred detection method for Cry proteins in environmental matrices. Concerns exist that ELISAs are capable of detecting fragments of Cry proteins, which may lead to an over-estimation of the concentration of these proteins in the enviro...

A Drosophila receptor tyrosine phosphatase expressed in the embryonic CNS and larval optic lobes is a member of the set of proteins bearing the "HRP" carbohydrate epitope.

PubMed

Desai, C J; Popova, E; Zinn, K

1994-12-01

Recent studies have defined several cell surface glycoproteins expressed in the developing nervous system of insect embryos that may be involved in axon outgrowth and guidance processes. These glycoproteins include the fasciclins and a group of receptor-linked protein tyrosine phosphatases (R-PTPs). In embryos, the fasciclins are localized to axonal subsets, while the R-PTPs appear to be expressed on most or all CNS axons. To identify other neuronal cell surface glycoproteins in the Drosophila embryo, we have taken a biochemical approach. This is based on the observation that antisera against horseradish peroxidase (HRP) recognize a carbohydrate epitope that is selectively expressed in the insect nervous system. A large number of neuronal glycoproteins (denoted "HRP proteins") apparently bear the HRP carbohydrate epitope. We have used polyclonal anti-HRP antibodies to purify these proteins from Drosophila embryos, and have obtained protein sequences from seven HRP protein bands. These data define three major HRP proteins as neurotactin, fasciclin I, and an R-PTP, DPTP69D. Western blotting data suggest that fasciclin II, neuroglian, DPTP10D, and DPTP99A are also HRP proteins. We show that DPTP69D, like the previously characterized R-PTPs, is localized to CNS axons in the embryo. In third instar larvae, DPTP69D expression is restricted to subsets of neuronal processes in the brain, ventral nerve cord, and eye disk. In the optic lobes, DPTP69D is localized to the neuropils of the lamina and medulla, and to an array of parallel thick bundles that may be the transmedullary fibers of the developing lobula complex.

A Plasmodium falciparum PHIST protein binds the virulence factor PfEMP1 and comigrates to knobs on the host cell surface

PubMed Central

Oberli, Alexander; Slater, Leanne M.; Cutts, Erin; Brand, Françoise; Mundwiler-Pachlatko, Esther; Rusch, Sebastian; Masik, Martin F. G.; Erat, Michèle C.; Beck, Hans-Peter; Vakonakis, Ioannis

2014-01-01

Uniquely among malaria parasites, Plasmodium falciparum-infected erythrocytes (iRBCs) develop membrane protrusions, known as knobs, where the parasite adhesion receptor P. falciparum erythrocyte membrane protein 1 (PfEMP1) clusters. Knob formation and the associated iRBC adherence to host endothelium are directly linked to the severity of malaria and are functional manifestations of protein export from the parasite to the iRBC. A family of exported proteins featuring Plasmodium helical interspersed subtelomeric (PHIST) domains has attracted attention, with members being implicated in host-parasite protein interactions and differentially regulated in severe disease and among parasite isolates. Here, we show that PHIST member PFE1605w binds the PfEMP1 intracellular segment directly with Kd = 5 ± 0.6 μM, comigrates with PfEMP1 during export, and locates in knobs. PHIST variants that do not locate in knobs (MAL8P1.4) or bind PfEMP1 30 times more weakly (PFI1780w) used as controls did not display the same pattern. We resolved the first crystallographic structure of a PHIST protein and derived a partial model of the PHIST-PfEMP1 interaction from nuclear magnetic resonance. We propose that PFE1605w reinforces the PfEMP1-cytoskeletal connection in knobs and discuss the possible role of PHIST proteins as interaction hubs in the parasite exportome.—Oberli, A., Slater, L. M., Cutts, E., Brand, F., Mundwiler-Pachlatko, E., Rusch, S., Masik, M. F. G., Erat, M. C., Beck, H.-P., Vakonakis, I. A Plasmodium falciparum PHIST protein binds the virulence factor PfEMP1 and comigrates to knobs on the host cell surface. PMID:24983468

The 2.5 Å Structure of the Enterococcus Conjugation Protein TraM resembles VirB8 Type IV Secretion Proteins*

PubMed Central

Goessweiner-Mohr, Nikolaus; Grumet, Lukas; Arends, Karsten; Pavkov-Keller, Tea; Gruber, Christian C.; Gruber, Karl; Birner-Gruenberger, Ruth; Kropec-Huebner, Andrea; Huebner, Johannes; Grohmann, Elisabeth; Keller, Walter

2013-01-01

Conjugative plasmid transfer is the most important means of spreading antibiotic resistance and virulence genes among bacteria and therefore presents a serious threat to human health. The process requires direct cell-cell contact made possible by a multiprotein complex that spans cellular membranes and serves as a channel for macromolecular secretion. Thus far, well studied conjugative type IV secretion systems (T4SS) are of Gram-negative (G−) origin. Although many medically relevant pathogens (e.g., enterococci, staphylococci, and streptococci) are Gram-positive (G+), their conjugation systems have received little attention. This study provides structural information for the transfer protein TraM of the G+ broad host range Enterococcus conjugative plasmid pIP501. Immunolocalization demonstrated that the protein localizes to the cell wall. We then used opsonophagocytosis as a novel tool to verify that TraM was exposed on the cell surface. In these assays, antibodies generated to TraM recruited macrophages and enabled killing of pIP501 harboring Enteroccocus faecalis cells. The crystal structure of the C-terminal, surface-exposed domain of TraM was determined to 2.5 Å resolution. The structure, molecular dynamics, and cross-linking studies indicated that a TraM trimer acts as the biological unit. Despite the absence of sequence-based similarity, TraM unexpectedly displayed a fold similar to the T4SS VirB8 proteins from Agrobacterium tumefaciens and Brucella suis (G−) and to the transfer protein TcpC from Clostridium perfringens plasmid pCW3 (G+). Based on the alignments of secondary structure elements of VirB8-like proteins from mobile genetic elements and chromosomally encoded T4SS from G+ and G− bacteria, we propose a new classification scheme of VirB8-like proteins. PMID:23188825

Hydrogen-bond dynamics at the bio-water interface in hydrated proteins: a molecular-dynamics study.

PubMed

Nandi, Prithwish K; English, Niall J; Futera, Zdenek; Benedetto, Antonio

2016-12-21

Water is fundamental to the biochemistry of enzymes. It is well known that without a minimum amount of water, enzymes are not biologically active. Bare minimal solvation for biological function corresponds to about a single layer of water covering enzymes' surfaces. Many contradictory studies on protein-hydration-water-coupled dynamics have been published in recent decades. Following prevailing wisdom, a dynamical crossover in hydration water (at around 220 K for hydrated lysozymes) can trigger larger-amplitude motions of the protein, activating, in turn, biological functions. Here, we present a molecular-dynamics-simulation study on a solvated model protein (hen egg-white lysozyme), in which we determine, inter alia, the relaxation dynamics of the hydrogen-bond network between the protein and its hydration water molecules on a residue-per-residue basis. Hydrogen-bond breakage/formation kinetics is rather heterogeneous in temperature dependence (due to the heterogeneity of the free-energy surface), and is driven by the magnitude of thermal motions of various different protein residues which provide enough thermal energy to overcome energy barriers to rupture their respective hydrogen bonds with water. In particular, arginine residues exhibit the highest number of such hydrogen bonds at low temperatures, losing almost completely such bonding above 230 K. This suggests that hydration water's dynamical crossover, observed experimentally for hydrated lysozymes at ∼220 K, lies not at the origin of the protein residues' larger-amplitude motions, but rather arises as a consequence thereof. This highlights the need for new experimental investigations, and new interpretations to link protein dynamics to functions, in the context of key interrelationships with the solvation layer.

Characterization of the Bacteroides fragilis bfr Gene Product Identifies a Bacterial DPS-Like Protein and Suggests Evolutionary Links in the Ferritin Superfamily

PubMed Central

Gauss, George H.; Reott, Michael A.; Rocha, Edson R.; Young, Mark J.; Douglas, Trevor

2012-01-01

A factor contributing to the pathogenicity of Bacteroides fragilis, the most common anaerobic species isolated from clinical infections, is the bacterium's extreme aerotolerance, which allows survival in oxygenated tissues prior to anaerobic abscess formation. We investigated the role of the bacterioferritin-related (bfr) gene in the B. fragilis oxidative stress response. The bfr mRNA levels are increased in stationary phase or in response to O2 or iron. In addition, bfr null mutants exhibit reduced aerotolerance, and the bfr gene product protects DNA from hydroxyl radical cleavage in vitro. Crystallographic studies revealed a protein with a dodecameric structure and greater similarity to an archaeal DNA protection in starved cells (DPS)-like protein than to the 24-subunit bacterioferritins. Similarity to the DPS-like (DPSL) protein extends to the subunit and includes a pair of conserved cysteine residues juxtaposed to a buried dimetal binding site within the four-helix bundle. Compared to archaeal DPSLs, however, this bacterial DPSL protein contains several unique features, including a significantly different conformation in the C-terminal tail that alters the number and location of pores leading to the central cavity and a conserved metal binding site on the interior surface of the dodecamer. Combined, these characteristics confirm this new class of miniferritin in the bacterial domain, delineate the similarities and differences between bacterial DPSL proteins and their archaeal homologs, allow corrected annotations for B. fragilis bfr and other dpsl genes within the bacterial domain, and suggest an evolutionary link within the ferritin superfamily that connects dodecameric DPS to the (bacterio)ferritin 24-mer. PMID:22020642

Molecular modeling of retinoschisin with functional analysis of pathogenic mutations from human X-linked retinoschisis

PubMed Central

Sergeev, Y.V.; Caruso, R.C.; Meltzer, M.R.; Smaoui, N.; MacDonald, I.M.; Sieving, P.A.

2010-01-01

Gene mutations that encode retinoschisin (RS1) cause X-linked retinoschisis (XLRS), a form of juvenile macular and retinal degeneration that affects males. RS1 is an adhesive protein which is proposed to preserve the structural and functional integrity of the retina, but there is very little evidence of the mechanism by which protein changes are related to XLRS disease. Here, we report molecular modeling of the RS1 protein and consider perturbations caused by mutations found in human XLRS subjects. In 60 XLRS patients who share 27 missense mutations, we then evaluated possible correlations of the molecular modeling with retinal function as determined by the electroretinogram (ERG) a- and b-waves. The b/a-wave ratio reflects visual-signal transfer in retina. We sorted the ERG b/a-ratios by patient age and by the mutation impact on protein structure. The majority of RS1 mutations caused minimal structure perturbation and targeted the protein surface. These patients' b/a-ratios were similar across younger and older subjects. Maximum structural perturbations from either the removal or insertion of cysteine residues or changes in the hydrophobic core were associated with greater difference in the b/a-ratio with age, with a significantly smaller ratio at younger ages, analogous to the ERG changes with age observed in mice with no RS1-protein expression due to a recombinant RS1-knockout gene. The molecular modeling suggests an association between the predicted structural alteration and/or damage to retinoschisin and the severity of XLRS as measured by the ERG analogous to the RS1-knockout mouse. PMID:20061330

Modified properties of a glycated and cross-linked soy protein isolate by transglutaminase and an oligochitosan of 5 kDa.

PubMed

Fu, Miao; Zhao, Xin-Huai

2017-01-01

Soy protein is an important protein ingredient for the food industry; however, its properties can be improved by enzymatic and chemical modifications. This study applied a new enzymatic glycation and cross-linking to modify soy protein isolate (SPI), using an oligochitosan of 5 kDa and transglutaminase. Properties of the obtained glycated and cross-linked SPI (GC-SPI) were unknown and thus assessed. GC-SPI contained glucosamine of 13.6 g kg -1 protein, but less reactable &bond;NH 2 than SPI (0.42 vs. 0.50 mol kg -1 protein). Infrared spectra and circular dichroism results showed that GC-SPI other than SPI and cross-linked SPI had more &bond;OH in molecules, and was more disordered in secondary structure. In comparison with SPI, GC-SPI showed enhanced water-binding capacity, could form aggregates with enlarged hydrodynamic radius (180.2 vs. 82.9 nm) and negative zeta-potential (-31.2 vs. -27.7 mV) in dispersion, but exhibited lower thermal stability (e.g. greater mass loss) upon heating at a temperature above 288 °C. GC-SPI also had lower in vitro proteolytic digestibility than SPI due to the protein cross-linking. Oligochitosan of 5 kDa and transglutaminase can be used to glycate and cross-link SPI. This approach is applicable to generate potential protein ingredient with good hydration and dispersive stabilisation. © 2016 Society of Chemical Industry. © 2016 Society of Chemical Industry.

Complex formation between the protein components of methane monooxygenase from Methylosinus trichosporium OB3b. Identification of sites of component interaction.

PubMed

Fox, B G; Liu, Y; Dege, J E; Lipscomb, J D

1991-01-05

Kinetic, spectroscopic, and chemical evidence for the formation of specific catalytically essential complexes between the three protein components of the soluble form of methane monooxygenase from Methylosinus trichosporium OB3b is reported. The effects of the concentrations of the reductase and component B on the hydroxylation activity of the reconstituted enzyme system has been numerically simulated based on a kinetic model which assumes formation of multiple high affinity complexes with the hydroxylase component during catalysis. The formation of several of these complexes has been directly demonstrated. By using EPR spectroscopy, the binding of approximately 2 mol of component B/mol of hydroxylase (subunit structure (alpha beta gamma)2) is shown to significantly change the electronic environment of the mu-(H/R)-oxo-bridged binuclear iron cluster of the hydroxylase in both the mixed valent (Fe(II).Fe(III)) and fully reduced (Fe(II).Fe(II)) states. Protein-protein complexes between the reductase and component B as well as between the reductase and hydroxylase have been shown to form by monitoring quenching of the tryptophan fluorescence spectrum of either the component B (KD approximately 0.4 microM) or hydroxylase (two binding sites, KDa approximately 10 nM, KDb approximately 8 microM). The observed KD values are in agreement with the best fit values from the kinetic simulation. Through the use of the covalent zero length cross-linking reagent 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC), the binding sites of the component B and reductase were shown to be on the hydroxylase alpha and beta subunits, respectively. The alpha and beta subunits of the hydroxylase are cross-linked by EDC suggesting that they are juxtaposed. EDC also caused the rapid loss of the ability of the monomeric component B to stimulate the hydroxylation reaction suggesting that cross-linking of reactive groups on the protein surface had occurred. This effect was inhibited by the presence of hydroxylase and was accompanied by a loss of the ability of the component B to bind to the hydroxylase. Thus, formation of a component B-hydroxylase complex is apparently required for effective catalysis linked to NADH oxidation. When present in concentrations greater than required to saturate the initial hydroxylase complex, component B inhibited both the rate of the enzymic reaction and the cross-linking of the reductase to the hydroxylase. This suggests that a second complex involving component B can form that negatively regulates catalysis by preventing formation of the reductase-hydroxylase complex.

Envelope Protein Dynamics in Paramyxovirus Entry

PubMed Central

Plattet, Philippe; Plemper, Richard K.

2013-01-01

ABSTRACT Paramyxoviruses include major pathogens with significant global health and economic impact. This large family of enveloped RNA viruses infects cells by employing two surface glycoproteins that tightly cooperate to fuse their lipid envelopes with the target cell plasma membrane, an attachment and a fusion (F) protein. Membrane fusion is believed to depend on receptor-induced conformational changes within the attachment protein that lead to the activation and subsequent refolding of F. While structural and mechanistic studies have considerably advanced our insight into paramyxovirus cell adhesion and the structural basis of F refolding, how precisely the attachment protein links receptor engagement to F triggering remained poorly understood. Recent reports based on work with several paramyxovirus family members have transformed our understanding of the triggering mechanism of the membrane fusion machinery. Here, we review these recent findings, which (i) offer a broader mechanistic understanding of the paramyxovirus cell entry system, (ii) illuminate key similarities and differences between entry strategies of different paramyxovirus family members, and (iii) suggest new strategies for the development of novel therapeutics. PMID:23820396

Envelope protein dynamics in paramyxovirus entry.

PubMed

Plattet, Philippe; Plemper, Richard K

2013-07-02

Paramyxoviruses include major pathogens with significant global health and economic impact. This large family of enveloped RNA viruses infects cells by employing two surface glycoproteins that tightly cooperate to fuse their lipid envelopes with the target cell plasma membrane, an attachment and a fusion (F) protein. Membrane fusion is believed to depend on receptor-induced conformational changes within the attachment protein that lead to the activation and subsequent refolding of F. While structural and mechanistic studies have considerably advanced our insight into paramyxovirus cell adhesion and the structural basis of F refolding, how precisely the attachment protein links receptor engagement to F triggering remained poorly understood. Recent reports based on work with several paramyxovirus family members have transformed our understanding of the triggering mechanism of the membrane fusion machinery. Here, we review these recent findings, which (i) offer a broader mechanistic understanding of the paramyxovirus cell entry system, (ii) illuminate key similarities and differences between entry strategies of different paramyxovirus family members, and (iii) suggest new strategies for the development of novel therapeutics.

Origins of Allostery and Evolvability in Proteins: A Case Study.

PubMed

Raman, Arjun S; White, K Ian; Ranganathan, Rama

2016-07-14

Proteins display the capacity for adaptation to new functions, a property critical for evolvability. But what structural principles underlie the capacity for adaptation? Here, we show that adaptation to a physiologically distinct class of ligand specificity in a PSD95, DLG1, ZO-1 (PDZ) domain preferentially occurs through class-bridging intermediate mutations located distant from the ligand-binding site. These mutations provide a functional link between ligand classes and demonstrate the principle of "conditional neutrality" in mediating evolutionary adaptation. Structures show that class-bridging mutations work allosterically to open up conformational plasticity at the active site, permitting novel functions while retaining existing function. More generally, the class-bridging phenotype arises from mutations in an evolutionarily conserved network of coevolving amino acids in the PDZ family (the sector) that connects the active site to distant surface sites. These findings introduce the concept that allostery in proteins could have its origins not in protein function but in the capacity to adapt. Copyright © 2016 Elsevier Inc. All rights reserved.

Sensitive and Facile Electrochemiluminescent Immunoassay for Detecting Genetically Modified Rapeseed Based on Novel Carbon Nanoparticles.

PubMed

Gao, Hongfei; Wen, Luke; Wu, Yuhua; Yan, Xiaohong; Li, Jun; Li, Xiaofei; Fu, Zhifeng; Wu, Gang

2018-05-23

A highly sensitive electrochemiluminescent (ECL) immunoassay targeting PAT/ bar protein was facilely developed for genetically modified (GM) rapeseed detection using carbon nanoparticles (CNPs) originally prepared from printer toner. In this work, CNPs linked with antibody for PAT/ bar protein were used to modify a working electrode. After an immunoreaction between the PAT/ bar protein and its antibody, the immunocomplex formed on the electrode receptor region resulted in an inhibition of electron transfer between the electrode surface and the ECL substance, thus led to a decrease of ECL response. Under the optimal conditions, the ECL responses linearly decreased as the increase of the PAT/ bar protein concentration and the GM rapeseed RF3 content in the ranges of 0.10-10 ng/mL and 0.050-1.0%, with the limits of detection of 0.050 ng/mL and 0.020% (S/N = 3). These results open a facile, sensitive, and rapid approach for the safety control of agricultural GM rape.

Compounds from silicones alter enzyme activity in curing barnacle glue and model enzymes.

PubMed

Rittschof, Daniel; Orihuela, Beatriz; Harder, Tilmann; Stafslien, Shane; Chisholm, Bret; Dickinson, Gary H

2011-02-17

Attachment strength of fouling organisms on silicone coatings is low. We hypothesized that low attachment strength on silicones is, in part, due to the interaction of surface available components with natural glues. Components could alter curing of glues through bulk changes or specifically through altered enzyme activity. GC-MS analysis of silicone coatings showed surface-available siloxanes when the coatings were gently rubbed with a cotton swab for 15 seconds or given a 30 second rinse with methanol. Mixtures of compounds were found on 2 commercial and 8 model silicone coatings. The hypothesis that silicone components alter glue curing enzymes was tested with curing barnacle glue and with commercial enzymes. In our model, barnacle glue curing involves trypsin-like serine protease(s), which activate enzymes and structural proteins, and a transglutaminase which cross-links glue proteins. Transglutaminase activity was significantly altered upon exposure of curing glue from individual barnacles to silicone eluates. Activity of purified trypsin and, to a greater extent, transglutaminase was significantly altered by relevant concentrations of silicone polymer constituents. Surface-associated silicone compounds can disrupt glue curing and alter enzyme properties. Altered curing of natural glues has potential in fouling management.

Compounds from Silicones Alter Enzyme Activity in Curing Barnacle Glue and Model Enzymes

PubMed Central

Rittschof, Daniel; Orihuela, Beatriz; Harder, Tilmann; Stafslien, Shane; Chisholm, Bret; Dickinson, Gary H.

2011-01-01

Background Attachment strength of fouling organisms on silicone coatings is low. We hypothesized that low attachment strength on silicones is, in part, due to the interaction of surface available components with natural glues. Components could alter curing of glues through bulk changes or specifically through altered enzyme activity. Methodology/Principal Findings GC-MS analysis of silicone coatings showed surface-available siloxanes when the coatings were gently rubbed with a cotton swab for 15 seconds or given a 30 second rinse with methanol. Mixtures of compounds were found on 2 commercial and 8 model silicone coatings. The hypothesis that silicone components alter glue curing enzymes was tested with curing barnacle glue and with commercial enzymes. In our model, barnacle glue curing involves trypsin-like serine protease(s), which activate enzymes and structural proteins, and a transglutaminase which cross-links glue proteins. Transglutaminase activity was significantly altered upon exposure of curing glue from individual barnacles to silicone eluates. Activity of purified trypsin and, to a greater extent, transglutaminase was significantly altered by relevant concentrations of silicone polymer constituents. Conclusions/Significance Surface-associated silicone compounds can disrupt glue curing and alter enzyme properties. Altered curing of natural glues has potential in fouling management. PMID:21379573

In vitro and in vivo evaluation of bone morphogenetic protein-2 (BMP-2) immobilized collagen-coated polyetheretherketone (PEEK)

NASA Astrophysics Data System (ADS)

Du, Ya-Wei; Zhang, Li-Nan; Ye, Xin; Nie, He-Min; Hou, Zeng-Tao; Zeng, Teng-Hui; Yan, Guo-Ping; Shang, Peng

2015-03-01

Polyetheretherketone (PEEK) is regarded as one of the most potential candidates of biomaterials in spinal implant applications. However, as a bioinert material, PEEK plays a limited role in osteoconduction and osseointegration. In this study, recombinant human bone morphogenetic protein-2 (rhBMP-2) was immobilized onto the surface of collagen-coated PEEK in order to prepare a multi-functional material. After adsorbed onto the PEEK surface by hydrophobic interaction, collagen was cross-linked with N-(3-dimethylaminopropyl)-N'-ethyl carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS). EDC/NHS system also contributed to the immobilization of rhBMP-2. Water contact angle tests, XPS and SEM clearly demonstrated the surface changes. ELISA tests quantified the amount of rhBMP-2 immobilized and the release over a period of 30 d. In vitro evaluation proved that the osteogenesis differentiation rate was higher when cells were cultured on modified PEEK discs than on regular ones. In vivo tests were conducted and positive changes of major parameters were presented. This report demonstrates that the rhBMP-2 immobilized method for PEEK modification increase bioactivity in vitro and in vivo, suggesting its practicability in orthopedic and spinal clinical applications.

Introduction of a specific binding domain on myoglobin surface by new chemical modification.

PubMed

Hayashi, T; Ando, T; Matsuda, T; Yonemura, H; Yamada, S; Hisaeda, Y

2000-11-01

A new myoglobin, reconstituted with a modified zinc protoporphyrin, having a total of four ammonium groups at the terminal of the two propionate side chains was constructed to introduce a substrate binding site. The protein with a positively charged patch on the surface formed a stable complex with negatively charged substrates, such as hexacyanoferrate(III) and anthraquinonesulfonate via an electrostatic interaction. The complexation was monitored by fluorescence quenching due to singlet electron transfer from the photoexcited reconstituted zinc myoglobin to the substrates. The binding properties were evaluated by Stern-Volmer plots from the fluorescence quenching of the zinc myoglobin by a quencher. Particularly, anthraquinone-2,7-disulfonic acid showed a high affinity with a binding constant of 1.5 x 10(5) M(-1) in 10 mM phosphate buffer, pH 7.0. In contrast, the plots upon the addition of anthraquinone-2-sulfonic acid at different ionic strengths indicated that the complex was formed not only by an electrostatic interaction but also by a hydrophobic contact. The findings from the fluorescence studies conclude that the present system is a useful model for discussion of electron transfer via non-covalently linked donor-acceptor pairing on the protein surface.

Structural and functional analyses of DM43, a snake venom metalloproteinase inhibitor from Didelphis marsupialis serum.

PubMed

Neves-Ferreira, Ana G C; Perales, Jonas; Fox, Jay W; Shannon, John D; Makino, Débora L; Garratt, Richard C; Domont, Gilberto B

2002-04-12

DM43, an opossum serum protein inhibitor of snake venom metalloproteinases, has been completely sequenced, and its disulfide bond pattern has been experimentally determined. It shows homology to human alpha(1)B-glycoprotein, a plasma protein of unknown function and a member of the immunoglobulin supergene family. Size exclusion and dynamic laser light scattering data indicated that two monomers of DM43, each composed of three immunoglobulin-like domains, associated to form a homodimer in solution. Analysis of its glycan moiety showed the presence of N-acetylglucosamine, mannose, galactose, and sialic acid, most probably forming four biantennary N-linked chains. DM43 inhibited the fibrinogenolytic activities of bothrolysin and jararhagin and formed 1:1 stoichiometric stable complexes with both metalloproteinases. DM43 was ineffective against atrolysin C or A. No complex formation was detected between DM43 and jararhagin C, indicating the essential role of the metalloproteinase domain for interaction. Homology modeling based on the crystal structure of a killer cell inhibitory receptor suggested the existence of an I-type Ig fold, a hydrophobic dimerization surface and six surface loops potentially forming the metalloproteinase-binding surface on DM43.

Structure of the Plexin Ectodomain Bound by Semaphorin-Mimicking Antibodies

PubMed Central

Omiya, Ryusuke; Matoba, Kyoko; Baba, Takeshi; Suzuki, Sachiyo; Segawa, Hiroaki; Kumanogoh, Atsushi; Iwasaki, Kenji; Hattori, Kunihiro; Takagi, Junichi

2016-01-01

Semaphorin family proteins act on cells to mediate both repulsive and attractive guidance via binding to plexin family receptors, thereby playing fundamental roles in the morphogenesis and homeostasis of various tissues. Although semaphorin-plexin signaling is implicated in various diseases and is thus a target of intensive research, our mechanistic understanding of how semaphorins activate plexins on the cell surface is limited. Here, we describe unique anti-plexin-A1 antibodies that can induce a collapsed morphology in mouse dendritic cells as efficiently as the semaphorin 3A (Sema3A) ligand. Precise epitope analysis indicates that these “semaphorin-mimicking” antibodies dimerize cell-surface plexin-A1 by binding to the N-terminal sema domain of the plexin at sites away from the interface used by the Sema3A ligand. Structural analysis of plexin-A1 fragments using negative stain electron microscopy further revealed that this agonistic capacity is closely linked to the location and orientation of antibody binding. In addition, the full-length plexin-A1 ectodomain exhibited a highly curved “C” shape, reinforcing the very unusual dimeric receptor conformation of this protein at the cell surface when engaged with Sema3A or agonistic antibodies. PMID:27258772

Phage display on the base of filamentous bacteriophages: application for recombinant antibodies selection.

PubMed

Tikunova, N V; Morozova, V V

2009-10-01

The display of peptides and proteins on the surface of filamentous bacteriophage is a powerful methodology for selection of peptides and protein domains, including antibodies. An advantage of this methodology is the direct physical link between the phenotype and the genotype, as an analyzed polypeptide and its encoding DNA fragment exist in one phage particle. Development of phage display antibody libraries provides repertoires of phage particles exposing antibody fragments of great diversity. The biopanning procedure facilitates selection of antibodies with high affinity and specificity for almost any target. This review is an introduction to phage display methodology. It presents recombinant antibodies display in more details:, construction of phage libraries of antibody fragments and different strategies for the biopanning procedure.

Human Vitronectin-Derived Peptide Covalently Grafted onto Titanium Surface Improves Osteogenic Activity: A Pilot In Vivo Study on Rabbits.

PubMed

Cacchioli, Antonio; Ravanetti, Francesca; Bagno, Andrea; Dettin, Monica; Gabbi, Carlo

2009-10-01

Peptide and protein exploitation for the biochemical functionalization of biomaterial surfaces allowed fabricating biomimetic devices able to evoke and promote specific and advantageous cell functions in vitro and in vivo. In particular, cell adhesion improvement to support the osseointegration of implantable devices has been thoroughly investigated. This study was aimed at checking the biological activity of the (351-359) human vitronectin precursor (HVP) sequence, mapped on the human vitronectin protein; the peptide was covalently linked to the surface of titanium cylinders, surgically inserted in the femurs of New Zealand white rabbits and analyzed at short experimental time points (4, 9, and 16 days after surgery). To assess the osteogenic activity of the peptide, three vital fluorochromic bone markers were used (calcein green, xylenol orange, and calcein blue) to stain the areas of newly grown bone. Static and dynamic histomorphometric parameters were measured at the bone-implant interface and at different distances from the surface. The biological role of the (351-359)HVP sequence was checked by comparing peptide-grafted samples and controls, analyzing how and how much its effects change with time across the bone regions surrounding the implant surface. The results obtained reveal a major activity of the investigated peptide 4 days after surgery, within the bone region closest to the implant surface, and larger bone to implant contact 9 and 16 days after surgery. Thus, improved primary fixation of endosseous devices can be foreseen, resulting in an increased osteointegration.

Juvenile hormone-binding proteins of Melanoplus bivittatus identified by EFDA photoaffinity labeling

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

Winder, B.S.

1988-01-01

Proteins that bind juvenile hormone in the hemolymph and fat body of the grasshopper, Melanoplus bivittatus were identified by photoaffinity labeling with radiolabeled epoxyfarnesyl diazoacetate ({sup 3}H-EFDA), and were characterized by electrophoretic analysis. A protocol was developed which allowed detection of {sup 3}H-EFDA that was covalently linked to proteins upon exposure to ultraviolet light at 254 nm. Quantification of protein-linked {sup 3}H-EFDA by liquid scintillation spectrometry took advantage of the differential solubility of unlinked {sup 3}H-EFDA in toluene alone, and of the protein-linked {sup 3}H-EFDA in toluene plus the detergent, Triton X-100. Competition between EFDA and juvenile hormone (JH) formore » binding to JH-specific binding sites was measured by hydroxyapatite protein binding assays in the presence of radiolabeled JH or EFDA and competing non-radiolabeled hormone. The protein-linked EFDA was detected on fluorograms of SDS or nondenaturing polyacrylamide gels (PAGE), and by liquid scintillation spectrometry of membranes to which the proteins had been electrophoretically transferred. Proteins which specifically bound JH were identified by photolabeling proteins in the presence and absence of nonlabeled JH-III.« less

Olfactomedin-1 Has a V-shaped Disulfide-linked Tetrameric Structure*

PubMed Central

Pronker, Matti F.; Bos, Trusanne G. A. A.; Sharp, Thomas H.; Thies-Weesie, Dominique M. E.; Janssen, Bert J. C.

2015-01-01

Olfactomedin-1 (Olfm1; also known as noelin and pancortin) is a member of the olfactomedin domain-containing superfamily and a highly expressed neuronal glycoprotein important for nervous system development. It binds a number of secreted proteins and cell surface-bound receptors to induce cell signaling processes. Using a combined approach of x-ray crystallography, solution scattering, analytical ultracentrifugation, and electron microscopy we determined that full-length Olfm1 forms disulfide-linked tetramers with a distinctive V-shaped architecture. The base of the “V” is formed by two disulfide-linked dimeric N-terminal domains. Each of the two V legs consists of a parallel dimeric disulfide-linked coiled coil with a C-terminal β-propeller dimer at the tips. This agrees with our crystal structure of a C-terminal coiled-coil segment and β-propeller combination (Olfm1coil-Olf) that reveals a disulfide-linked dimeric arrangement with the β-propeller top faces in an outward exposed orientation. Similar to its family member myocilin, Olfm1 is stabilized by calcium. The dimer-of-dimers architecture suggests a role for Olfm1 in clustering receptors to regulate signaling and sheds light on the conformation of several other olfactomedin domain family members. PMID:25903135

Studies of the permeation properties of glomerular basement membrane: cross-linking renders glomerular basement membrane permeable to protein.

PubMed

Walton, H A; Byrne, J; Robinson, G B

1992-03-20

Cross-linking glomerular basement membrane (GBM) has been shown to render it more permeable to protein. Isolated pig GBM was cross-linked with dimethylmalonimidate which reacts selectively with lysine epsilon-NH2 groups or with glutaraldehyde, a less selective cross-linking agent. Studies of the ultrafiltration properties of these materials in vitro using cytochrome c, myoglobin, bovine serum albumin and immunoglobulin showed that cross-linking had markedly increased solvent and protein fluxes as compared with native membranes particularly at higher pressures. Filtration studies with serum demonstrated that the cross-linked membranes were more permeable to serum proteins. Thickness measurements under pressure indicated that cross-linked membrane was less compressed than native membrane as pressure was increased. Pore theory did not provide a suitable model for analysis of the results, but analysis of the results using the fibre-matrix hypothesis indicated that cross-linking had the effect of bundling together the fibres (type IV collagen) in the GBM matrix. The effect of cross-linking on filtration could be explained by a combination of contraction of the membrane, fibre bundling and increased rigidity compared with native membrane. Cross-linking of GBM might lead to long-term damage of the glomerular capillary wall in nephritis, so promoting proteinuria.

Expression of decay-accelerating factor (CD55), membrane cofactor protein (CD46) and CD59 in the human astroglioma cell line, D54-MG, and primary rat astrocytes.

PubMed

Yang, C; Jones, J L; Barnum, S R

1993-09-01

In this report, we have shown the expression of the complement regulatory proteins decay-accelerating factor (DAF, CD55), membrane cofactor protein (MCP, CD46) and CD59 on human D54-MG astroglioma cells by several methods, including immunofluorescence, flow cytometry and Western blotting and Northern blot analysis. These studies demonstrate that all three proteins are structurally and antigenically similar to their counterparts expressed on HepG2 and SW480 cells (hepatocyte and epithelial cell lines, respectively). D54-MG cells express mRNA for all three proteins of the appropriate size(s). The phosphatidylinositol-specific enzyme, PIPLC, cleaved DAF from the surface of D54-MG cells, demonstrating that DAF is linked by a glycophospholipid anchor as has been shown for other cell types. Flow cytometry demonstrates that primary rat astrocytes also constitutively express all three regulatory proteins. These data are the first to demonstrate the expression of CD59 on astrocytes, and the presence of all three regulatory proteins on astrocytes suggests that regulation of complement activation in the central nervous system is important in neural host defense mechanisms.

Reassembly of S-layer proteins

NASA Astrophysics Data System (ADS)

Pum, Dietmar; Sleytr, Uwe B.

2014-08-01

Crystalline bacterial cell surface layers (S-layers) represent the outermost cell envelope component in a broad range of bacteria and archaea. They are monomolecular arrays composed of a single protein or glycoprotein species and represent the simplest biological membranes developed during evolution. They are highly porous protein mesh works with unit cell sizes in the range of 3 to 30 nm, and pore sizes of 2 to 8 nm. S-layers are usually 5 to 20 nm thick (in archaea, up to 70 nm). S-layer proteins are one of the most abundant biopolymers on earth. One of their key features, and the focus of this review, is the intrinsic capability of isolated native and recombinant S-layer proteins to form self-assembled mono- or double layers in suspension, at solid supports, the air-water interface, planar lipid films, liposomes, nanocapsules, and nanoparticles. The reassembly is entropy-driven and a fascinating example of matrix assembly following a multistage, non-classical pathway in which the process of S-layer protein folding is directly linked with assembly into extended clusters. Moreover, basic research on the structure, synthesis, genetics, assembly, and function of S-layer proteins laid the foundation for their application in novel approaches in biotechnology, biomimetics, synthetic biology, and nanotechnology.

Large-scale recrystallization of the S-layer of Bacillus coagulans E38-66 at the air/water interface and on lipid films.

PubMed Central

Pum, D; Weinhandl, M; Hödl, C; Sleytr, U B

1993-01-01

S-layer protein isolated from Bacillus coagulans E38-66 could be recrystallized into large-scale coherent monolayers at an air/water interface and on phospholipid films spread on a Langmuir-Blodgett trough. Because of the asymmetry in the physiochemical surface properties of the S-layer protein, the subunits were associated with their more hydrophobic outer face with the air/water interface and oriented with their negatively charged inner face to the zwitterionic head groups of the dipalmitoylphosphatidylcholine and dipalmitoylphosphatidylethanolamine (DPPE) monolayer films. The dynamic crystal growth at both types of interfaces was first initiated at several distant nucleation points. The individual monocrystalline areas grew isotropically in all directions until the front edge of neighboring crystals was met. The recrystallized S-layer protein and the S-layer-DPPE layer could be chemically cross-linked from the subphase with glutaraldehyde. Images PMID:8478338

Antimicrobial Functions of Lactoferrin Promote Genetic Conflicts in Ancient Primates and Modern Humans.

PubMed

Barber, Matthew F; Kronenberg, Zev; Yandell, Mark; Elde, Nels C

2016-05-01

Lactoferrin is a multifunctional mammalian immunity protein that limits microbial growth through sequestration of nutrient iron. Additionally, lactoferrin possesses cationic protein domains that directly bind and inhibit diverse microbes. The implications for these dual functions on lactoferrin evolution and genetic conflicts with microbes remain unclear. Here we show that lactoferrin has been subject to recurrent episodes of positive selection during primate divergence predominately at antimicrobial peptide surfaces consistent with long-term antagonism by bacteria. An abundant lactoferrin polymorphism in human populations and Neanderthals also exhibits signatures of positive selection across primates, linking ancient host-microbe conflicts to modern human genetic variation. Rapidly evolving sites in lactoferrin further correspond to molecular interfaces with opportunistic bacterial pathogens causing meningitis, pneumonia, and sepsis. Because microbes actively target lactoferrin to acquire iron, we propose that the emergence of antimicrobial activity provided a pivotal mechanism of adaptation sparking evolutionary conflicts via acquisition of new protein functions.

Structure of Rotavirus Outer-Layer Protein VP7 Bound with a Neutralizing Fab

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

Aoki, Scott T.; Settembre, Ethan C.; Trask, Shane D.

2009-06-17

Rotavirus outer-layer protein VP7 is a principal target of protective antibodies. Removal of free calcium ions (Ca{sup 2+}) dissociates VP7 trimers into monomers, releasing VP7 from the virion, and initiates penetration-inducing conformational changes in the other outer-layer protein, VP4. We report the crystal structure at 3.4 angstrom resolution of VP7 bound with the Fab fragment of a neutralizing monoclonal antibody. The Fab binds across the outer surface of the intersubunit contact, which contains two Ca{sup 2+} sites. Mutations that escape neutralization by other antibodies suggest that the same region bears the epitopes of most neutralizing antibodies. The monovalent Fab ismore » sufficient to neutralize infectivity. We propose that neutralizing antibodies against VP7 act by stabilizing the trimer, thereby inhibiting the uncoating trigger for VP4 rearrangement. A disulfide-linked trimer is a potential subunit immunogen.« less

Plant immunity: a lesson from pathogenic bacterial effector proteins.

PubMed

Cui, Haitao; Xiang, Tingting; Zhou, Jian-Min

2009-10-01

Phytopathogenic bacteria inject an array of effector proteins into host cells to alter host physiology and assist the infection process. Some of these effectors can also trigger disease resistance as a result of recognition in the plant cell by cytoplasmic immune receptors. In addition to effector-triggered immunity, plants immunity can be triggered upon the detection of Pathogen/Microbe-Associated Molecular Patterns by surface-localized immune receptors. Recent progress indicates that many bacterial effector proteins use a variety of biochemical properties to directly attack key components of PAMP-triggered immunity and effector-triggered immunity, providing new insights into the molecular basis of plant innate immunity. Emerging evidence indicate that the evolution of disease resistance in plants is intimately linked to the mechanism by which bacterial effectors promote parasitism. This review focuses on how these studies have conceptually advanced our understanding of plant-pathogen interactions.

Generation of Micropatterned Substrates Using Micro Photopatterning

PubMed Central

Doyle, Andrew D.

2010-01-01

Micro photopatterning (µPP) has been developed to rapidly test and generate different patterns for extracellular matrix adsorption without being hindered with the process of making physical stamps through nanolithography techniques. It uses two-photon excitation guided through a point-scanning confocal microscope to locally photoablate poly(vinyl) alcohol (PVA) thin films in user-defined computer-controlled patterns. PVA thin films are ideal for surface blocking, being hydrophilic substrates that deter protein adsorption and cell attachment. Because gold substrates are not used during µPP, all live-cell fluorescent imaging techniques including total internal reflection fluorescence microscopy of GFP–linked proteins can be performed with minimal loss of fluorescence signal. Furthermore, because µPP does not require physical stamps for pattern generation, multiple ECMs or other proteins can be localized within microns of each other. This unit details the setup of µPP as well as giving troubleshooting techniques. PMID:20013752

Absence of an N-Linked Glycosylation Motif in the Glycoprotein of the Live-Attenuated Argentine Hemorrhagic Fever Vaccine, Candid #1, Results in Its Improper Processing, and Reduced Surface Expression.

PubMed

Manning, John T; Seregin, Alexey V; Yun, Nadezhda E; Koma, Takaaki; Huang, Cheng; Barral, José; de la Torre, Juan C; Paessler, Slobodan

2017-01-01

Junin virus (JUNV), a highly pathogenic New World arenavirus, is the causative agent of Argentine hemorrhagic fever (AHF). The live-attenuated Candid #1 (Can) strain currently serves as a vaccine for at-risk populations. We have previously shown that the Can glycoprotein (GPC) gene is the primary gene responsible for attenuation in a guinea pig model of AHF. However, the mechanisms through which the GPC contributes to the attenuation of the Can strain remain unknown. A more complete understanding of the mechanisms underlying the attenuation and immunogenicity of the Can strain will potentially allow for the rational design of additional safe and novel vaccines. Here, we provide a detailed comparison of both RNA and protein expression profiles between both inter- and intra-segment chimeric JUNV recombinant clones expressing combinations of genes from the Can strain and the pathogenic Romero (Rom) strain. The recombinant viruses that express Can GPC, which were shown to be attenuated in guinea pigs, displayed different RNA levels and GPC processing patterns as determined by Northern and Western blot analyses, respectively. Analysis of recombinant viruses containing amino acid substitutions selected at different mouse brain passages during the generation of Can revealed that altered Can GPC processing was primarily due to the T168A substitution within G1, which eliminates an N-linked glycosylation motif. Incorporation of the T168A substitution in the Rom GPC resulted in a Can-like processing pattern of Rom GPC. In addition, JUNV GPCs containing T168A substitution were retained within the endoplasmic reticulum (ER) and displayed significantly lower cell surface expression than wild-type Rom GPC. Interestingly, the reversion A168T in Can GPC significantly increased GPC expression at the cell surface. Our results demonstrate that recombinant JUNV (rJUNV) expressing Can GPC display markedly different protein expression and elevated genomic RNA expression when compared to viruses expressing Rom GPC. Additionally, our findings indicate that the N-linked glycosylation motif at amino acid positions 166-168 is important for trafficking of JUNV GPC to the cell surface, and the elimination of this motif interferes with the GPC release from the ER.

A closer look at the complex hydrophilic/hydrophobic interactions forces at the human hair surface

NASA Astrophysics Data System (ADS)

Baghdadli, N.; Luengo, G. S.; Recherche, L.

2008-03-01

The complex chemical structure of the hair surface is far from being completely understood. Current understanding is based on Rivett's model1 that was proposed to explain the macroscopic hydrophobic nature of the surface of natural hair. In this model covalently-linked fatty acids are chemically grafted to the amorphous protein (keratin) through a thio-ester linkage2,3. Nevertheless, experience like wetting and electrical properties of human hair surface4 shows that the complexity of the hair surface is not fully understand based on this model in literature. Recent studies in our laboratory show for the first time microscopic evidence of the heterogeneous physico-chemical character of the hair surface. By using Chemical Force Microscopy, the presence of hydrophobic and ionic species are detected and localized, before and after a cosmetic treatment (bleaching). Based on force curve analysis the mapping of the local distribution of hydrophilic and hydrophobic groups of hair surface is obtained. A discussion on a more plausible hair model and its implications will be presented based on these new results.

Highly sensitive photoelectrochemical biosensor for kinase activity detection and inhibition based on the surface defect recognition and multiple signal amplification of metal-organic frameworks.

PubMed

Wang, Zonghua; Yan, Zhiyong; Wang, Feng; Cai, Jibao; Guo, Lei; Su, Jiakun; Liu, Yang

2017-11-15

A turn-on photoelectrochemical (PEC) biosensor based on the surface defect recognition and multiple signal amplification of metal-organic frameworks (MOFs) was proposed for highly sensitive protein kinase activity analysis and inhibitor evaluation. In this strategy, based on the phosphorylation reaction in the presence of protein kinase A (PKA), the Zr-based metal-organic frameworks (UiO-66) accommodated with [Ru(bpy) 3 ] 2+ photoactive dyes in the pores were linked to the phosphorylated kemptide modified TiO 2 /ITO electrode through the chelation between the Zr 4+ defects on the surface of UiO-66 and the phosphate groups in kemptide. Under visible light irradiation, the excited electrons from [Ru(bpy) 3 ] 2+ adsorbed in the pores of UiO-66 injected into the TiO 2 conduction band to generate photocurrent, which could be utilized for protein kinase activities detection. The large surface area and high porosities of UiO-66 facilitated a large number of [Ru(bpy) 3 ] 2+ that increased the photocurrent significantly, and afforded a highly sensitive PEC analysis of kinase activity. The detection limit of the as-proposed PEC biosensor was 0.0049UmL -1 (S/N!=!3). The biosensor was also applied for quantitative kinase inhibitor evaluation and PKA activities detection in MCF-7 cell lysates. The developed visible-light PEC biosensor provides a simple detection procedure and a cost-effective manner for PKA activity assays, and shows great potential in clinical diagnosis and drug discoveries. Copyright © 2017 Elsevier B.V. All rights reserved.

A highly sensitive immunoassay for atrazine based on covalently linking the small molecule hapten to a urea-glutaraldehyde network on a polystyrene surface.

PubMed

Sai, Na; Sun, Wenjing; Wu, Yuntang; Sun, Zhong; Yu, Guanggui; Huang, Guowei

2016-11-01

A new enzyme-linked immunosorbent assay (ELISA) for atrazine was developed based on covalent bonding of the small molecule hapten, 2-mercaptopropionic acid-4-ethylamino-6-isopropylamino-1,3,5-triazine (MPA-atrazine), to urea-glutaraldehyde (UGA)-treated microtiter plates. In this assay, the microtiter plate surface was treated with the UGA network to both introduce amino groups, which were used to cross-link with the hapten carboxylate groups, and efficiently prevent non-specific adsorption of antibodies, which successfully eliminated the time-consuming routine blocking step. Compared with HNO 3 -H 2 SO 4 -APTES-hapten coated ELISA (modified with a HNO 3 -H 2 SO 4 -APTES mixture and covalent-linked hapten) and conventional ELISA (coated with hapten-carrier protein conjugates), the novel ELISA format increased the sensitivity by approximately 3.5-fold and 7.5-fold, respectively, and saved 2.5h and 34h of coating hapten time, respectively. The method's 50% inhibition concentration for atrazine was 5.54ngmL -1 , and the limit of detection was 0.16ngmL -1 after optimization of reaction conditions. Furthermore, the ELISA was adapted for analysis of atrazine in corn, rice, and water samples, demonstrating recoveries of 90%-108%. Thus, the assay provides a convenient alternative to conventional, laborious immunoassays for routine supervision of residue detection in food and the environment. Copyright © 2016 Elsevier B.V. All rights reserved.

Brain Morphology Links Systemic Inflammation to Cognitive Function in Midlife Adults

PubMed Central

Marsland, Anna L.; Gianaros, Peter J.; Kuan, Dora C-H.; Sheu, Lei K.; Krajina, Katarina; Manuck, Stephen B.

2015-01-01

Background Inflammation is linked to cognitive decline in midlife, but the neural basis for this link is unclear. One possibility is that inflammation associates with adverse changes in brain morphology, which accelerates cognitive aging and later dementia risk. Clear evidence is lacking, however, regarding whether inflammation relates to cognition in midlife via changes in brain morphology. Accordingly, the current study examines whether associations of inflammation with cognitive function are mediated by variation in cortical gray matter volume among midlife adults. Methods Plasma levels of interleukin (IL)-6 and C-reactive protein (CRP), relatively stable markers of peripheral systemic inflammation, were assessed in 408 community volunteers aged 30–54 years. All participants underwent structural neuroimaging to assess global and regional brain morphology and completed neuropsychological tests sensitive to early changes in cognitive function. Measurements of brain morphology (regional tissue volumes and cortical thickness and surface area) were derived using Freesurfer. Results Higher peripheral inflammation was associated with poorer spatial reasoning, short term memory, verbal proficiency, learning and memory, and executive function, as well as lower cortical gray and white matter volumes, hippocampal volume and cortical surface area. Mediation models with age, sex and intracranial volume as covariates showed cortical gray matter volume to partially mediate the association of inflammation with cognitive performance. Exploratory analyses of body mass suggested that adiposity may be a source of the inflammation linking brain morphology to cognition. Conclusions Inflammation and adiposity might relate to cognitive decline via influences on brain morphology. PMID:25882911

High confidence proteomic analysis of yeast LDs identifies additional droplet proteins and reveals connections to dolichol synthesis and sterol acetylation.

PubMed

Currie, Erin; Guo, Xiuling; Christiano, Romain; Chitraju, Chandramohan; Kory, Nora; Harrison, Kenneth; Haas, Joel; Walther, Tobias C; Farese, Robert V

2014-07-01

Accurate protein inventories are essential for understanding an organelle's functions. The lipid droplet (LD) is a ubiquitous intracellular organelle with major functions in lipid storage and metabolism. LDs differ from other organelles because they are bounded by a surface monolayer, presenting unique features for protein targeting to LDs. Many proteins of varied functions have been found in purified LD fractions by proteomics. While these studies have become increasingly sensitive, it is often unclear which of the identified proteins are specific to LDs. Here we used protein correlation profiling to identify 35 proteins that specifically enrich with LD fractions of Saccharomyces cerevisiae Of these candidates, 30 fluorophore-tagged proteins localize to LDs by microscopy, including six proteins, several with human orthologs linked to diseases, which we newly identify as LD proteins (Cab5, Rer2, Say1, Tsc10, YKL047W, and YPR147C). Two of these proteins, Say1, a sterol deacetylase, and Rer2, a cis-isoprenyl transferase, are enzymes involved in sterol and polyprenol metabolism, respectively, and we show their activities are present in LD fractions. Our results provide a highly specific list of yeast LD proteins and reveal that the vast majority of these proteins are involved in lipid metabolism. Copyright © 2014 by the American Society for Biochemistry and Molecular Biology, Inc.

Glycoprotein biosynthesis in animal cells grown in suspension culture. Assembly of lipid-linked saccharides and formation of protein-bound 'high-mannose' oligosaccharides.

PubMed Central

Bailey, D S; Burke, J; Sinclair, R; Mukherjee, B B

1981-01-01

Glycoprotein biosynthesis was studied with mouse L-cells grown in suspension culture. Glucose-deprived cells incorporated [3H]mannose into 'high-mannose' protein-bound oligosaccharides and a few relatively high-molecular-weight lipid-linked oligosaccharides. The latter were retained by DEAE-cellulose and turned over quite slowly during pulse--chase experiments. Increased heterogeneity in size of lipid-linked oligosaccharides developed during prolonged glucose deprivation. Sequential elongation of lipid-linked oligosaccharides was also observed, and conditions that prevented the assembly of the higher lipid-linked oligosaccharides also prevented the formation of the larger protein-bound 'high-mannose' oligosaccharides. In parallel experiments, [3H]mannose was incorporated into a total polyribosome fraction, suggesting that mannose residues were transferred co-translationally to nascent protein. Membrane preparations from these cells catalysed the assembly from UDP-N-acetyl-D-[6-3H]glucosamine and GDP-D-[U-14C]mannose of polyisoprenyl diphosphate derivatives whose oligosaccharide moieties were heterogeneous in size. Elongation of the N-acetyl-D-[6-3H]glucosamine-initiated glycolipids with mannose residues produced several higher lipid-linked oligosaccharides similar to those seen during glucose deprivation in vivo. Glucosylation of these mannose-containing oligosaccharides from UDP-D-[6-3H]glucose was restricted to those of a relatively high molecular weight. Protein-bound saccharides formed in vitro were mainly smaller in size than those assembled on the lipid acceptors. These results support the involvement of lipid-linked saccharides in the synthesis of asparagine-linked glycoproteins, but show both in vivo and in vitro that protein-bound 'high-mannose' oligosaccharide formation can occur independently of higher lipid-linked oligosaccharide synthesis. PMID:7306042

Targeted Resequencing and Functional Testing Identifies Low-Frequency Missense Variants in the Gene Encoding GARP as Significant Contributors to Atopic Dermatitis Risk.

PubMed

Manz, Judith; Rodríguez, Elke; ElSharawy, Abdou; Oesau, Eva-Maria; Petersen, Britt-Sabina; Baurecht, Hansjörg; Mayr, Gabriele; Weber, Susanne; Harder, Jürgen; Reischl, Eva; Schwarz, Agatha; Novak, Natalija; Franke, Andre; Weidinger, Stephan

2016-12-01

Gene-mapping studies have consistently identified a susceptibility locus for atopic dermatitis and other inflammatory diseases on chromosome band 11q13.5, with the strongest association observed for a common variant located in an intergenic region between the two annotated genes C11orf30 and LRRC32. Using a targeted resequencing approach we identified low-frequency and rare missense mutations within the LRRC32 gene encoding the protein GARP, a receptor on activated regulatory T cells that binds latent transforming growth factor-β. Subsequent association testing in more than 2,000 atopic dermatitis patients and 2,000 control subjects showed a significant excess of these LRRC32 variants in individuals with atopic dermatitis. Structural protein modeling and bioinformatic analysis predicted a disruption of protein transport upon these variants, and overexpression assays in CD4 + CD25 - T cells showed a significant reduction in surface expression of the mutated protein. Consistently, flow cytometric (FACS) analyses of different T-cell subtypes obtained from atopic dermatitis patients showed a significantly reduced surface expression of GARP and a reduced conversion of CD4 + CD25 - T cells into regulatory T cells, along with lower expression of latency-associated protein upon stimulation in carriers of the LRRC32 A407T variant. These results link inherited disturbances of transforming growth factor-β signaling with atopic dermatitis risk. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

Protein-Glycan Quinary Interactions in Crowding Environment Unveiled by NMR Spectroscopy.

PubMed

Diniz, Ana; Dias, Jorge S; Jiménez-Barbero, Jesús; Marcelo, Filipa; Cabrita, Eurico J

2017-09-21

Protein-glycan interactions as modulators for quinary structures in crowding environments were explored. The interaction between human galectin 3 (Gal-3) and distinct macromolecular crowders, such as bovine and human serum albumin (BSA and HSA), Ficoll 70 and PEG3350, was scrutinized. The molecular recognition event of the specific ligand, lactose, by Gal-3 in crowding conditions was evaluated. Gal-3 interactions were monitored by NMR analysing chemical shift perturbation (CSP) and line broadening of 1 H 15 N-HSQC signals. The intensity of the Gal-3 1 H 15 N-HSQC signals decreased in the presence of all crowders, due to the increase in the solution viscosity and to the formation of large protein complexes. When glycosylated containing samples of BSA and HSA were used, signal broadening was more severe than that observed in the presence of the more viscous solutions of PEG3350 and Ficoll 70. However, for the samples containing glycoproteins, the signal intensity of 1 H 15 N-HSQC recovered upon addition of lactose. We show that serum proteins interact with Gal-3, through their α2,3-linked sialylgalactose moieties exposed at their surfaces, competing with lactose for the same binding site. The quinary interaction between Gal-3 and serum glycoproteins, could help to co-localize Gal-3 at the cell surface, and may play a role in adhesion and signalling functions of this protein. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

HLA class I is most tightly linked to levels of tapasin compared with other antigen-processing proteins in glioblastoma.

PubMed

Thuring, Camilla; Follin, Elna; Geironson, Linda; Freyhult, Eva; Junghans, Victoria; Harndahl, Mikkel; Buus, Søren; Paulsson, Kajsa M

2015-09-15

Tumour cells can evade the immune system by dysregulation of human leukocyte antigens (HLA-I). Low quantity and/or altered quality of HLA-I cell surface expression is the result of either HLA-I alterations or dysregulations of proteins of the antigen-processing machinery (APM). Tapasin is an APM protein dedicated to the maturation of HLA-I and dysregulation of tapasin has been linked to higher malignancy in several different tumours. We studied the expression of APM components and HLA-I, as well as HLA-I tapasin-dependency profiles in glioblastoma tissues and corresponding cell lines. Tapasin displayed the strongest correlation to HLA-I heavy chain but also clustered with β2-microglobulin, transporter associated with antigen processing (TAP) and LMP. Moreover, tapasin also correlated to survival of glioblastoma patients. Some APM components, for example, TAP1/TAP2 and LMP2/LMP7, showed variable but coordinated expression, whereas ERAP1/ERAP2 displayed an imbalanced expression pattern. Furthermore, analysis of HLA-I profiles revealed variable tapasin dependence of HLA-I allomorphs in glioblastoma patients. Expression of APM proteins is highly variable between glioblastomas. Tapasin stands out as the APM component strongest correlated to HLA-I expression and we proved that HLA-I profiles in glioblastoma patients include tapasin-dependent allomorphs. The level of tapasin was also correlated with patient survival time. Our results support the need for individualisation of immunotherapy protocols.

Sperm-binding fibronectin type II-module proteins are genetically linked and functionally related.

PubMed

Ekhlasi-Hundrieser, Mahnaz; Schäfer, Bettina; Philipp, Ute; Kuiper, Heidi; Leeb, Tosso; Mehta, Meenal; Kirchhoff, Christiane; Töpfer-Petersen, Edda

2007-05-01

Fibronectin type II (Fn2) module-containing proteins in the male genital tract are characterized by different numbers of Fn2 modules. Predominantly two classes exist which are distinct by having either two or four Fn2 modules. Minor variants with three Fn2 modules were also found in the human and the porcine epididymis. To reveal their relationship, mRNAs and proteins of representatives of these classes were studied in human, in Sus scrofa, and in rodents. Adult boars expressed members of both classes, i.e. ELSPBP1 and pB1, in subsequent regions of the epididymis, and both were under androgenic control. Human and rodent epididymides, on the other hand, alternatively contained only representatives of one of these two classes, i.e. ELSPBP1 in the human and two different pB1-related counterparts in rodents. ELSPBP1 and pB1-related genomic sequences were closely linked in chromosomal regions HSA 19q and SSC 6 q11-q21; conserved synteny between these regions is well established. On the other hand, in a syntenic region on mouse chromosome 7, ELSPBP1-related sequences were lacking. Tight binding to the sperm membrane via a choline-mediated mechanism was a common feature of the two classes of Fn2-module proteins, suggesting related function(s). However, differences in their regionalized expression patterns along the male genital tract as well as in association sites on the sperm surface suggested a species-specific sequential order in sperm binding.

Biosynthesis and processing of bovine cartilage link proteins.

PubMed

Hering, T M; Sandell, L J

1990-02-05

We have examined posttranslational modifications which are responsible for converting an apparently single precursor (Hering, T. M., and Sandell, L. J. (1988) J. Biol. Chem. 263, 1030-1036) to the two major forms of link protein in bovine articular cartilage. Resistance to endoglycosidases H and F suggests that Asn-linked oligosaccharides of link protein secreted by bovine chondrocytes in culture are of the complex or hybrid type. There is no evidence for O-linked oligosaccharides. There is no apparent precursor-product relationship between link protein (LP)1 and LP2, since after a short pulse with [3H]leucine two forms are present, consistent with the existence of two glycosylation sites. An immunoprecipitate of LP1 from pulse-labeled chondrocytes was observed to show a decrease in electrophoretic mobility and increased microheterogeneity during transit through the Golgi, whereas LP2 did not change. During processing both LP1 and LP2 become endoglycosidase H resistant. LP1, but not LP2, can be biosynthetically labeled with [35S]sulfate. Incorporation of [35S]sulfate is inhibited by tunicamycin, indicating that the sulfate is associated with Asn-linked carbohydrate. Sulfation may be important for normal processing, secretion, or degradation of link protein and with sialylation may confer considerable charge heterogeneity upon LP1. We conclude that there are considerable biochemical differences between glycoproteins LP1 and LP2 which may provide a basis for functional differences.

Novel Soluble Dietary Fiber-Tannin Self-Assembled Film: A Promising Protein Protective Material.

PubMed

Song, Guo-Bin; Xu, Juan; Zheng, Hua; Feng, Ying; Zhang, Wen-Wen; Li, Kun; Ge, Shuang-shuang; Li, Kai; Zhang, Hong

2015-06-24

In this experiment, a natural promising protein protective film was fabricated through soluble dietary fiber (SDF)-tannin nanocluster self-assembly. FT-IR, XRD, and DSC tests were employed to investigate the interaction between the SDF and tannins before and after cross-linking induced by calcium ion. On the other hand, referring to the SEM and TEM results, the self-assembly process of the protein protective film could be indicated as follows: first, calcium ion, with its cross-ability, served as the "nucleus"; SDF and tannins were combined to prepare the nanoscale SDF-tannin clusters; then, the clusters were homogeneously deposited on the surface of protein to form a protective film by self-assembling hydrogen bond between tannin component of clusters as "adhesive" and protein in aqueous solutions under very mild conditions. Film thickness could also be controlled by tannin of different concentrations ranging from 114 to 1384 μm. Antibacterial test and in vitro cytotoxicity test proved that the film had a broad spectrum of antimicrobial properties and excellent cell biocompatibility, respectively, which might open up new applications in the food preservation and biomedical fields.

Interaction of Tim23 with Tim50 Is essential for protein translocation by the mitochondrial TIM23 complex.

PubMed

Gevorkyan-Airapetov, Lada; Zohary, Keren; Popov-Celeketic, Dusan; Mapa, Koyeli; Hell, Kai; Neupert, Walter; Azem, Abdussalam; Mokranjac, Dejana

2009-02-20

The TIM23 complex is the major translocase of the mitochondrial inner membrane responsible for the import of essentially all matrix proteins and a number of inner membrane proteins. Tim23 and Tim50, two essential proteins of the complex, expose conserved domains into the intermembrane space that interact with each other. Here, we describe in vitro reconstitution of this interaction using recombinantly expressed and purified intermembrane space domains of Tim50 and Tim23. We established two independent methods, chemical cross-linking and surface plasmon resonance, to track their interaction. In addition, we identified mutations in Tim23 that abolish its interaction with Tim50 in vitro. These mutations also destabilized the interaction between the two proteins in vivo, leading to defective import of preproteins via the TIM23 complex and to cell death at higher temperatures. This is the first study to describe the reconstitution of the Tim50-Tim23 interaction in vitro and to identify specific residues of Tim23 that are vital for the interaction with Tim50.

The Campylobacter jejuni/coli cjaA (cj0982c) gene encodes an N-glycosylated lipoprotein localized in the inner membrane.

PubMed

Wyszyńska, Agnieszka; Zycka, Joanna; Godlewska, Renata; Jagusztyn-Krynicka, Elzbieta K

2008-09-01

The Campylobacter coli 72Dz/92 cjaA gene (orthologue of cj0982c of C. jejuni NCTC 11168) product is a highly immunogenic, amino acid-binding protein. CjaA was palmitic acid-modified when processed in E. coli. In addition, site-directed mutagenesis of the Cys residue of the LAAC motif of its signal sequence confirmed that CjaA is a lipoprotein when processed in Campylobacter. Localization of the protein appeared to be host dependent. In Campylobacter, CjaA was recovered mainly as an inner-membrane protein, whereas in E. coli most of the protein was present in the periplasmic space. Interestingly, antiserum raised against Campylobacter glycine-extracted material also recognized CjaA produced by Campylobacter and Escherichia coli, indicating that at least part of the protein may be surface exposed. Site-directed mutagenesis of the Asn residues of two putative N-linked glycosylation sites (NIS and NFT) showed that CjaA is glycosylated and that only the first N-X-S/T sequeon serves as a glycan acceptor.

Endorepellin causes endothelial cell disassembly of actin cytoskeleton and focal adhesions through α2β1 integrin

PubMed Central

Bix, Gregory; Fu, Jian; Gonzalez, Eva M.; Macro, Laura; Barker, Amy; Campbell, Shelly; Zutter, Mary M.; Santoro, Samuel A.; Kim, Jiyeun K.; Höök, Magnus; Reed, Charles C.; Iozzo, Renato V.

2004-01-01

Endorepellin, the COOH-terminal domain of the heparan sulfate proteoglycan perlecan, inhibits several aspects of angiogenesis. We provide evidence for a novel biological axis that links a soluble fragment of perlecan protein core to the major cell surface receptor for collagen I, α2β1 integrin, and provide an initial investigation of the intracellular signaling events that lead to endorepellin antiangiogenic activity. The interaction between endorepellin and α2β1 integrin triggers a unique signaling pathway that causes an increase in the second messenger cAMP; activation of two proximal kinases, protein kinase A and focal adhesion kinase; transient activation of p38 mitogen-activated protein kinase and heat shock protein 27, followed by a rapid down-regulation of the latter two proteins; and ultimately disassembly of actin stress fibers and focal adhesions. The end result is a profound block of endothelial cell migration and angiogenesis. Because perlecan is present in both endothelial and smooth muscle cell basement membranes, proteolytic activity during the initial stages of angiogenesis could liberate antiangiogenic fragments from blood vessels' walls, including endorepellin. PMID:15240572

Pilus hijacking by a bacterial coaggregation factor critical for oral biofilm development.

PubMed

Reardon-Robinson, Melissa E; Wu, Chenggang; Mishra, Arunima; Chang, Chungyu; Bier, Naomi; Das, Asis; Ton-That, Hung

2014-03-11

The formation of dental plaque, a highly complex biofilm that causes gingivitis and periodontitis, requires specific adherence among many oral microbes, including the coaggregation of Actinomyces oris with Streptococcus oralis that helps to seed biofilm development. Here, we report the discovery of a key coaggregation factor for this process. This protein, which we named coaggregation factor A (CafA), is one of 14 cell surface proteins with the LPXTG motif predicted in A. oris MG1, whose function was hitherto unknown. By systematic mutagenesis of each of these genes and phenotypic characterization, we found that the Actinomyces/Streptococcus coaggregation is only abolished by deletion of cafA. Subsequent biochemical and cytological experiments revealed that CafA constitutes the tip of a unique form of the type 2 fimbria long known for its role in coaggregation. The direct and predominant role of CafA in adherence is evident from the fact that CafA or an antibody against CafA inhibits coaggregation, whereas the shaft protein FimA or a polyclonal antibody against FimA has no effect. Remarkably, FimA polymerization was blocked by deletion of genes for both CafA and FimB, the previously described tip protein of the type 2 fimbria. Together, these results indicate that some surface proteins not linked to a pilus gene cluster in Gram-positive bacteria may hijack the pilus. These unique tip proteins displayed on a common pilus shaft may serve distinct physiological functions. Furthermore, the pilus shaft assembly in Gram-positive bacteria may require a tip, as is true for certain Gram-negative bacterial pili.

Rosetta stone method for detecting protein function and protein-protein interactions from genome sequences

DOEpatents

Eisenberg, David; Marcotte, Edward M.; Pellegrini, Matteo; Thompson, Michael J.; Yeates, Todd O.

2002-10-15

A computational method system, and computer program are provided for inferring functional links from genome sequences. One method is based on the observation that some pairs of proteins A' and B' have homologs in another organism fused into a single protein chain AB. A trans-genome comparison of sequences can reveal these AB sequences, which are Rosetta Stone sequences because they decipher an interaction between A' and B. Another method compares the genomic sequence of two or more organisms to create a phylogenetic profile for each protein indicating its presence or absence across all the genomes. The profile provides information regarding functional links between different families of proteins. In yet another method a combination of the above two methods is used to predict functional links.

NMDA Receptor Autoantibodies in Autoimmune Encephalitis Cause a Subunit-Specific Nanoscale Redistribution of NMDA Receptors.

PubMed

Ladépêche, Laurent; Planagumà, Jesús; Thakur, Shreyasi; Suárez, Irina; Hara, Makoto; Borbely, Joseph Steven; Sandoval, Angel; Laparra-Cuervo, Lara; Dalmau, Josep; Lakadamyali, Melike

2018-06-26

Anti-N-methyl-D-aspartate receptor (NMDAR) encephalitis is a severe neuropsychiatric disorder mediated by autoantibodies against the GluN1 subunit of the NMDAR. Patients' antibodies cause cross-linking and internalization of NMDAR, but the synaptic events leading to depletion of NMDAR are poorly understood. Using super-resolution microscopy, we studied the effects of the autoantibodies on the nanoscale distribution of NMDAR in cultured neurons. Our findings show that, under control conditions, NMDARs form nanosized objects and patients' antibodies increase the clustering of synaptic and extrasynaptic receptors inside the nano-objects. This clustering is subunit specific and predominantly affects GluN2B-NMDARs. Following internalization, the remaining surface NMDARs return to control clustering levels but are preferentially retained at the synapse. Monte Carlo simulations using a model in which antibodies induce NMDAR cross-linking and disruption of interactions with other proteins recapitulated these results. Finally, activation of EphB2 receptor partially antagonized the antibody-mediated disorganization of the nanoscale surface distribution of NMDARs. Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.

Integrated antibacterial and antifouling surfaces via cross-linking chitosan-g-eugenol/zwitterionic copolymer on electrospun membranes.

PubMed

Li, Zhenguang; Hu, Wenhong; Zhao, Yunhui; Ren, Lixia; Yuan, Xiaoyan

2018-04-27

Integrated antibacterial and antifouling surfaces in favor of avoiding implant-related infections are necessarily required for biomaterials when they contact with the body fluid. In this work, an antibacterial and antifouling membrane was developed via cross-linking chitosan-g-eugenol and the zwitterionic copolymer poly(sulfobetaine methylacrylate-co-2-aminoethyl methacrylate) on the electrospun polycarbonate urethane substrate using genipin as a cross-linker. Antibacterial assays demonstrated that the prepared membranes had efficient antibacterial activity with 92.8 ± 2.5% and 95.2 ± 1.3% growth inhibition rates against Escherichia coli and Staphylococcus aureus, respectively. The investigations on antifouling activity and hemocompatibility of the membranes showed significant resistances to bacterial attachment, non-specific protein adsorption and platelet adhesion, and presented lower hemolytic activity and good anticoagulant activity as well. Moreover, cell culture assays indicated that the prepared membranes exerted no obvious cytotoxicity with more than 80% of relative L929 fibroblast viability. Therefore, the membranes with integrated antibacterial and antifouling properties could be potentially applied in promising indwelling devices. Copyright © 2018 Elsevier B.V. All rights reserved.

One-pot synthesis of redox-responsive polymers-coated mesoporous silica nanoparticles and their controlled drug release.

PubMed

Sun, Jiao-Tong; Piao, Ji-Gang; Wang, Long-Hai; Javed, Mohsin; Hong, Chun-Yan; Pan, Cai-Yuan

2013-09-01

A versatile one-pot strategy for the preparation of reversibly cross-linked polymer-coated mesoporous silica nanoparticles (MSNs) via surface reversible addition-fragmentation chain transfer (RAFT) polymerization is presented for the first time in this paper. The less reactive monomer oligo(ethylene glycol) acrylate (OEGA) and the more reactive cross-linker N,N'-cystaminebismethacrylamide (CBMA) are chosen to be copolymerized on the external surfaces of RAFT agent-functionalized MSNs to form the cross-linked polymer shells. Owing to the reversible cleavage and restoration of disulfide bonds via reduction/oxidation reactions, the polymer shells can control the on/off switching of the nanopores and regulate the drug loading and release. The redox-responsive release of doxorubicin (DOX) from this drug carrier is realized. The protein adsorption, in vitro cytotoxicity assays, and endocytosis studies demonstrate that this biocompatible vehicle is a potential candidate for delivering drugs. It is expected that this versatile grafting strategy may help fabricate satisfying MSN-based drug delivery systems for clinical application. © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Functional anatomy and immunological interactions of ocular surface and adnexa.

PubMed

Paulsen, Friedrich

2008-01-01

This chapter gives an overview about the structures and physiology of the ocular surface and its adnexa and focuses in a second part on the possible meaning of eye-associated lymphoid tissue (EALT) in a context with the development of dry eye. Sections deal with (1) anatomy of the ocular surface, lacrimal gland, eyelid and nasolacrimal ducts. (2) The meaning and importance of the lacrimal functional unit and the function of the mucosal innate immune system are briefly summarized. (3) Finally, the occurrence and the possible function of EALT is discussed with regard to tolerance induction and dry eye. The epithelial surface of the eye and its specialized glandular infoldings produce the components of the tear film, which include water, protective antimicrobials, cytokines, lipids as well as mucins and trefoil factor family (TFF) peptides. Antimicrobials, mucins and TFF peptides perform a number of essential functions which, collectively, provide protection of the ocular surface. Their production changes in cases of dry eye. The development of EALT is a common feature frequently occurring in symptomatically normal conjunctiva and nasolacrimal ducts. The production of antimicrobials, mucins and TFF peptides can be linked with cell signaling, tear film rheology, and antimicrobial defense at the ocular surface. Changes in the production of such peptides and proteins in cases of dry eye support the assumption that these peptides and proteins are involved in the pathophysiological events that occur at the ocular surface and lacrimal apparatus. Whether special types of bacteria, viruses, or other factors, e.g., immune deviation, are responsible for the development of EALT in humans requires further investigation in prospective and experimental studies.

Design of a nanocomposite substrate inducing adult stem cell assembly and progression toward an Epiblast-like or Primitive Endoderm-like phenotype via mechanotransduction.

PubMed

Morena, Francesco; Armentano, Ilaria; Montanucci, Pia; Argentati, Chiara; Fortunati, Elena; Montesano, Simona; Bicchi, Ilaria; Pescara, Teresa; Pennoni, Ilaria; Mattioli, Samantha; Torre, Luigi; Latterini, Loredana; Emiliani, Carla; Basta, Giuseppe; Calafiore, Riccardo; Kenny, Josè Maria; Martino, Sabata

2017-11-01

This work shows that the active interaction between human umbilical cord matrix stem cells and Poly (l-lactide)acid (PLLA) and PLLA/Multi Walled Carbon Nanotubes (MWCNTs) nanocomposite films results in the stem cell assembly as a spheroid conformation and affects the stem cell fate transition. We demonstrated that spheroids directly respond to a tunable surface and the bulk properties (electric, dielectric and thermal) of plain and nanocomposite PLLA films by triggering a mechanotransduction axis. This stepwise process starts from tethering of the cells' focal adhesion proteins to the surface, together with the adherens junctions between cells. Both complexes transmit traction forces to F-Actin stress fibres that link Filamin-A and Myosin-IIA proteins, generating a biological scaffold, with increased stiffening conformation from PLLA to PLLA/MWCNTs, and enable the nucleoskeleton proteins to boost chromatin reprogramming processes. Herein, the opposite expression of NANOG and GATA6 transcription factors, together with other lineage specification related proteins, steer spheroids toward an Epiblast-like or Primitive Endoderm-like lineage commitment, depending on the absence or presence of 1 wt% MWCNTs, respectively. This work represents a pioneering effort to create a stem cell/material interface that can model the stem cell fate transition under growth culture conditions. Copyright © 2017 Elsevier Ltd. All rights reserved.

Role of Protein Kinase C in Endothelin Converting Enzyme-1 trafficking and shedding from endothelial cells

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

Kuruppu, Sanjaya, E-mail: Sanjaya.Kuruppu@med.monash.edu.au; Tochon-Danguy, Natalie; Ian Smith, A.

2010-07-23

Research highlights: {yields} PKC activation increases the trafficking of ECE-1 to the cell surface. {yields} This in turn leads to an increase in the amount of ECE-1 shed. {yields} Only the catalytically active C-terminal region is shed from the cell surface. -- Abstract: This study aimed to determine the consequences of Protein Kinase C (PKC) mediated Endothelin Converting Enzyme-1 (ECE-1) phosphorylation and its relationship to ECE-1 expression and shedding. The proteins on the surface of EA.hy926 cells were labelled with EZ-Link NHS-SS-Biotin both prior to (control) and following stimulation by 2 {mu}M phorbol 12-myristate 13-acetate (PMA) which activates PKC. Themore » biotinylated proteins were isolated using neutravidin beads, resolved by gel electrophoresis and analysed by western blotting using anti-ECE-1 antibodies. Significant increase in ECE-1 expression at the cell surface was observed following stimulation by PMA, compared to unstimulated control cells (170 {+-} 32.3% of control, n = 5). The ECE-1 activity (expressed as {mu}M substrate cleaved/min) was determined by monitoring the cleavage of a quenched fluorescent substrate. The specificity of cleavage was confirmed using the ECE-1 inhibitor (CGS35066). The stimulation of cells by PMA (1 {mu}M, 6 h) significantly increased the ECE-1 activity (0.28 {+-} 0.02; n = 3) compared to the control (0.07 {+-} 0.02; n = 3). This increase was prevented by prior incubation with the PKC inhibitor bisindolymaleimide (BIM; 2 {mu}M for 1 h; 0.10 {+-} 0.01; n = 3). Treatment with PMA also increased the activity of ECE-1 in the media (0.18 {+-} 0.01; n = 3) compared to control (0.08 {+-} 0.01; n = 3). In addition, this study confirmed by western immunoblotting that only the extracellular region of ECE-1 is released from the cell surface. These data indicate for the first time that PKC activation induces the trafficking and shedding of ECE to and from the cell surface, respectively.« less

Production of unnaturally linked chimeric proteins using a combination of sortase-catalyzed transpeptidation and click chemistry.

PubMed

Witte, Martin D; Theile, Christopher S; Wu, Tongfei; Guimaraes, Carla P; Blom, Annet E M; Ploegh, Hidde L

2013-09-01

Chimeric proteins, including bispecific antibodies, are biological tools with therapeutic applications. Genetic fusion and ligation methods allow the creation of N-to-C and C-to-N fused recombinant proteins, but not unnaturally linked N-to-N and C-to-C fusion proteins. This protocol describes a simple procedure for the production of such chimeric proteins, starting from correctly folded proteins and readily available peptides. By equipping the N terminus or C terminus of the proteins of interest with a set of click handles using sortase A, followed by a strain-promoted click reaction, unnatural N-to-N and C-to-C linked (hetero) fusion proteins are established. Examples of proteins that have been conjugated via this method include interleukin-2, interferon-α, ubiquitin, antibodies and several single-domain antibodies. If the peptides, sortase A and the proteins of interest are in hand, the unnaturally N-to-N and C-to-C fused proteins can be obtained in 3-4 d.

Assigning protein functions by comparative genome analysis protein phylogenetic profiles

DOEpatents

Pellegrini, Matteo; Marcotte, Edward M.; Thompson, Michael J.; Eisenberg, David; Grothe, Robert; Yeates, Todd O.

2003-05-13

A computational method system, and computer program are provided for inferring functional links from genome sequences. One method is based on the observation that some pairs of proteins A' and B' have homologs in another organism fused into a single protein chain AB. A trans-genome comparison of sequences can reveal these AB sequences, which are Rosetta Stone sequences because they decipher an interaction between A' and B. Another method compares the genomic sequence of two or more organisms to create a phylogenetic profile for each protein indicating its presence or absence across all the genomes. The profile provides information regarding functional links between different families of proteins. In yet another method a combination of the above two methods is used to predict functional links.

Molecular Characterization of the S-Layer Gene, sbpA, of Bacillus sphaericus CCM 2177 and Production of a Functional S-Layer Fusion Protein with the Ability To Recrystallize in a Defined Orientation while Presenting the Fused Allergen

PubMed Central

Ilk, Nicola; Völlenkle, Christine; Egelseer, Eva M.; Breitwieser, Andreas; Sleytr, Uwe B.; Sára, Margit

2002-01-01

The nucleotide sequence encoding the crystalline bacterial cell surface (S-layer) protein SbpA of Bacillus sphaericus CCM 2177 was determined by a PCR-based technique using four overlapping fragments. The entire sbpA sequence indicated one open reading frame of 3,804 bp encoding a protein of 1,268 amino acids with a theoretical molecular mass of 132,062 Da and a calculated isoelectric point of 4.69. The N-terminal part of SbpA, which is involved in anchoring the S-layer subunits via a distinct type of secondary cell wall polymer to the rigid cell wall layer, comprises three S-layer-homologous motifs. For screening of amino acid positions located on the outer surface of the square S-layer lattice, the sequence encoding Strep-tag I, showing affinity to streptavidin, was linked to the 5′ end of the sequence encoding the recombinant S-layer protein (rSbpA) or a C-terminally truncated form (rSbpA31-1068). The deletion of 200 C-terminal amino acids did not interfere with the self-assembly properties of the S-layer protein but significantly increased the accessibility of Strep-tag I. Thus, the sequence encoding the major birch pollen allergen (Bet v1) was fused via a short linker to the sequence encoding the C-terminally truncated form rSpbA31-1068. Labeling of the square S-layer lattice formed by recrystallization of rSbpA31-1068/Bet v1 on peptidoglycan-containing sacculi with a Bet v1-specific monoclonal mouse antibody demonstrated the functionality of the fused protein sequence and its location on the outer surface of the S-layer lattice. The specific interactions between the N-terminal part of SbpA and the secondary cell wall polymer will be exploited for an oriented binding of the S-layer fusion protein on solid supports to generate regularly structured functional protein lattices. PMID:12089001

Visualization of Host-Polerovirus Interaction Topologies Using Protein Interaction Reporter Technology.

PubMed

DeBlasio, Stacy L; Chavez, Juan D; Alexander, Mariko M; Ramsey, John; Eng, Jimmy K; Mahoney, Jaclyn; Gray, Stewart M; Bruce, James E; Cilia, Michelle

2016-02-15

Demonstrating direct interactions between host and virus proteins during infection is a major goal and challenge for the field of virology. Most protein interactions are not binary or easily amenable to structural determination. Using infectious preparations of a polerovirus (Potato leafroll virus [PLRV]) and protein interaction reporter (PIR), a revolutionary technology that couples a mass spectrometric-cleavable chemical cross-linker with high-resolution mass spectrometry, we provide the first report of a host-pathogen protein interaction network that includes data-derived, topological features for every cross-linked site that was identified. We show that PLRV virions have hot spots of protein interaction and multifunctional surface topologies, revealing how these plant viruses maximize their use of binding interfaces. Modeling data, guided by cross-linking constraints, suggest asymmetric packing of the major capsid protein in the virion, which supports previous epitope mapping studies. Protein interaction topologies are conserved with other species in the Luteoviridae and with unrelated viruses in the Herpesviridae and Adenoviridae. Functional analysis of three PLRV-interacting host proteins in planta using a reverse-genetics approach revealed a complex, molecular tug-of-war between host and virus. Structural mimicry and diversifying selection-hallmarks of host-pathogen interactions-were identified within host and viral binding interfaces predicted by our models. These results illuminate the functional diversity of the PLRV-host protein interaction network and demonstrate the usefulness of PIR technology for precision mapping of functional host-pathogen protein interaction topologies. The exterior shape of a plant virus and its interacting host and insect vector proteins determine whether a virus will be transmitted by an insect or infect a specific host. Gaining this information is difficult and requires years of experimentation. We used protein interaction reporter (PIR) technology to illustrate how viruses exploit host proteins during plant infection. PIR technology enabled our team to precisely describe the sites of functional virus-virus, virus-host, and host-host protein interactions using a mass spectrometry analysis that takes just a few hours. Applications of PIR technology in host-pathogen interactions will enable researchers studying recalcitrant pathogens, such as animal pathogens where host proteins are incorporated directly into the infectious agents, to investigate how proteins interact during infection and transmission as well as develop new tools for interdiction and therapy. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

Visualization of Host-Polerovirus Interaction Topologies Using Protein Interaction Reporter Technology

PubMed Central

DeBlasio, Stacy L.; Chavez, Juan D.; Alexander, Mariko M.; Ramsey, John; Eng, Jimmy K.; Mahoney, Jaclyn; Gray, Stewart M.; Bruce, James E.

2015-01-01

ABSTRACT Demonstrating direct interactions between host and virus proteins during infection is a major goal and challenge for the field of virology. Most protein interactions are not binary or easily amenable to structural determination. Using infectious preparations of a polerovirus (Potato leafroll virus [PLRV]) and protein interaction reporter (PIR), a revolutionary technology that couples a mass spectrometric-cleavable chemical cross-linker with high-resolution mass spectrometry, we provide the first report of a host-pathogen protein interaction network that includes data-derived, topological features for every cross-linked site that was identified. We show that PLRV virions have hot spots of protein interaction and multifunctional surface topologies, revealing how these plant viruses maximize their use of binding interfaces. Modeling data, guided by cross-linking constraints, suggest asymmetric packing of the major capsid protein in the virion, which supports previous epitope mapping studies. Protein interaction topologies are conserved with other species in the Luteoviridae and with unrelated viruses in the Herpesviridae and Adenoviridae. Functional analysis of three PLRV-interacting host proteins in planta using a reverse-genetics approach revealed a complex, molecular tug-of-war between host and virus. Structural mimicry and diversifying selection—hallmarks of host-pathogen interactions—were identified within host and viral binding interfaces predicted by our models. These results illuminate the functional diversity of the PLRV-host protein interaction network and demonstrate the usefulness of PIR technology for precision mapping of functional host-pathogen protein interaction topologies. IMPORTANCE The exterior shape of a plant virus and its interacting host and insect vector proteins determine whether a virus will be transmitted by an insect or infect a specific host. Gaining this information is difficult and requires years of experimentation. We used protein interaction reporter (PIR) technology to illustrate how viruses exploit host proteins during plant infection. PIR technology enabled our team to precisely describe the sites of functional virus-virus, virus-host, and host-host protein interactions using a mass spectrometry analysis that takes just a few hours. Applications of PIR technology in host-pathogen interactions will enable researchers studying recalcitrant pathogens, such as animal pathogens where host proteins are incorporated directly into the infectious agents, to investigate how proteins interact during infection and transmission as well as develop new tools for interdiction and therapy. PMID:26656710

Ascidian sperm glycosylphosphatidylinositol-anchored CRISP-like protein as a binding partner for an allorecognizable sperm receptor on the vitelline coat.

PubMed

Urayama, Satoshi; Harada, Yoshito; Nakagawa, Yoko; Ban, Susumu; Akasaka, Mari; Kawasaki, Nana; Sawada, Hitoshi

2008-08-01

Although ascidians are hermaphroditic, many species including Halocynthia roretzi are self-sterile. We previously reported that a vitelline coat polymorphic protein HrVC70, consisting of 12 EGF (epidermal growth factor)-like repeats, is a candidate allorecognition protein in H. roretzi, because the isolated HrVC70 shows higher affinity to nonself-sperm than to self-sperm. Here, we show that a sperm 35-kDa glycosylphosphatidylinositol-anchored CRISP (cysteine-rich secretory protein)-like protein HrUrabin in a low density detergent-insoluble membrane fraction is a physiological binding partner for HrVC70. We found that HrVC70 specifically interacts with HrUrabin, which had been separated by SDS-PAGE and transferred onto a nitrocellulose membrane. HrUrabin has an N-linked sugar chain, essential for binding to HrVC70. HrUrabin mRNA is expressed in the testis but not in the ovary, and the protein appears to be localized on the surface of sperm head and tail. Anti-HrUrabin antibody, which neutralizes the interaction between HrUrabin and HrVC70, potently inhibited fertilization and allorecognizable sperm-binding to HrVC70-agarose. However, no significant difference in the binding ability of HrUrabin to HrVC70 was observed in autologous and allogeneic combinations by Far Western analyses. These results indicate that sperm-egg binding in H. roretzi is mediated by the molecular interaction between HrUrabin on the sperm surface and HrVC70 on the vitelline coat, but that HrUrabin per se is unlikely to be a direct allorecognition protein.

Factor H-IgG Chimeric Proteins as a Therapeutic Approach against the Gram-Positive Bacterial Pathogen Streptococcus pyogenes.

PubMed

Blom, Anna M; Magda, Michal; Kohl, Lisa; Shaughnessy, Jutamas; Lambris, John D; Ram, Sanjay; Ermert, David

2017-12-01

Bacteria can cause life-threatening infections, such as pneumonia, meningitis, or sepsis. Antibiotic therapy is a mainstay of treatment, although antimicrobial resistance has drastically increased over the years. Unfortunately, safe and effective vaccines against most pathogens have not yet been approved, and thus developing alternative treatments is important. We analyzed the efficiency of factor H (FH)6-7/Fc, a novel antibacterial immunotherapeutic protein against the Gram-positive bacterium Streptococcus pyogenes This protein is composed of two domains of complement inhibitor human FH (FH complement control protein modules 6 and 7) that bind to S. pyogenes , linked to the Fc region of IgG (FH6-7/Fc). FH6-7/Fc has previously been shown to enhance complement-dependent killing of, and facilitate bacterial clearance in, animal models of the Gram-negative pathogens Haemophilus influenzae and Neisseria meningitidis We hypothesized that activation of complement by FH6-7/Fc on the surface of Gram-positive bacteria such as S. pyogenes will enable professional phagocytes to eliminate the pathogen. We found that FH6-7/Fc alleviated S. pyogenes- induced sepsis in a transgenic mouse model expressing human FH ( S. pyogenes binds FH in a human-specific manner). Furthermore, FH6-7/Fc, which binds to protein H and selected M proteins, displaced FH from the bacterial surface, enhanced alternative pathway activation, and reduced bacterial blood burden by opsonophagocytosis in a C3-dependent manner in an ex vivo human whole-blood model. In conclusion, FH-Fc chimeric proteins could serve as adjunctive treatments against multidrug-resistant bacterial infections. Copyright © 2017 by The American Association of Immunologists, Inc.

Cholangiopathy and tumors of the pancreas, liver, and biliary tree in boys with X-linked immunodeficiency with hyper-IgM.

PubMed

Hayward, A R; Levy, J; Facchetti, F; Notarangelo, L; Ochs, H D; Etzioni, A; Bonnefoy, J Y; Cosyns, M; Weinberg, A

1997-01-15

We report an association between X-linked immunodeficiency with hyper-IgM (XHIM) and carcinomas affecting the liver, pancreas, biliary tree, and associated neuroectodermal endocrine cells. The tumors were fatal in eight of nine cases and in most instances were preceded by chronic cholangiopathy and/or cirrhosis. An additional group of subjects with XHIM had chronic inflammation of the liver or bile ducts but no malignancy. Many patients with XHIM were infected with cryptosporidia. CD40 is normally expressed on regenerating or inflammed bile duct epithelium. A CD40+ hepatocellular carcinoma cell line, HepG2, susceptible to cryptosporidia and CMV infection became resistant when cell surface CD40 was cross-linked by a CD40 ligand fusion protein. Apoptosis was triggered in HepG2 cells if protein synthesis was blocked by cycloheximide or if the cells were infected by cryptosporidia. Ligation of CD40 on biliary epithelium may contribute to defense against infection by intracellular pathogens. We propose that the CD40 ligand mutations that cause XHIM deprive the biliary epithelium of one line of defense against intracellular pathogens and that malignant transformation in the biliary tree follows chronic infection or inflammation. The resulting tumors may then progress without check by an effective immune response. Patients with XHIM who have abnormal liver function tests should be considered at increased risk for cholangiopathy or malignancy.

GARP is regulated by miRNAs and controls latent TGF-β1 production by human regulatory T cells.

PubMed

Gauthy, Emilie; Cuende, Julia; Stockis, Julie; Huygens, Caroline; Lethé, Bernard; Collet, Jean-François; Bommer, Guido; Coulie, Pierre G; Lucas, Sophie

2013-01-01

GARP is a transmembrane protein present on stimulated human regulatory T lymphocytes (Tregs), but not on other T lymphocytes (Th cells). It presents the latent form of TGF-β1 on the Treg surface. We report here that GARP favors the cleavage of the pro-TGF-β1 precursor and increases the amount of secreted latent TGF-β1. Stimulated Tregs, which naturally express GARP, and Th cells transfected with GARP secrete a previously unknown form of latent TGF-β1 that is disulfide-linked to GARP. These GARP/TGF-β1 complexes are possibly shed from the T cell surface. Secretion of GARP/TGF-β1 complexes was not observed with transfected 293 cells and may thus be restricted to the T cell lineage. We conclude that in stimulated human Tregs, GARP not only displays latent TGF-β1 at the cell surface, but also increases its secretion by forming soluble disulfide-linked complexes. Moreover, we identified six microRNAs (miRNAs) that are expressed at lower levels in Treg than in Th clones and that target a short region of the GARP 3' UTR. In transfected Th cells, the presence of this region decreased GARP levels, cleavage of pro-TGF-β1, and secretion of latent TGF-β1.

Protein-linked Ubiquitin Chain Structure Restricts Activity of Deubiquitinating Enzymes*

PubMed Central

Schaefer, Jonathan B.; Morgan, David O.

2011-01-01

The attachment of lysine 48 (Lys48)-linked polyubiquitin chains to proteins is a universal signal for degradation by the proteasome. Here, we report that long Lys48-linked chains are resistant to many deubiquitinating enzymes (DUBs). Representative enzymes from this group, Ubp15 from yeast and its human ortholog USP7, rapidly remove mono- and diubiquitin from substrates but are slow to remove longer Lys48-linked chains. This resistance is lost if the structure of Lys48-linked chains is disrupted by mutation of ubiquitin or if chains are linked through Lys63. In contrast to Ubp15 and USP7, Ubp12 readily cleaves the ends of long chains, regardless of chain structure. We propose that the resistance to many DUBs of long, substrate-attached Lys48-linked chains helps ensure that proteins are maintained free from ubiquitin until a threshold of ubiquitin ligase activity enables degradation. PMID:22072716

In Vivo Photo-Cross-Linking to Study T3S Interactions Demonstrated Using the Yersinia pestis T3S System.

PubMed

Henderson, Thomas A; Nilles, Matthew L

2017-01-01

Cross-linking of proteins is effective in determining protein-protein interactions. The use of photo-cross-linkers was developed to study protein interactions in several manners. One method involved the incorporation of photo-activatable cross-linking groups into chemically synthesized peptides. A second approach relies on incorporation of photo-activatable cross-linking groups into proteins using tRNAs with chemically bound photo-activatable amino acids with suppressor tRNAs translational systems to incorporate the tags into specific sites. A third system was made possible by the development of photoreactive amino acids that use the normal cellular tRNAs and aminoacyl tRNA synthetases. In this method, the third system is used to demonstrate its utility for the study of T3S system interactions. This method describes how two photo-activatable amino acids, photo-methionine and photo-leucine, that use the normal cellular machinery are incorporated into Yersinia pestis and used to study interactions in the T3S system. To demonstrate the system, the method was used to cross-link the T3S regulatory proteins LcrG and LcrV.

The First MS-Cleavable, Photo-Thiol-Reactive Cross-Linker for Protein Structural Studies

NASA Astrophysics Data System (ADS)

Iacobucci, Claudio; Piotrowski, Christine; Rehkamp, Anne; Ihling, Christian H.; Sinz, Andrea

2018-04-01

Cleavable cross-linkers are gaining increasing importance for chemical cross-linking/mass spectrometry (MS) as they permit a reliable and automated data analysis in structural studies of proteins and protein assemblies. Here, we introduce 1,3-diallylurea (DAU) as the first CID-MS/MS-cleavable, photo-thiol-reactive cross-linker. DAU is a commercially available, inexpensive reagent that efficiently undergoes an anti-Markovnikov hydrothiolation with cysteine residues in the presence of a radical initiator upon UV-A irradiation. Radical cysteine cross-linking proceeds via an orthogonal "click reaction" and yields stable alkyl sulfide products. DAU reacts at physiological pH and cross-linking reactions with peptides, and proteins can be performed at temperatures as low as 4 °C. The central urea bond is efficiently cleaved upon collisional activation during tandem MS experiments generating characteristic product ions. This improves the reliability of automated cross-link identification. Different radical initiators have been screened for the cross-linking reaction of DAU using the thiol-containing compounds cysteine and glutathione. Our concept has also been exemplified for the biologically relevant proteins bMunc13-2 and retinal guanylyl cyclase-activating protein-2. [Figure not available: see fulltext.

Virus-based surface patterning of biological molecules, probes, and inorganic materials.

PubMed

Ahn, Suji; Jeon, Seongho; Kwak, Eun-A; Kim, Jong-Man; Jaworski, Justyn

2014-10-01

An essential requirement for continued technological advancement in many areas of biology, physics, chemistry, and materials science is the growing need to generate custom patterned materials. Building from recent achievements in the site-specific modification of virus for covalent surface tethering, we show in this work that stable 2D virus patterns can be generated in custom geometries over large area glass surfaces to yield templates of biological, biochemical, and inorganic materials in high density. As a nanomaterial building block, filamentous viruses have been extensively used in recent years to produce materials with interesting properties, owing to their ease of genetic and chemical modification. By utilizing un-natural amino acids generated at specific locations on the filamentous fd bacteriophage protein coat, surface immobilization is carried out on APTES patterned glass resulting in precise geometries of covalently linked virus material. This technique facilitated the surface display of a high density of virus that were labeled with biomolecules, fluorescent probes, and gold nanoparticles, thereby opening the possibility of integrating virus as functional components for surface engineering. Copyright © 2014 Elsevier B.V. All rights reserved.

Super-Resolution Fluorescence Imaging of Spatial Organization of Proteins and Lipids in Natural Rubber.

PubMed

Wu, Jinrong; Qu, Wei; Huang, Guangsu; Wang, Siyuan; Huang, Cheng; Liu, Han

2017-06-12

Natural rubber (NR) with proteins and lipids has superior mechanical properties to its synthetic counterpart, polyisoprene rubber. However, it is a challenge to unravel the morphology of proteins and lipids. Here we used two-color stochastic optical reconstruction microscopy (STORM) to directly visualize the spatial organization of proteins and lipids in NR. We found that the proteins and lipids form an interdispersed stabilizing layer on the surface of NR latex particles. After drying, the proteins and lipids form aggregates of up to 300 nm in diameter. The aggregates physically interact with the terminal groups of polyisoprene chains, leading to the formation of a network, which contributes to the high elasticity and mechanical property of NR. If we remove proteins in NR, the large phospholipid aggregates disintegrate into small ones. However, it does not decompose the network but rather reduces the effective cross-linking density, thus the deproteinized NR is still elastic-like with decreased mechanical property. Removing both proteins and lipids wholly decomposes the network, thus, results in a liquid-like behavior of the rubber. The STORM measurements in this paper enable more insight into the structure-property relationship of NR, which also shows a great potential of STORM in studying the fine structure of polymeric materials and nanocomposites.

Digital Assays Part II: Digital Protein and Cell Assays.

PubMed

Basu, Amar S

2017-08-01

A digital assay is one in which the sample is partitioned into many containers such that each partition contains a discrete number of biological entities (0, 1, 2, 3, . . .). A powerful technique in the biologist's toolkit, digital assays bring a new level of precision in quantifying nucleic acids, measuring proteins and their enzymatic activity, and probing single-cell genotype and phenotype. Where part I of this review focused on the fundamentals of partitioning and digital PCR, part II turns its attention to digital protein and cell assays. Digital enzyme assays measure the kinetics of single proteins with enzymatic activity. Digital enzyme-linked immunoassays (ELISAs) quantify antigenic proteins with 2 to 3 log lower detection limit than conventional ELISA, making them well suited for low-abundance biomarkers. Digital cell assays probe single-cell genotype and phenotype, including gene expression, intracellular and surface proteins, metabolic activity, cytotoxicity, and transcriptomes (scRNA-seq). These methods exploit partitioning to 1) isolate single cells or proteins, 2) detect their activity via enzymatic amplification, and 3) tag them individually by coencapsulating them with molecular barcodes. When scaled, digital assays reveal stochastic differences between proteins or cells within a population, a key to understanding biological heterogeneity. This review is intended to give a broad perspective to scientists interested in adopting digital assays into their workflows.

Sugar-Terminated Nanoparticle Chaperones Are 102-105 Times Better Than Molecular Sugars in Inhibiting Protein Aggregation and Reducing Amyloidogenic Cytotoxicity.

PubMed

Pradhan, Nibedita; Shekhar, Shashi; Jana, Nihar R; Jana, Nikhil R

2017-03-29

Sugar-based osmolyte molecules are known to stabilize proteins under stress, but usually they have poor chaperone performance in inhibiting protein aggregation. Here, we show that the nanoparticle form of sugars molecule can enhance their chaperone performance typically by 10 2 -10 5 times, compared to molecular sugar. Sugar-based plate-like nanoparticles of 20-40 nm hydrodynamic size have been synthesized by simple heating of acidic aqueous solution of glucose/sucrose/maltose/trehalose. These nanoparticles have excitation-dependent green/yellow/orange emission and surface chemistry identical to the respective sugar molecule. Fibrillation of lysozyme/insulin/amyloid beta in extracellular space, aggregation of mutant huntingtin protein inside model neuronal cell, and cytotoxic effect of fibrils are investigated in the presence of these sugar nanoparticles. We found that sugar nanoparticles are 10 2 -10 5 times efficient than respective sugar molecules in inhibiting protein fibrillation and preventing cytotoxicity arising of fibrils. We propose that better performance of the nanoparticle form is linked to its stronger binding with fibril structure and enhanced cell uptake. This result suggests that nanoparticle form of osmolyte can be an attractive option in prevention and curing of protein aggregation-derived diseases.

A structural study of F-actin - filamin networks

NASA Astrophysics Data System (ADS)

Ahrens-Braunstein, Ashley; Nguyen, Lam; Hirst, Linda

2010-03-01

The cell's ability to move and contract is attributed to the semi-flexible filamentous protein, F -actin, one of the three filaments in the cytoskeleton. Actin bundling can be formed by a cross-linking actin binding protein (ABP) filamin. By examining filamin's cross-linking abilities at different concentrations and molar ratios, we can study the flexibility, structure and multiple network formations created when cross-linking F-actin with this protein. We have studied the phase diagram of this protein system using fluorescence microscopy, analyzing the network structures observed in the context of a coarse grained molecular dynamics simulation carried out by our group.

Production of unnaturally linked chimeric proteins using a combination of sortase-catalyzed transpeptidation and click chemistry

PubMed Central

Witte, Martin D.; Theile, Chris; Wu, Tongfei; Guimaraes, Carla P.; Blom, Annet E. M.; Ploegh, Hidde L.

2014-01-01

Chimeric proteins, including bi-specific antibodies, are biological tools with therapeutic applications. Genetic fusion and ligation methods allow the creation of N-to-C and C-toN fused recombinant proteins, but not the majority of non-template encoded fusions. The present protocol describes a simple procedure for the production of unnaturally linked N-to-N and C-to-C chimeric proteins. Equipping the N-terminus or C-terminus of the proteins of interest with a set of click handles using sortase A, followed by a click reaction, establishes unnatural N-to-N and C-to-C (hetero)dimer linked fusions. If the peptides, sortase A, and the proteins of interest are in hand, the unnaturally fused proteins can be obtained in 3–4 days. PMID:23989675

Comparative study on protein cross-linking and gel enhancing effect of microbial transglutaminase on surimi from different fish.

PubMed

Chanarat, Sochaya; Benjakul, Soottawat; H-Kittikun, Aran

2012-03-15

Microbial transglutaminase (MTGase) has been used to increase the gel strength of surimi. Nevertheless, its effectiveness varies with fish species. The aim of this study was to elucidate the effect of MTGase at different levels on protein cross-linking and gel property of surimi from threadfin bream, Indian mackerel and sardine in the presence and absence of endogenous transglutaminase. Breaking force of all surimi gels increased as MTGase levels (0-0.6 U g⁻¹) increased except for threadfin bream surimi gel, where the breaking force decreased at 0.6 U g⁻¹ (P < 0.05). In the presence of EDTA, the gel strengthening effect was lower, suggesting the combined effect of endogenous transglutaminase with MTGase. With the addition of MTGase, the gel with the highest increase in breaking force showed highest decrease in myosin heavy chain. When cross-linking activity of MTGase on natural actomyosin (NAM) was determined, the highest decreasing rate in ε-amino group content with the concomitant increased formation of cross-linked proteins was found in NAM from threadfin bream. The reactivity of muscle proteins toward MTGase-induced cross-linking was in agreement with surimi gel strengthening. The composition and properties of muscle proteins of varying fish species more likely determined protein cross-linking induced by MTGase, thereby affecting their gel properties.

Selective fibronectin adsorption against albumin and enhanced stem cell attachment on helium atmospheric pressure glow discharge treated titanium

NASA Astrophysics Data System (ADS)

Han, Inho; Vagaska, Barbora; Joo Park, Bong; Lee, Mi Hee; Jin Lee, Seung; Park, Jong-Chul

2011-06-01

Successful tissue integration of implanted medical devices depends on appropriate initial cellular response. In this study, the effect of helium atmospheric pressure glow discharge (He-APGD) treatment of titanium on selective protein adsorption and the initial attachment processes and focal adhesion formation of osteoprogenitor cells and stem cells were examined. Titanium disks were treated in a self-designed He-APGD system. Initial attachment of MC3T3-E1 mouse pre-osteoblasts and human mesenchymal stem cells (MSCs) was evaluated by MTT assay and plasma membrane staining followed by morphometric analysis. Fibronectin adsorption was investigated by Enzyme-Linked ImmunoSorbant Assay. MSCs cell attachment to treated and non-treated titanium disks coated with different proteins was verified also in serum-free culture. Organization of actin cytoskeleton and focal adhesions was evaluated microscopically. He-APGD treatment effectively modified the titanium surfaces by creating a super-hydrophilic surface, which promoted selectively higher adsorption of fibronectin, a protein of critical importance for cell/biomaterial interaction. In two different types of cells, the He-APGD treatment enhanced the number of attaching cells as well as their attachment area. Moreover, cells had higher organization of actin cytoskeleton and focal adhesions. Faster acceptance of the material by the progenitor cells in the early phases of tissue integration after the implantation may significantly reduce the overall healing time; therefore, titanium treatment with He-APGD seems to be an effective method of surface modification of titanium for improving its tissue inductive properties.

Polymorphisms in Fibronectin Binding Protein A of Staphylococcus Aureus are Associated with Infection of Cardiovascular Devices

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

Lower, Steven; Lamlertthon, Supaporn; Casillas-Ituarte, Nadia

Medical implants, like cardiovascular devices, improve the quality of life for countless individuals but may become infected with bacteria like Staphylococcus aureus. Such infections take the form of a bio-film, a structured community of bacterial cells adherent to the surface of a solid substrate. Every bio-film begins with an attractive force or bond between bacterium and substratum. We used atomic force microscopy to probe experimentally forces between a fibronectin-coated surface (i.e., proxy for an implanted cardiac device) and fibronectin-binding receptors on the surface of individual living bacteria from each of 80 clinical isolates of S. aureus. These isolates originated frommore » humans with infected cardiac devices (CDI; n = 26), uninfected cardiac devices (n = 20), and the anterior nares of asymptomatic subjects (n = 34). CDI isolates exhibited a distinct bindingforce signature and had speci!c single amino acid polymorphisms in fibronectin-binding protein A corresponding to E652D, H782Q, and K786N. In silico molecular dynamics simulations demonstrate that residues D652, Q782, and N786 in fibronectin-binding protein A form extra hydrogen bonds with fibronectin, complementing the higher binding force and energy measured by atomic force microscopy for the CDI isolates. This study is significant, because it links pathogenic bacteria biofilms from the length scale of bonds acting across a nanometer-scale space to the clinical presentation of disease at the human dimension.« less

Baculovirus display of functional antibody Fab fragments.

PubMed

Takada, Shinya; Ogawa, Takafumi; Matsui, Kazusa; Suzuki, Tasuku; Katsuda, Tomohisa; Yamaji, Hideki

2015-08-01

The generation of a recombinant baculovirus that displays antibody Fab fragments on the surface was investigated. A recombinant baculovirus was engineered so that the heavy chain (Hc; Fd fragment) of a mouse Fab fragment was expressed as a fusion to the N-terminus of baculovirus gp64, while the light chain of the Fab fragment was simultaneously expressed as a secretory protein. Following infection of Sf9 insect cells with the recombinant baculovirus, the culture supernatant was analyzed by enzyme-linked immunosorbent assay using antigen-coated microplates and either an anti-mouse IgG or an anti-gp64 antibody. A relatively strong signal was obtained in each case, showing antigen-binding activity in the culture supernatant. In western blot analysis of the culture supernatant using the anti-gp64 antibody, specific protein bands were detected at an electrophoretic mobility that coincided with the molecular weight of the Hc-gp64 fusion protein as well as that of gp64. Flow cytometry using a fluorescein isothiocyanate-conjugated antibody specific to mouse IgG successfully detected the Fab fragments on the surface of the Sf9 cells. These results suggest that immunologically functional antibody Fab fragments can be displayed on the surface of baculovirus particles, and that a fluorescence-activated cell sorter with a fluorescence-labeled antigen can isolate baculoviruses displaying specific Fab fragments. This successful baculovirus display of antibody Fab fragments may offer a novel approach for the efficient selection of specific antibodies.

Protein enriched pasta: structure and digestibility of its protein network.

PubMed

Laleg, Karima; Barron, Cécile; Santé-Lhoutellier, Véronique; Walrand, Stéphane; Micard, Valérie

2016-02-01

Wheat (W) pasta was enriched in 6% gluten (G), 35% faba (F) or 5% egg (E) to increase its protein content (13% to 17%). The impact of the enrichment on the multiscale structure of the pasta and on in vitro protein digestibility was studied. Increasing the protein content (W- vs. G-pasta) strengthened pasta structure at molecular and macroscopic scales but reduced its protein digestibility by 3% by forming a higher covalently linked protein network. Greater changes in the macroscopic and molecular structure of the pasta were obtained by varying the nature of protein used for enrichment. Proteins in G- and E-pasta were highly covalently linked (28-32%) resulting in a strong pasta structure. Conversely, F-protein (98% SDS-soluble) altered the pasta structure by diluting gluten and formed a weak protein network (18% covalent link). As a result, protein digestibility in F-pasta was significantly higher (46%) than in E- (44%) and G-pasta (39%). The effect of low (55 °C, LT) vs. very high temperature (90 °C, VHT) drying on the protein network structure and digestibility was shown to cause greater molecular changes than pasta formulation. Whatever the pasta, a general strengthening of its structure, a 33% to 47% increase in covalently linked proteins and a higher β-sheet structure were observed. However, these structural differences were evened out after the pasta was cooked, resulting in identical protein digestibility in LT and VHT pasta. Even after VHT drying, F-pasta had the best amino acid profile with the highest protein digestibility, proof of its nutritional interest.

Automated Hydrophobic Interaction Chromatography Column Selection for Use in Protein Purification

PubMed Central

Murphy, Patrick J. M.; Stone, Orrin J.; Anderson, Michelle E.

2011-01-01

In contrast to other chromatographic methods for purifying proteins (e.g. gel filtration, affinity, and ion exchange), hydrophobic interaction chromatography (HIC) commonly requires experimental determination (referred to as screening or "scouting") in order to select the most suitable chromatographic medium for purifying a given protein 1. The method presented here describes an automated approach to scouting for an optimal HIC media to be used in protein purification. HIC separates proteins and other biomolecules from a crude lysate based on differences in hydrophobicity. Similar to affinity chromatography (AC) and ion exchange chromatography (IEX), HIC is capable of concentrating the protein of interest as it progresses through the chromatographic process. Proteins best suited for purification by HIC include those with hydrophobic surface regions and able to withstand exposure to salt concentrations in excess of 2 M ammonium sulfate ((NH4)2SO4). HIC is often chosen as a purification method for proteins lacking an affinity tag, and thus unsuitable for AC, and when IEX fails to provide adequate purification. Hydrophobic moieties on the protein surface temporarily bind to a nonpolar ligand coupled to an inert, immobile matrix. The interaction between protein and ligand are highly dependent on the salt concentration of the buffer flowing through the chromatography column, with high ionic concentrations strengthening the protein-ligand interaction and making the protein immobile (i.e. bound inside the column) 2. As salt concentrations decrease, the protein-ligand interaction dissipates, the protein again becomes mobile and elutes from the column. Several HIC media are commercially available in pre-packed columns, each containing one of several hydrophobic ligands (e.g. S-butyl, butyl, octyl, and phenyl) cross-linked at varying densities to agarose beads of a specific diameter 3. Automated column scouting allows for an efficient approach for determining which HIC media should be employed for future, more exhaustive optimization experiments and protein purification runs 4. The specific protein being purified here is recombinant green fluorescent protein (GFP); however, the approach may be adapted for purifying other proteins with one or more hydrophobic surface regions. GFP serves as a useful model protein, due to its stability, unique light absorbance peak at 397 nm, and fluorescence when exposed to UV light 5. Bacterial lysate containing wild type GFP was prepared in a high-salt buffer, loaded into a Bio-Rad DuoFlow medium pressure liquid chromatography system, and adsorbed to HiTrap HIC columns containing different HIC media. The protein was eluted from the columns and analyzed by in-line and post-run detection methods. Buffer blending, dynamic sample loop injection, sequential column selection, multi-wavelength analysis, and split fraction eluate collection increased the functionality of the system and reproducibility of the experimental approach. PMID:21968976

Type 1 Does The Two-Step: Type 1 Secretion Substrates With A Functional Periplasmic Intermediate.

PubMed

Smith, Timothy J; Sondermann, Holger; O'Toole, George A

2018-06-04

Bacteria have evolved several secretion strategies for polling and responding to environmental flux and insult. Of these, the type 1 secretion system (T1SS) is known to secrete an array of biologically diverse proteins - from small < 10 kDa bacteriocins to gigantic adhesins with a mass over 1 MDa. For the last several decades T1SS have been characterized as a one-step translocation strategy whereby the secreted substrate is transported directly into the extracellular environment from the cytoplasm with no periplasmic intermediate. Recent phylogenetic, biochemical, and genetic evidence point to a distinct sub-group of T1SS machinery linked with a bacterial transglutaminase-like cysteine proteinase (BTLCP), which uses a two-step secretion mechanism. BTLCP-linked T1SS transport a class of repeats-in-toxin (RTX) adhesins that are critical for biofilm formation. The prototype of this RTX adhesin group, LapA of Pseudomonas fluorescens Pf0-1, uses a novel N-terminal retention module to anchor the adhesin at the cell surface as a secretion intermediate threaded through the outer membrane-localized, TolC-like protein LapE. This secretion intermediate is post-translationally cleaved by the BTLCP family LapG protein to release LapA from its cognate T1SS pore. Thus, secretion of LapA and related RTX adhesins into the extracellular environment appears to be a T1SS-mediated, two-step process that involves a periplasmic intermediate. In this review, we contrast the T1SS machinery and substrates of the BLTCP-linked two-step secretion process with those of the classical one-step T1SS to better understand the newly recognized and expanded role of this secretion machinery. Copyright © 2018 American Society for Microbiology.

A photo-cross-linking approach to monitor folding and assembly of newly synthesized proteins in a living cell.

PubMed

Miyazaki, Ryoji; Myougo, Naomi; Mori, Hiroyuki; Akiyama, Yoshinori

2018-01-12

Many proteins form multimeric complexes that play crucial roles in various cellular processes. Studying how proteins are correctly folded and assembled into such complexes in a living cell is important for understanding the physiological roles and the qualitative and quantitative regulation of the complex. However, few methods are suitable for analyzing these rapidly occurring processes. Site-directed in vivo photo-cross-linking is an elegant technique that enables analysis of protein-protein interactions in living cells with high spatial resolution. However, the conventional site-directed in vivo photo-cross-linking method is unsuitable for analyzing dynamic processes. Here, by combining an improved site-directed in vivo photo-cross-linking technique with a pulse-chase approach, we developed a new method that can analyze the folding and assembly of a newly synthesized protein with high spatiotemporal resolution. We demonstrate that this method, named the pulse-chase and in vivo photo-cross-linking experiment (PiXie), enables the kinetic analysis of the formation of an Escherichia coli periplasmic (soluble) protein complex (PhoA). We also used our new technique to investigate assembly/folding processes of two membrane complexes (SecD-SecF in the inner membrane and LptD-LptE in the outer membrane), which provided new insights into the biogenesis of these complexes. Our PiXie method permits analysis of the dynamic behavior of various proteins and enables examination of protein-protein interactions at the level of individual amino acid residues. We anticipate that our new technique will have valuable utility for studies of protein dynamics in many organisms. © 2018 by The American Society for Biochemistry and Molecular Biology, Inc.

Surface functionalization of a polymeric lipid bilayer for coupling a model biological membrane with molecules, cells, and microstructures.

PubMed

Morigaki, Kenichi; Mizutani, Kazuyuki; Saito, Makoto; Okazaki, Takashi; Nakajima, Yoshihiro; Tatsu, Yoshiro; Imaishi, Hiromasa

2013-02-26

We describe a stable and functional model biological membrane based on a polymerized lipid bilayer with a chemically modified surface. A polymerized lipid bilayer was formed from a mixture of two diacetylene-containing phospholipids, 1,2-bis(10,12-tricosadiynoyl)-sn-glycero-3-phosphocholine (DiynePC) and 1,2-bis(10,12-tricosadiynoyl)-sn-glycero-3-phosphoethanolamine (DiynePE). DiynePC formed a stable bilayer structure, whereas the ethanolamine headgroup of DiynePE enabled functional molecules to be grafted onto the membrane surface. Copolymerization of DiynePC and DiynePE resulted in a robust bilayer. Functionalization of the polymeric bilayer provided a route to a robust and biomimetic surface that can be linked with biomolecules, cells, and three-dimensional (3D) microstructures. Biotin and peptides were grafted onto the polymeric bilayer for attaching streptavidin and cultured mammalian cells by molecular recognition, respectively. Nonspecific adsorption of proteins and cells on polymeric bilayers was minimum. DiynePE was also used to attach a microstructure made of an elastomer (polydimethylsiloxan: PDMS) onto the membrane, forming a confined aqueous solution between the two surfaces. The microcompartment enabled us to assay the activity of a membrane-bound enzyme (cyochrome P450). Natural (fluid) lipid bilayers were incorporated together with membrane-bound proteins by lithographically polymerizing DiynePC/DiynePE bilayers. The hybrid membrane of functionalized polymeric bilayers and fluid bilayers offers a novel platform for a wide range of biomedical applications including biosensor, bioassay, cell culture, and cell-based assay.

Fast Modulation of μ-Opioid Receptor (MOR) Recycling Is Mediated by Receptor Agonists*

PubMed Central

Roman-Vendrell, Cristina; Yu, Y. Joy; Yudowski, Guillermo Ariel

2012-01-01

The μ-opioid receptor (MOR) is a member of the G protein-coupled receptor family and the main target of endogenous opioid neuropeptides and morphine. Upon activation by ligands, MORs are rapidly internalized via clathrin-coated pits in heterologous cells and dissociated striatal neurons. After initial endocytosis, resensitized receptors recycle back to the cell surface by vesicular delivery for subsequent cycles of activation. MOR trafficking has been linked to opioid tolerance after acute exposure to agonist, but it is also involved in the resensitization process. Several studies describe the regulation and mechanism of MOR endocytosis, but little is known about the recycling of resensitized receptors to the cell surface. To study this process, we induced internalization of MOR with [d-Ala2, N-Me-Phe4, Gly5-ol]-enkephalin (DAMGO) and morphine and imaged in real time single vesicles recycling receptors to the cell surface. We determined single vesicle recycling kinetics and the number of receptors contained in them. Then we demonstrated that rapid vesicular delivery of recycling MORs to the cell surface was mediated by the actin-microtubule cytoskeleton. Recycling was also dependent on Rab4, Rab11, and the Ca2+-sensitive motor protein myosin Vb. Finally, we showed that recycling is acutely modulated by the presence of agonists and the levels of cAMP. Our work identifies a novel trafficking mechanism that increases the number of cell surface MORs during acute agonist exposure, effectively reducing the development of opioid tolerance. PMID:22378794

Optical fiber immunosensor based on a poly(pyrrole-benzophenone) film for the detection of antibodies to viral antigen.

PubMed

Konry, T; Novoa, A; Shemer-Avni, Y; Hanuka, N; Cosnier, S; Lepellec, Arielle; Marks, R S

2005-03-15

We describe herein a newly developed optical microbiosensor for the diagnosis of hepatitis C virus (HCV) by using a novel photoimmobilization methodology based on a photoactivable electrogenerated polymer film deposited upon surface-conductive fiber optics, which are then used to link a biological receptor to the fiber tip through light mediation. This fiber-optic electroconductive surface modification is done by the deposition of a thin layer of indium tin oxide on the silica surface of the fiber optics. Monomers are then electropolymerized onto the conductive metal oxide surface; thereafter, the fibers are immersed in a solution containing HCV-E2 envelope protein antigen and illuminated with UV light (wavelength approximately 345 nm). As a result of the photochemical reaction, a thin layer of the antigen becomes covalently bound to the benzophenone-modified surface. The photochemically modified fiber optics were tested as immunosensors for the detection of anti-E2 protein antibody analyte that was measured through chemiluminescence reaction. The biosensor was tested for sensitivity, specificity, and overall practicality. Our results suggest that the detection of anti-E2 antibodies with this microbiosensor may enhance significantly HCV serological standard testing especially among patients during dialysis, which were diagnosed as HCV negative, by standard immunological tests, but were known to carry the virus. If transformed into an easy to use procedure, this assay might be used in the future as an important clinical tool for HCV screening in blood banks.

Mapping protein-RNA interactions by RCAP, RNA-cross-linking and peptide fingerprinting.

PubMed

Vaughan, Robert C; Kao, C Cheng

2015-01-01

RNA nanotechnology often feature protein RNA complexes. The interaction between proteins and large RNAs are difficult to study using traditional structure-based methods like NMR or X-ray crystallography. RCAP, an approach that uses reversible-cross-linking affinity purification method coupled with mass spectrometry, has been developed to map regions within proteins that contact RNA. This chapter details how RCAP is applied to map protein-RNA contacts within virions.

Improvement of interfacial interactions using natural polyphenol-inspired tannic acid-coated nanoclay enhancement of soy protein isolate biofilms

NASA Astrophysics Data System (ADS)

Wang, Zhong; Kang, Haijiao; Zhang, Wei; Zhang, Shifeng; Li, Jianzhang

2017-04-01

In this study, a novel and economic surface modification technique for montmorillonite (MMT) nanosheets, a biocompatible coupling cross-linking agent, was developed on an attempt at improving the interfacial adhesion with soy protein isolate (SPI) matrix. Inspired by natural polyphenol, the "green dip-coating" method using tannic acid (TA) to surface-modify MMT (TA@MMT). SPI nanocomposite films modified with MMT or TA@MMT, as well as the control ones, were prepared via the casting method. The TA layer was successfully coated on the MMT surface through the (FeIII) ions coordination chemistry and the synthetic samples were characterized by the Fourier transform infrared (FT-IR) spectroscopy, X-ray photoelectron spectroscopy (XPS), X-ray diffraction (XRD), thermogravimetric analysis (TGA), atomic force microscopy (AFM), and transmission electron microscopy (TEM). The compatibility and interfacial interactions between modified MMT and SPI matrix were greatly enhanced by the TA-FeIII coating on the MMT surface. The mechanical properties, water resistance, and thermal stability of the resultant biofilm were increased accordingly. Compared with that of the unmodified SPI film, the tensile strength of the nanocomposite films modified by the green dip-coating was increased by 113.3%. These SPI-based nanocomposite films showed the favorable potential in terms of food packing applications due to their efficient barriers to water vapor and UV and/or visible light.

Towards an in vitro model mimicking the foreign body response: tailoring the surface properties of biomaterials to modulate extracellular matrix.

PubMed

Damanik, Febriyani F R; Rothuizen, Tonia C; van Blitterswijk, Clemens; Rotmans, Joris I; Moroni, Lorenzo

2014-09-19

Despite various studies to minimize host reaction following a biomaterial implantation, an appealing strategy in regenerative medicine is to actively use such an immune response to trigger and control tissue regeneration. We have developed an in vitro model to modulate the host response by tuning biomaterials' surface properties through surface modifications techniques as a new strategy for tissue regeneration applications. Results showed tunable surface topography, roughness, wettability, and chemistry by varying treatment type and exposure, allowing for the first time to correlate the effect of these surface properties on cell attachment, morphology, strength and proliferation, as well as proinflammatory (IL-1β, IL-6) and antiinflammatory cytokines (TGF-β1, IL-10) secreted in medium, and protein expression of collagen and elastin. Surface microstructuring, derived from chloroform partial etching, increased surface roughness and oxygen content. This resulted in enhanced cell adhesion, strength and proliferation as well as a balance of soluble factors for optimum collagen and elastin synthesis for tissue regeneration. By linking surface parameters to cell activity, we could determine the fate of the regenerated tissue to create successful soft tissue-engineered replacement.

Towards an in vitro model mimicking the foreign body response: tailoring the surface properties of biomaterials to modulate extracellular matrix

NASA Astrophysics Data System (ADS)

Damanik, Febriyani F. R.; Rothuizen, Tonia C.; van Blitterswijk, Clemens; Rotmans, Joris I.; Moroni, Lorenzo

2014-09-01

Despite various studies to minimize host reaction following a biomaterial implantation, an appealing strategy in regenerative medicine is to actively use such an immune response to trigger and control tissue regeneration. We have developed an in vitro model to modulate the host response by tuning biomaterials' surface properties through surface modifications techniques as a new strategy for tissue regeneration applications. Results showed tunable surface topography, roughness, wettability, and chemistry by varying treatment type and exposure, allowing for the first time to correlate the effect of these surface properties on cell attachment, morphology, strength and proliferation, as well as proinflammatory (IL-1β, IL-6) and antiflammatory cytokines (TGF-β1, IL-10) secreted in medium, and protein expression of collagen and elastin. Surface microstructuring, derived from chloroform partial etching, increased surface roughness and oxygen content. This resulted in enhanced cell adhesion, strength and proliferation as well as a balance of soluble factors for optimum collagen and elastin synthesis for tissue regeneration. By linking surface parameters to cell activity, we could determine the fate of the regenerated tissue to create successful soft tissue-engineered replacement.

Extracellular matrix protein in calcified endoskeleton: a potential additive for crystal growth and design

NASA Astrophysics Data System (ADS)

Azizur Rahman, M.; Fujimura, Hiroyuki; Shinjo, Ryuichi; Oomori, Tamotsu

2011-06-01

In this study, we demonstrate a key function of extracellular matrix proteins (ECMPs) on seed crystals, which are isolated from calcified endoskeletons of soft coral and contain only CaCO 3 without any living cells. This is the first report that an ECMP protein extracted from a marine organism could potentially influence in modifying the surface of a substrate for designing materials via crystallization. We previously studied with the ECMPs from a different type of soft coral ( Sinularia polydactyla) without introducing any seed crystals in the process , which showed different results. Thus, crystallization on the seed in the presence of ECMPs of present species is an important first step toward linking function to individual proteins from soft coral. For understanding this interesting phenomenon, in vitro crystallization was initiated in a supersaturated solution on seed particles of calcite (1 0 4) with and without ECMPs. No change in the crystal growth shape occurred without ECMPs present during the crystallization process. However, with ECMPs, the morphology and phase of the crystals in the crystallization process changed dramatically. Upon completion of crystallization with ECMPs, an attractive crystal morphology was found. Scanning electron microscopy (SEM) was utilized to observe the crystal morphologies on the seeds surface. The mineral phases of crystals nucleated by ECMPs on the seeds surface were examined by Raman spectroscopy. Although 50 mM Mg 2+ is influential in making aragonite in the crystallization process, the ECMPs significantly made calcite crystals even when 50 mM Mg 2+ was present in the process. Crystallization with the ECMP additive seems to be a technically attractive strategy to generate assembled micro crystals that could be used in crystals growth and design in the Pharmaceutical and biotechnology industries.