The role of lipid composition on the interaction between a tryptophan-rich protein and model bacterial membranes

The interaction between tryptophan-rich puroindoline proteins and model bacterial membranes at the air-liquid interface has been investigated by FTIR spectroscopy, surface pressure measurements and Brewster angle microscopy. The role of different lipid constituents on the interactions between lipid membrane and protein was studied using wild type (Pin-b) and mutant (Trp44 to Arg44 mutant, Pin-bs) puroindoline proteins. The results show differences in the lipid selectivity of the two proteins in terms of preferential binding to specific lipid head groups in mixed lipid systems. Pin-b wild type was able to penetrate mixed layers of phosphatidylethanolamine (PE) and phosphatidylglycerol (PG) head groups more deeply compared to the mutant Pin-bs. Increasing saturation of the lipid tails increased penetration and adsorption of Pin-b wild type, but again the response of the mutant form differed. The results provide insight as to the role of membrane architecture, lipid composition and fluidity, on antimicrobial activity of proteins. Data show distinct differences in the lipid binding behavior of Pin-b as a result of a single residue mutation, highlighting the importance of hydrophobic and charged amino acids in antimicrobial protein and peptide activity.

Selected wheat seed defense proteins exhibit competitive binding to model microbial lipid interfaces

Puroindolines (Pins) and purothionins (Pths) are basic, amphiphilic, cysteine-rich wheat proteins that play a role in plant defense against microbial pathogens. We have examined the co-adsorption and sequential addition of Pins (Pin-a, Pin-b and a mutant form of Pin-b with Trp-44 to Arg-44 substitution) and β-purothionin (β-Pth) model anionic lipid layers, using a combination of surface pressure measurements, external reflection FTIR spectroscopy and neutron reflectometry. Results highlighted differences in the protein binding mechanisms, and in the competitive binding and penetration of lipid layers between respective Pins and β-Pth. Pin-a formed a blanket-like layer of protein below the lipid surface that resulted in the reduction or inhibition of β-Pth penetration of the lipid layer. Wild-type Pin-b participated in co-operative binding with β-Pth, whereas the mutant Pin-b did not bind to the lipid layer in the presence of β-Pth. The results provide further insight into the role of hydrophobic and cationic amino acid residues in antimicrobial activity.

Lipid binding interactions of antimicrobial plant seed defence proteins: puroindoline-a and β-purothionin

The indolines and thionins are basic, amphiphilic and cysteine-rich proteins found in cereals; puroindoline-a (Pin-a) and β-purothionin (β-Pth) are members of these families in wheat (Triticum aestivum). Pin-a and β-Pth have been suggested to play a significant role in seed defence against microbial pathogens, making the interaction of these proteins with model bacterial membranes an area of potential interest. We have examined the binding of these proteins to lipid monolayers composed of 1,2-dipalmitoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (DPPG) using a combination of neutron reflectometry, Brewster angle microscopy, and infrared spectroscopy. Results showed that both Pin-a and β-Pth interact strongly with condensed phase DPPG monolayers, but the degree of penetration was different. β-Pth was shown to penetrate the lipid acyl chain region of the monolayer and remove lipids from the air/liquid interface during the adsorption process, suggesting this protein may be able to both form membrane spanning ion channels and remove membrane phospholipids in its lytic activity. Conversely, Pin-a was shown to interact mainly with the head-group region of the condensed phase DPPG monolayer and form a 33 Å thick layer below the lipid film. The differences between the interfacial structures formed by these two proteins may be related to the differing composition of the Pin-a and β-Pth hydrophobic regions.

Promoter analysis and immunolocalisation show that puroindoline genes are exclusively expressed in starchy endosperm cells of wheat grain

The purolindolines are small cysteine-rich proteins which are present in the grain of wheat. They have a major impact on the utilisation of the grain as they are the major determinants of grain texture, which affects both milling and baking properties. Bread and durum wheats were transformed with constructs comprising the promoter regions of the Puroindoline a (Pina) and Puroindoline b (Pinb) genes fused to the uidA (GUS) reporter gene. Nine lines showing 3:1 segregation for the transgene and comprising all transgene/species combinations were selected for detailed analysis of transgene expression during grain development. This showed that transgene expression occurred only in the starchy endosperm cells and was not observed in any other seed or vegetative tissues. The location of the puroindoline proteins in these cells was confirmed by tissue printing of developing grain, using a highly specific monoclonal antibody for detection and an antibody to the aleurone-localised 8S globulin as a control. This provides clear evidence that puroindolines are only synthesised and accumulated in the starchy endosperm cells of the wheat grain.

Spatial patterns of gluten protein and polymer distribution in wheat grain

The starchy endosperm is the major storage tissue in the mature wheat grain and exhibits quantitative and qualitative gradients in composition, with the outermost cell layers being rich in protein, mainly gliadins, and the inner cells being low in protein but enriched in high-molecular-weight (HMW) subunits of glutenin. We have used sequential pearling to produce flour fractions enriched in particular cell layers to determine the protein gradients in four different cultivars grown at two nitrogen levels. The results show that the steepness of the protein gradient is determined by both genetic and nutritional factors, with three high-protein breadmaking cultivars being more responsive to the N treatment than a low-protein cultivar suitable for livestock feed. Nitrogen also affected the relative abundances of the three main classes of wheat prolamins: the sulfur-poor ω-gliadins showed the greatest response to nitrogen and increased evenly across the grain; the HMW subunits also increased in response to nitrogen but proportionally more in the outer layers of the starchy endosperm than near the core, while the sulfur-rich prolamins showed the opposite trend.

In vitro batch cultures of gut microbiota from healthy and ulcerative colitis (UC) subjects suggest that sulphate-reducing bacteria levels are raised in UC and by a protein-rich diet.

Imbalances in gut microbiota composition during ulcerative colitis (UC) indicate a role for the microbiota in propagating the disorder. Such effects were investigated using in vitro batch cultures (with/without mucin, peptone or starch) inoculated with faecal slurries from healthy or UC patients; the growth of five bacterial groups was monitored along with short-chain fatty acid (SCFA) production. Healthy cultures gave two-fold higher growth and SCFA levels with up to ten-fold higher butyrate production. Starch gave the highest growth and SCFA production (particularly butyrate), indicating starch-enhanced saccharolytic activity. Sulphate-reducing bacteria (SRB) were the predominant bacterial group (of five examined) for UC inocula whereas they were the minority group for the healthy inocula. Furthermore, SRB growth was stimulated by peptone presumably due to the presence of sulphur-rich amino acids. The results suggest raised SRB levels in UC, which could contribute to the condition through release of toxic sulphide.

AtTRB1, a telomeric DNA-binding protein from Arabidopsis, is concentrated in the nucleolus and shows highly dynamic association with chromatin

AtTRB1, 2 and 3 are members of the SMH (single Myb histone) protein family, which comprises double-stranded DNA-binding proteins that are specific to higher plants. They are structurally conserved, containing a Myb domain at the N-terminus, a central H1/H5-like domain and a C-terminally located coiled-coil domain. AtTRB1, 2 and 3 interact through their Myb domain specifically with telomeric double-stranded DNA in vitro, while the central H1/H5-like domain interacts non-specifically with DNA sequences and mediates protein–protein interactions. Here we show that AtTRB1, 2 and 3 preferentially localize to the nucleus and nucleolus during interphase. Both the central H1/H5-like domain and the Myb domain from AtTRB1 can direct a GFP fusion protein to the nucleus and nucleolus. AtTRB1–GFP localization is cell cycle-regulated, as the level of nuclear-associated GFP diminishes during mitotic entry and GFP progressively re-associates with chromatin during anaphase/telophase. Using fluorescence recovery after photobleaching and fluorescence loss in photobleaching, we determined the dynamics of AtTRB1 interactions in vivo. The results reveal that AtTRB1 interaction with chromatin is regulated at two levels at least, one of which is coupled with cell-cycle progression, with the other involving rapid exchange.

Probing protein−tannin interactions by isothermal titration microcalorimetry

Isothermal titration microcalorimetry (ITC) has been applied to investigate protein−tannin interactions. Two hydrolyzable tannins were studied, namely myrabolan and tara tannins, for their interaction with bovine serum albumin (BSA), a model globular protein, and gelatin, a model proline-rich random coil protein. Calorimetry data indicate that protein−tannin interaction mechanisms are dependent upon the nature of the protein involved. Tannins apparently interact nonspecifically with the globular BSA, leading to binding saturation at estimated tannin/BSA molar ratios of 48:1 for tara- and 178:1 for myrabolan tannins. Tannins bind to the random coil protein gelatin by a two-stage mechanism. The energetics of the first stage show evidence for cooperative binding of tannins to the protein, while the second stage indicates gradual saturation of binding sites as observed for interaction with BSA. The structure and flexibility of the tannins themselves alters the stoichiometry of the interaction, but does not appear to have any significant affect on the overall binding mechanism observed. This study demonstrates the potential of ITC for providing an insight into the nature of protein−tannin interactions.

Probing protein-tannin interactions by isothermal titration microcalorimetry

Isothermal titration microcalorimetry (ITC) has been applied to investigate protein-tannin interactions. Two hydrolyzable tannins were studied, namely myrabolan and tara tannins, for their interaction with bovine serum albumin (BSA), a model globular protein, and gelatin, a model proline-rich random coil protein. Calorimetry data indicate that protein-tannin interaction mechanisms are dependent upon the nature of the protein involved. Tannins apparently interact nonspecifically with the globular BSA, leading to binding saturation at estimated tannin/BSA molar ratios of 48:1 for tara- and 178:1 for myrabolan tannins. Tannins bind to the random coil protein gelatin by a two-stage mechanism. The energetics of the first stage show evidence for cooperative binding of tannins to the protein, while the second stage indicates gradual saturation of binding sites as observed for interaction with BSA. The structure and flexibility of the tannins themselves alters the stoichiometry of the interaction, but does not appear to have any significant affect on the overall binding mechanism observed. This study demonstrates the potential of ITC for providing an insight into the nature of protein-tannin interactions.

Chemical composition and in vitro fermentation of tannin-rich tree fruits

Dry and mature tree fruits are a potential source of protein for goats in the semi-arid areas of southern Africa, but their chemical composition and feeding value is largely unknown. This study presents the chemical composition and in vitro fermentation of indehiscent whole fruits and separated seed and hull fractions from Acacia nilotica, Acacia erubescens, Acacia sieberiana, Acacia erioloba, Piliostigma thonningii and Dichrostachys cinerea trees. Results indicate that the N contents of whole fruits ranged between 13.5 g/kg DM (A. nilotica) and 27.1 g/kg DM (A. erubescens). Seeds had a higher N content than hulls for all tree species. A. nilotica, D. cinerea and P thonningii fruits had high levels of extractable phenolics (758, 458 and 299 g/kg DM, respectively). Soluble phenolics (SPh) and ytterbium precipitable phenolics (YbPh) levels were negatively correlated to in vitro gas production but positively correlated to in vitro organic matter degradability (iOMD). Partition factors for whole fruits at 48 h ranged between 3.6 mg/ml for A. erioloba and 7.8 mg/ml for A. nilotica. Seeds of A. erioloba, A. erubescens and P thonningii were consistently fermented more efficiently throughout the incubation period compared to their whole fruits or hulls. Estimating in vitro degradability of phenolic-rich substrates through filtration procedures can give erroneous results due to the loss of soluble phenolics, which are not necessarily degradable. The feeding value of fruits from D. cinerea and A. nilotica tree species may be reduced due to the presence of high levels of phenolics. (C) 2007 Elsevier B.V. All rights reserved.

Contribution of redox status to hepatitis C virus E2 envelope protein function and antigenicity

Disulfide bonding contributes to the function and antigenicity of many viral envelope glycoproteins. We assessed here its significance for the hepatitis C virus E2 envelope protein and a counterpart deleted for hypervariable region-1 (HVR1). All 18 cysteine residues of the antigens were involved in disulfides. Chemical reduction of up to half of these disulfides was compatible with anti-E2 monoclonal antibody reaction, CD81 receptor binding, and viral entry, whereas complete reduction abrogated these properties. The addition of 5,5'-dithiobis-2-nitrobenzoic acid had no effect on viral entry. Thus, E2 function is only weakly dependent on its redox status, and cell entry does not require redox catalysts, in contrast to a number of enveloped viruses. Because E2 is a major neutralizing antibody target, we examined the effect of disulfide bonding on E2 antigenicity. We show that reduction of three disulfides, as well as deletion of HVR1, improved antibody binding for half of the patient sera tested, whereas it had no effect on the remainder. Small scale immunization of mice with reduced E2 antigens greatly improved serum reactivity with reduced forms of E2 when compared with immunization using native E2, whereas deletion of HVR1 only marginally affected the ability of the serum to bind the redox intermediates. Immunization with reduced E2 also showed an improved neutralizing antibody response, suggesting that potential epitopes are masked on the disulfide-bonded antigen and that mild reduction may increase the breadth of the antibody response. Although E2 function is surprisingly independent of its redox status, its disulfide bonds mask antigenic domains. E2 redox manipulation may contribute to improved vaccine design.

A novel method for production of lipid hydroperoxide- or oxysterol-rich low-density lipoprotein

LDL oxidation may be important in atherosclerosis. Extensive oxidation of LDL by copper induces increased uptake by macrophages, but results in decomposition of hydroperoxides, making it more difficult to investigate the effects of hydroperoxides in oxidised LDL on cell function. We describe here a simple method of oxidising LDL by dialysis against copper ions at 4 degrees C, which inhibits the decomposition of hydroperoxides, and allows the production of LDL rich in hydroperoxides (626 +/- 98 nmol/mg LDL protein) but low in oxysterols (3 +/- 1 nmol 7-ketocholesterol/mg LDL protein), whilst allowing sufficient modification (2.6 +/- 0.5 relative electrophoretic mobility) for rapid uptake by macrophages (5.49 +/- 0.75 mu g I-125-labelled hydroperoxide-rich LDL vs. 0.46 +/- 0.04 mu g protein/mg cell protein in 18 h for native LDL). By dialysing under the same conditions, but at 37 degrees C, the hydroperoxides are decomposed extensively and the LDL becomes rich in oxysterols. This novel method of oxidising LDL with high yield to either a hydroperoxide- or oxysterol-rich form by simply altering the temperature of dialysis may provide a useful tool for determining the effects of these different oxidation products on cell function. (C) 2007 Elsevier Ireland Ltd. All rights reserved.

Proteornics analysis unravels the functional repertoire of coronavirus nonstructural protein 3

Severe acute respiratory syndrome (SARS) coronavirus infection and growth are dependent on initiating signaling and enzyme actions upon viral entry into the host cell. Proteins packaged during virus assembly may subsequently form the first line of attack and host manipulation upon infection. A complete characterization of virion components is therefore important to understanding the dynamics of early stages of infection. Mass spectrometry and kinase profiling techniques identified nearly 200 incorporated host and viral proteins. We used published interaction data to identify hubs of connectivity with potential significance for virion formation. Surprisingly, the hub with the most potential connections was not the viral M protein but the nonstructurall protein 3 (nsp3), which is one of the novel virion components identified by mass spectrometry. Based on new experimental data and a bioinformatics analysis across the Coronaviridae, we propose a higher-resolution functional domain architecture for nsp3 that determines the interaction capacity of this protein. Using recombinant protein domains expressed in Escherichia coli, we identified two additional RNA-binding domains of nsp3. One of these domains is located within the previously described SARS-unique domain, and there is a nucleic acid chaperone-like domain located immediately downstream of the papain-like proteinase domain. We also identified a novel cysteine-coordinated metal ion-binding domain. Analyses of interdomain interactions and provisional functional annotation of the remaining, so-far-uncharacterized domains are presented. Overall, the ensemble of data surveyed here paint a more complete picture of nsp3 as a conserved component of the viral protein processing machinery, which is intimately associated with viral RNA in its role as a virion component.

Fas ligand-induced apoptosis is regulated by nitric oxide through the inhibition of fas receptor clustering and the nitrosylation of protein kinase Cepsilon

Apoptosis induced by the death-inducing ligand FasL (CD95L) is a major mechanism of cell death. Trophoblast cells express the Fas receptor yet survive in an environment that is rich in the ligand. We report that basal nitric oxide (NO) production is responsible for the resistance of trophoblasts to FasL-induced apoptosis. In this study we demonstrate that basal NO production resulted in the inhibition of receptor clustering following ligand binding. In addition NO also protected cells through the selective nitrosylation, and inhibition, of protein kinase Cepsilon (PKCepsilon) but not PKCalpha. In the absence of NO production PKCepsilon interacted with, and phosphorylated, the anti-apoptotic protein cFLIP. The interaction is predominantly with the short form of cFLIP and its phosphorylation reduces its recruitment to the death-inducing signaling complex (DISC) that is formed following binding of a death-inducing ligand to its receptor. Inhibition of cFLIP recruitment to the DISC leads to increased activation of caspase 8 and subsequently to apoptosis. Inhibition of PKCepsilon using siRNA significantly reversed the sensitivity to apoptosis induced by inhibition of NO synthesis suggesting that NO-mediated inhibition of PKCepsilon plays an important role in the regulation of Fas-induced apoptosis.

Nuclear magnetic resonance structure of the N-terminal domain of nonstructural protein 3 from the severe acute respiratory syndrome coronavirus

This paper describes the structure determination of nsp3a, the N-terminal domain of the severe acute respiratory syndrome coronavirus (SARS-CoV) nonstructural protein 3. nsp3a exhibits a ubiquitin-like globular fold of residues 1 to 112 and a flexibly extended glutamic acid-rich domain of residues 113 to 183. In addition to the four beta-strands and two alpha-helices that are common to ubiquitin-like folds, the globular domain of nsp3a contains two short helices representing a feature that has not previously been observed in these proteins. Nuclear magnetic resonance chemical shift perturbations showed that these unique structural elements are involved in interactions with single-stranded RNA. Structural similarities with proteins involved in various cell-signaling pathways indicate possible roles of nsp3a in viral infection and persistence.