No impact of malaria infection or immunisation on the expression of the immune GTPase GIMAP1 at the protein or mRNA levels (Article no. 53)

GIMAP (GTPase of the immunity-associated protein family) proteins are a family of putative GTPases believed to be regulators of cell death in lymphomyeloid cells. GIMAP1 was the first reported member of this gene family, identified as a gene up-regulated at the RNA level in the spleens of mice infected with the malarial parasite, Plasmodium chabaudi. Methods A monoclonal antibody against mouse GIMAP1 was developed and was used to analyse the expression of the endogenous protein in tissues of normal mice and in defined sub-populations of cells prepared from lymphoid tissues using flow cytometry. It was also used to assess the expression of GIMAP1 protein after infection and/or immunization of mice with P. chabaudi. Real-time PCR analysis was employed to measure the expression of GIMAP1 for comparison with the protein level analysis. Results GIMAP1 protein expression was detected in all lineages of lymphocytes (T, B, NK), in F4/80+ splenic macrophages and in some lymphoid cell lines. Additional evidence is presented suggesting that the strong expression by mature B cells of GIMAP1 and other GIMAP genes and proteins seen in mice may be a species-dependent characteristic. Unexpectedly, no increase was found in the expression of GIMAP1 in P. chabaudi infected mice at either the mRNA or protein level, and this remained so despite applying a number of variations to the protocol. Conclusion The model of up-regulation of GIMAP1 in response to infection/immunization with P. chabaudi is not a robustly reproducible experimental system. The GIMAP1 protein is widely expressed in lymphoid cells, with an interesting increase in expression in the later stages of B cell development. Alternative approaches will be required to define the functional role of this GTPase in immune cells.

Epidermal growth factor-stimulated extravillous cytotrophoblast motility is mediated by the activation of PI3-K, Akt and both p38 and p42/44 mitogen-activated protein kinases

BACKGROUND: Trophoblast invasion is a temporally and spatially regulated scheme of events that can dictate pregnancy outcome. Evidence suggests that the potent mitogen epidermal growth factor (EGF) regulates cytotrophoblast (CTB) differentiation and invasion during early pregnancy. METHODS AND RESULTS: In the present study, the first trimester extravillous CTB cell line SGHPL-4 was used to investigate the signalling pathways involved in the motile component of EGF-mediated CTB migration/invasion. EGF induced the phosphorylation of the phosphatidylinositol 3-kinase (PI3-K)-dependent proteins, Akt and GSK-3β as well as both p42/44 MAPK and p38 mitogen-activated protein kinases (MAPK). EGF-stimulated motility was significantly reduced following the inhibition of PI3-K (P < 0.001), Akt (P < 0.01) and both p42/44 MAPK (P < 0.001) and p38 MAPKs (P < 0.001) but not the inhibition of GSK-3β. Further analysis indicated that the p38 MAPK inhibitor SB 203580 inhibited EGF-stimulated phosphorylation of Akt on serine 473, which may be responsible for the effect SB 203580 has on CTB motility. Although Akt activation leads to GSK-3β phosphorylation and the subsequent expression of β-catenin, activation of this pathway by 1-azakenpaullone was insufficient to stimulate the motile phenotype. CONCLUSION: We demonstrate a role for PI3-K, p42/44 MAPK and p38 MAPK in the stimulation of CTB cell motility by EGF, however activation of β-catenin alone was insufficient to stimulate cell motility.

Intrinsic disorder prediction from the analysis of multiple protein fold recognition models

Motivation: Intrinsic protein disorder is functionally implicated in numerous biological roles and is, therefore, ubiquitous in proteins from all three kingdoms of life. Determining the disordered regions in proteins presents a challenge for experimental methods and so recently there has been much focus on the development of improved predictive methods. In this article, a novel technique for disorder prediction, called DISOclust, is described, which is based on the analysis of multiple protein fold recognition models. The DISOclust method is rigorously benchmarked against the top.ve methods from the CASP7 experiment. In addition, the optimal consensus of the tested methods is determined and the added value from each method is quantified. Results: The DISOclust method is shown to add the most value to a simple consensus of methods, even in the absence of target sequence homology to known structures. A simple consensus of methods that includes DISOclust can significantly outperform all of the previous individual methods tested.

Dietary glycated protein modulates the colonic microbiota towards a more detrimental composition in ulcerative colitis patients and non-ulcerative colitis subjects

AIM: To investigate the effect of native, heated and glycated bovine serum albumin (BSA) on the ulcerative colitis (UC) and non-UC colonic microbiota in vitro. METHODS AND RESULTS: Continuous flow culture (CFC) models of the human colonic microbiota inoculated with faeces from UC and non-UC volunteers were maintained on BSA as growth substrate. Changes in bacterial populations and short-chain fatty acids were determined. UC and non-UC microbiota differed significantly in microbial populations, with elevated numbers of sulfate-reducing bacteria (SRB) and clostridia in the microbiota from UC patients. Compared with native BSA, glycated BSA modulated the gut microbiota of UC patients in vitro towards a more detrimental community structure with significant increases in putatively harmful bacteria (clostridia, bacteroides and SRB; P < 0.009) and decreases in dominant and putatively beneficial bacterial groups (eubacteria and bifidobacteria; P < 0.0004). The levels of beneficial short-chain fatty acids were significantly decreased by heated or glycated BSA, but were increased significantly by native BSA. CONCLUSION: The UC colonic microbiota maintained in CFC was significantly modified by glycated BSA. SIGNIFICANCE AND IMPACT OF THE STUDY: Results suggest that dietary glycated protein may impact upon the composition and activity of the colonic microbiota, an important environmental variable in UC.

Benchmarking consensus model quality assessment for protein fold recognition

Background: Selecting the highest quality 3D model of a protein structure from a number of alternatives remains an important challenge in the field of structural bioinformatics. Many Model Quality Assessment Programs (MQAPs) have been developed which adopt various strategies in order to tackle this problem, ranging from the so called "true" MQAPs capable of producing a single energy score based on a single model, to methods which rely on structural comparisons of multiple models or additional information from meta-servers. However, it is clear that no current method can separate the highest accuracy models from the lowest consistently. In this paper, a number of the top performing MQAP methods are benchmarked in the context of the potential value that they add to protein fold recognition. Two novel methods are also described: ModSSEA, which based on the alignment of predicted secondary structure elements and ModFOLD which combines several true MQAP methods using an artificial neural network. Results: The ModSSEA method is found to be an effective model quality assessment program for ranking multiple models from many servers, however further accuracy can be gained by using the consensus approach of ModFOLD. The ModFOLD method is shown to significantly outperform the true MQAPs tested and is competitive with methods which make use of clustering or additional information from multiple servers. Several of the true MQAPs are also shown to add value to most individual fold recognition servers by improving model selection, when applied as a post filter in order to re-rank models. Conclusion: MQAPs should be benchmarked appropriately for the practical context in which they are intended to be used. Clustering based methods are the top performing MQAPs where many models are available from many servers; however, they often do not add value to individual fold recognition servers when limited models are available. Conversely, the true MQAP methods tested can often be used as effective post filters for re-ranking few models from individual fold recognition servers and further improvements can be achieved using a consensus of these methods.

Assembly rules for protein networks derived from phylogenetic-statistical analysis of whole genomes

Background: We report an analysis of a protein network of functionally linked proteins, identified from a phylogenetic statistical analysis of complete eukaryotic genomes. Phylogenetic methods identify pairs of proteins that co-evolve on a phylogenetic tree, and have been shown to have a high probability of correctly identifying known functional links. Results: The eukaryotic correlated evolution network we derive displays the familiar power law scaling of connectivity. We introduce the use of explicit phylogenetic methods to reconstruct the ancestral presence or absence of proteins at the interior nodes of a phylogeny of eukaryote species. We find that the connectivity distribution of proteins at the point they arise on the tree and join the network follows a power law, as does the connectivity distribution of proteins at the time they are lost from the network. Proteins resident in the network acquire connections over time, but we find no evidence that 'preferential attachment' - the phenomenon of newly acquired connections in the network being more likely to be made to proteins with large numbers of connections - influences the network structure. We derive a 'variable rate of attachment' model in which proteins vary in their propensity to form network interactions independently of how many connections they have or of the total number of connections in the network, and show how this model can produce apparent power-law scaling without preferential attachment. Conclusion: A few simple rules can explain the topological structure and evolutionary changes to protein-interaction networks: most change is concentrated in satellite proteins of low connectivity and small phenotypic effect, and proteins differ in their propensity to form attachments. Given these rules of assembly, power law scaled networks naturally emerge from simple principles of selection, yielding protein interaction networks that retain a high-degree of robustness on short time scales and evolvability on longer evolutionary time scales.

Study of tyrosine kinases and protein tyrosine phosphorylation

In recent years, our increased understanding of the complex signal transduction mechanisms that regulate cellular function has fueled huge advances in all aspects of biomedical science and cell biology. Platelet and megakaryocyte function is no exception to this. In the last 10 yr our understanding of the receptor biochemistry and the systems that they control has been pivotal in the development of new strategies to inhibit platelet function and thereby prevent thrombosis. Experimental techniques have become more and more elegant, however; the basic toolbox that a researcher requires to study signaling in platelets and megakaryoctes is described in this and several subsequent chapters.

Dietary glycated protein modulates the colonic microbiota towards a more detrimental composition in ulcerative colitis patients and non-ulcerative colitis subjects

Aim: To investigate the effect of native, heated and glycated bovine serum albumin (BSA) on the ulcerative colitis (UC) and non-UC colonic microbiota in vitro. Methods and Results: Continuous flow culture (CFC) models of the human colonic microbiota inoculated with faeces from UC and non-UC volunteers were maintained on BSA as growth substrate. Changes in bacterial populations and short-chain fatty acids were determined. UC and non-UC microbiota differed significantly in microbial populations, with elevated numbers of sulfate-reducing bacteria (SRB) and clostridia in the microbiota from UC patients. Compared with native BSA, glycated BSA modulated the gut microbiota of UC patients in vitro towards a more detrimental community structure with significant increases in putatively harmful bacteria (clostridia, bacteroides and SRB; P < 0.009) and decreases in dominant and putatively beneficial bacterial groups (eubacteria and bifidobacteria; P < 0.0004). The levels of beneficial short-chain fatty acids were significantly decreased by heated or glycated BSA, but were increased significantly by native BSA. Conclusion: The UC colonic microbiota maintained in CFC was significantly modified by glycated BSA. Significance and Impact of the Study: Results suggest that dietary glycated protein may impact upon the composition and activity of the colonic microbiota, an important environmental variable in UC.

Some functional properties of fractionated soy protein isolates obtained by microfiltration

Five soy proteins isolate (SPI) fractions were produced using two microfiltration membranes with different pore sizes. Fractionation was carried out on SPI produced by isoelectric precipitation of a crude protein extract. The five fractions were two retentates and two permeates from the two membranes, the fifth fraction was obtained as the retentate on the smaller-po re- sized membrane fed with the permeate from the larger-pore-sized membrane. Solubility, foaming and emulsifying properties of the collected fractionates were investigated. It was observed that in the pH range 3-8 the retentates featured superior solubility compared with permeates. There was no significant difference (p > 0.0 1) in solubility between the retentates and SPI at pH >= 6. Foaming characteristics of the fractions followed the same trend as solubility with regard to foam expansion. There was, however, no particular trend observed with regards to foam stability. Emulsions stabilised by the retentates exhibited higher values (p<0.01) of emulsion stability index (ESI) and emulsifying activity index (EAI) than those stabilised with permeates. Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) profiles indicated that the fractions exhibiting high functionality in terms of solubility, foaming and emulsifying properties were also richer in 7S globulin soy protein subunits. Isoelectric focussing (IEF) profiles showed that retentates were richer in species with isoelectric points (pl) between 5.2 and 5.6 while permeates featured more prominently at pis between 4.5 and 4.8. (C) 2006 Elsevier Ltd. All rights reserved.

Agonist binding, agonist affinity and agonist efficacy at G protein-coupled receptors

Measurements of affinity and efficacy are fundamental for work on agonists both in drug discovery and in basic studies on receptors. In this review I wish to consider methods for measuring affinity and efficacy at G protein coupled receptors (GPCRs). Agonist affinity may be estimated in terms of the dissociation constant for agonist binding to a receptor using ligand binding or functional assays. It has, however, been suggested that measurements of affinity are always contaminated by efficacy so that it is impossible to separate the two parameters. Here I show that for many GPCRs, if receptor/G protein coupling is suppressed, experimental measurements of agonist affinity using ligand binding (K-obs) provide quite accurate measures of the agonist microscopic dissociation constant (K-A). Also in pharmacological functional studies, good estimates of agonist dissociation constants are possible. Efficacy can be quantitated in several ways based on functional data ( maximal effect of the agonist (E-max), ratio of agonist dissociation constant to concentration of agonist giving half maximal effect in functional assay ( K-obs/ EC50), a combined parameter EmaxKobs/EC50). Here I show that EmaxKobs/EC50 provides the best assessment of efficacy for a range of agonists across the full range of efficacy for full to partial agonists. Considerable evidence now suggests that ligand efficacy may be dependent on the pathway used to assess it. The efficacy of a ligand may, therefore, be multidimensional. It is still, however, necessary to have accurate measures of efficacy in different pathways.

Expression and regulation of 80K/MARCKS, a major substrate of protein kinase C, in the developing rat heart

Objective: Protein kinase C (PKC) plays a pivotal role in modulating the growth and differentiation of many cell types including the cardiac myocyte. However, little is known about molecules that act immediately downstream of PKC in the heart. In this study we have investigated the expression of 80K/MARCKS, a major PKC substrate, in whole ventricles and in cardiac myocytes from developing rat hearts. Methods: Poly A+ RNA was prepared from neonatal (2-day) and adult (42-day) cardiac myocytes and whole ventricular tissue and mRNA expression determined by reverse transcription-polymerase chain reaction (RT-PCR) using primers designed to identify a 420 bp fragment in the 80K/MARCKS gene. Protein extracts were prepared from either 2-day and 42-day cardiac myocytes or from whole ventricular tissue at 2, 5–11, 14, 17, 21, 28 and 42 days of age. Protein expression was determined by immunoblotting with an 80K/MARCKS antipeptide antibody and PKC activity was determined by measuring the amount of γ32P-ATP transferred to a specific peptide substrate. Results: RT-PCR analysis of 80K/MARCKS mRNA in neonatal (2-day) and adult (42-day) cardiac myocytes showed the expression of this gene in both cell types. Immunoblotting revealed maximum 80K/MARCKS protein expression in whole ventricular tissue at 5 days (a 75% increase above values at 2 days), followed by a transient decrease in expression during the 6–8-day period (61% of the protein expressed at 2 days for 8-day tissue) with levels returning to 5 day levels by 11 days of age. 80K/MARCKS protein was present in cardiac myocytes at 2 days of age whereas it was not detectable in adult cells. In addition, PKC activity levels increased to 160% of levels present at 2 days in 8-day-old ventricles with PKC activity levels returning to 5-day levels by 9 days of age. This was then followed by a steady decline in both 80K/MARCKS protein expression and PKC activity through to adulthood. Conclusions: Expression of the PKC substrate, 80K/MARCKS, in cardiac myocytes changes significantly during development and the transient loss of immunoreactive protein during the 6–8-day developmental period may reflect 80K/MARCKS phosphorylation and subsequent down-regulation as a result of the concomitant up-regulation of PKC activity at this time.

Computational chemistry for elucidating protein function: energetics and dynamics of myoglobin-ligand systems

State-of-the-art computational methodologies are used to investigate the energetics and dynamics of photodissociated CO and NO in myoglobin (Mb···CO and Mb···NO). This includes the combination of molecular dynamics, ab initio MD, free energy sampling, and effective dynamics methods to compare the results with studies using X-ray crystallography and ultrafast spectroscopy metho ds. It is shown that modern simulation techniques along with careful description of the intermolecular interactions can give quantitative agreement with experiments on complex molecular systems. Based on this agreement predictions for as yet uncharacterized species can be made.

CO migration in native and mutant myoglobin: atomistic simulations for the understanding of protein function

Molecular dynamics simulations of the events after the photodissociation of CO in the myoglobin mutant L29F in which leucine is replaced by phenylalanine are reported. Using both classical and mixed quantum-classical molecular dynamics calculations, we observed the rapid motion of CO away from the distal heme pocket to other regions of the protein, in agreement with recent experimental results. The experimentally observed and calculated infrared spectra of CO after dissociation are also in good agreement. We compared the results with data from simulations of WT myoglobin. As the time resolution of experimental techniques is increased, theoretical methods and models can be validated at the atomic scale by direct comparison with experiment.

Evaluation of peak-picking algorithms for protein mass spectrometry

Peak picking is an early key step in MS data analysis. We compare three commonly used approaches to peak picking and discuss their merits by means of statistical analysis. Methods investigated encompass signal-to-noise ratio, continuous wavelet transform, and a correlation-based approach using a Gaussian template. Functionality of the three methods is illustrated and discussed in a practical context using a mass spectral data set created with MALDI-TOF technology. Sensitivity and specificity are investigated using a manually defined reference set of peaks. As an additional criterion, the robustness of the three methods is assessed by a perturbation analysis and illustrated using ROC curves.

Platelet endothelial cell adhesion molecule-1 regulates collagen-stimulated platelet function by modulating the association of phosphatidylinositol 3-kinase with Grb-2-associated binding protein-1 and linker for activation of T cells

Background: Platelet activation by collagen depends on signals transduced by the glycoprotein (GP)VI–Fc receptor (FcR)-chain collagen receptor complex, which involves recruitment of phosphatidylinositol 3-kinase (PI3K) to phosphorylated tyrosines in the linker for activation of T cells (LAT). An interaction between the p85 regulatory subunit of PI3K and the scaffolding molecule Grb-2-associated binding protein-1 (Gab1), which is regulated by binding of the Src homology 2 domain-containing protein tyrosine phosphatase-2 (SHP-2) to Gab1, has been shown in other cell types to sustain PI3K activity to elicit cellular responses. Platelet endothelial cell adhesion molecule-1 (PECAM-1) functions as a negative regulator of platelet reactivity and thrombosis, at least in part by inhibiting GPVI–FcR-chain signaling via recruitment of SHP-2 to phosphorylated immunoreceptor tyrosine-based inhibitory motifs in PECAM-1. Objective: To investigate the possibility that PECAM-1 regulates the formation of the Gab1–p85 signaling complexes, and the potential effect of such interactions on GPVI-mediated platelet activation in platelets. Methods: The ability of PECAM-1 signaling to modulate the LAT signalosome was investigated with immunoblotting assays on human platelets and knockout mouse platelets. Results: PECAM-1-associated SHP-2 in collagen-stimulated platelets binds to p85, which results in diminished levels of association with both Gab1 and LAT and reduced collagen-stimulated PI3K signaling. We therefore propose that PECAM-1-mediated inhibition of GPVI-dependent platelet responses result, at least in part, from recruitment of SHP-2–p85 complexes to tyrosine-phosphorylated PECAM-1, which diminishes the association of PI3K with activatory signaling molecules, such as Gab1 and LAT.