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.

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.

The FunFOLD2 server for the prediction of protein-ligand interactions

The FunFOLD2 server is a new independent server that integrates our novel protein–ligand binding site and quality assessment protocols for the prediction of protein function (FN) from sequence via structure. Our guiding principles were, first, to provide a simple unified resource to make our function prediction software easily accessible to all via a simple web interface and, second, to produce integrated output for predictions that can be easily interpreted. The server provides a clean web interface so that results can be viewed on a single page and interpreted by non-experts at a glance. The output for the prediction is an image of the top predicted tertiary structure annotated to indicate putative ligand-binding site residues. The results page also includes a list of the most likely binding site residues and the types of predicted ligands and their frequencies in similar structures. The protein–ligand interactions can also be interactively visualized in 3D using the Jmol plug-in. The raw machine readable data are provided for developers, which comply with the Critical Assessment of Techniques for Protein Structure Prediction data standards for FN predictions. The FunFOLD2 webserver is freely available to all at the following web site: http://www.reading.ac.uk/bioinf/FunFOLD/FunFOLD_form_2_0.html.

Genetic, structural, and molecular insights into the function of ras of complex proteins domains

Ras of complex proteins (ROC) domains were identified in 2003 as GTP binding modules in large multidomain proteins from Dictyostelium discoideum. Research into the function of these domains exploded with their identification in a number of proteins linked to human disease, including leucine-rich repeat kinase 2 (LRRK2) and death-associated protein kinase 1 (DAPK1) in Parkinson’s disease and cancer, respectively. This surge in research has resulted in a growing body of data revealing the role that ROC domains play in regulating protein function and signaling pathways. In this review, recent advances in the structural informa- tion available for proteins containing ROC domains, along with insights into enzymatic function and the integration of ROC domains as molecular switches in a cellular and organismal context, are explored.

Regulation of gene and protein expression in cardiac myocyte hypertrophy and apoptosis

Considerable efforts have been expended in elucidating the inter-cellular and intra-cellular signaling pathways which elicit cardiac myocyte hypertrophy or apoptosis, and in identifying the changes which are associated with the end-stage of the response. The challenge now is to link the two. Although some of the signaling effects will be the acute modulation of existing protein function, long-term effects which bring about and maintain the hypertrophic state or which culminate in cell death are mediated at the level of gene and protein expression. With the advances in micro-array technology and genome sequencing, it is now possible to obtain a picture of the global gene expression profile in myocytes or in whole heart which dictates the proteins which could be made. This is not the final picture since additional regulation at the level of translation modulates the relative proportions of each protein that can be made from the transcriptome. Even here, further regulation of protein stability and turnover means that ultimately it is still necessary to examine the proteome to determine what may cause the functional changes in a cell. Thus, in order to gain a full picture of events which regulate the response and gain some insight into possible points of intervention for therapy, it is necessary to examine gene expression, mRNA translation and protein expression in concert.

In silico identification and characterization of protein-ligand binding sites

Protein–ligand binding site prediction methods aim to predict, from amino acid sequence, protein–ligand interactions, putative ligands, and ligand binding site residues using either sequence information, structural information, or a combination of both. In silico characterization of protein–ligand interactions has become extremely important to help determine a protein’s functionality, as in vivo-based functional elucidation is unable to keep pace with the current growth of sequence databases. Additionally, in vitro biochemical functional elucidation is time-consuming, costly, and may not be feasible for large-scale analysis, such as drug discovery. Thus, in silico prediction of protein–ligand interactions must be utilized to aid in functional elucidation. Here, we briefly discuss protein function prediction, prediction of protein–ligand interactions, the Critical Assessment of Techniques for Protein Structure Prediction (CASP) and the Continuous Automated EvaluatiOn (CAMEO) competitions, along with their role in shaping the field. We also discuss, in detail, our cutting-edge web-server method, FunFOLD for the structurally informed prediction of protein–ligand interactions. Furthermore, we provide a step-by-step guide on using the FunFOLD web server and FunFOLD3 downloadable application, along with some real world examples, where the FunFOLD methods have been used to aid functional elucidation.

The use of proteomics to identify novel therapeutic targets for the treatment of disease

The completion of the Human Genome Project has revealed a multitude of potential avenues for the identification of therapeutic targets. Extensive sequence information enables the identification of novel genes but does not facilitate a thorough understanding of how changes in gene expression control the molecular mechanisms underlying the development and regulation of a cell or the progression of disease. Proteomics encompasses the study of proteins expressed by a population of cells, and evaluates changes in protein expression, post-translational modifications, protein interactions, protein structure and splice variants, all of which are imperative for a complete understanding of protein function within the cell. From the outset, proteomics has been used to compare the protein profiles of cells in healthy and diseased states and as such can be used to identify proteins associated with disease development and progression. These candidate proteins might provide novel targets for new therapeutic agents or aid the development of assays for disease biomarkers. This review provides an overview of the current proteomic techniques available and focuses on their application in the search for novel therapeutic targets for the treatment of disease.

Proteins and their interacting partners: an introduction to protein–ligand binding site prediction methods

Elucidating the biological and biochemical roles of proteins, and subsequently determining their interacting partners, can be difficult and time consuming using in vitro and/or in vivo methods, and consequently the majority of newly sequenced proteins will have unknown structures and functions. However, in silico methods for predicting protein–ligand binding sites and protein biochemical functions offer an alternative practical solution. The characterisation of protein–ligand binding sites is essential for investigating new functional roles, which can impact the major biological research spheres of health, food, and energy security. In this review we discuss the role in silico methods play in 3D modelling of protein–ligand binding sites, along with their role in predicting biochemical functionality. In addition, we describe in detail some of the key alternative in silico prediction approaches that are available, as well as discussing the Critical Assessment of Techniques for Protein Structure Prediction (CASP) and the Continuous Automated Model EvaluatiOn (CAMEO) projects, and their impact on developments in the field. Furthermore, we discuss the importance of protein function prediction methods for tackling 21st century problems.

A role for the thiol isomerase. protein ERP5 in platelet function

Formation and rearrangement of disulfide bonds during the correct folding of nascent proteins is modulated by a family of enzymes known as thiol isomerases, which include protein disulfide isomerase (PDI), endoplasmic reticulum protein 5 (ERP5), and ERP57. Recent evidence supports an alternative role for this family of proteins on the surface of cells, where they are involved in receptor 'remodeling and recognition. In platelets, blocking PDI with inhibitory antibodies inhibits a number of platelet activation pathways, including aggregation, secretion, and fibrinogen binding. Analysis of human platelet membrane fractions identified the presence of the thiol isomerase protein ERP5. Further study showed that ERP5 is resident mainly on platelet intracellular membranes, although it is rapidly recruited to the cell, surface in response to a range of platelet agonists. Blocking cell-surface ERP5 using inhibitory antibodies leads to a decrease in platelet aggregation in response to agonists, and a decrease in fibrinogen binding and P-selectin exposure. It is Possible that this is based on the disruption of integrin function, as we observed that ERP5 becomes physically associated with the integrin beta(3) subunit during platelet stimulation. These results provide new insights into the involvement of thiol isomerases and regulation of platelet activation. (C) 2005 by The American Society of Hematology.

Function of the hydration layer around an antifreeze protein revealed by atomistic molecular dynamics simulations

Atomistic molecular dynamics simulations are used to investigate the mechanism by which the antifreeze protein from the spruce budworm, Choristoneura fumiferana, binds to ice. Comparison of structural and dynamic properties of the water around the three faces of the triangular prism-shaped protein in aqueous solution reveals that at low temperature the water structure is ordered and the dynamics slowed down around the ice-binding face of the protein, with a disordering effect observed around the other two faces. These results suggest a dual role for the solvation water around the protein. The preconfigured solvation shell around the ice-binding face is involved in the initial recognition and binding of the antifreeze protein to ice by lowering the barrier for binding and consolidation of the protein:ice interaction surface. Thus, the antifreeze protein can bind to the molecularly rough ice surface by becoming actively involved in the formation of its own binding site. Also, the disruption of water structure around the rest of the protein helps prevent the adsorbed protein becoming covered by further ice growth.

PIGOK: linking protein identity to gene ontology and function

Here we introduce a computer database that allows for the rapid retrieval of physicochemical properties, Gene Ontology, and Kyoto Encyclopedia of Genes and Genomes information about a protein or a list of proteins. We applied PIGOK analyzing Schizosaccharomyces pombe proteins displaying differential expression under oxidative stress and identified their biological functions and pathways. The database is available on the Internet at http://pc4-133.ludwig.ucl.ac.uk/pigok.html.

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.

Roles of proteolysis in regulation of G protein-coupled receptor function

The enzymatic activity of peptidases must be tightly regulated to prevent uncontrolled hydrolysis of peptide bonds, which could have devastating effects in biological systems. Peptidases are often generated as inactive propeptidases, secreted with endogenous inhibitors or they are compartmentalized. Propeptidases become active after proteolytic removal of N-terminal activation peptides by other peptidases. Some peptidases only become active towards substrates only at certain pHs, thus confining activity to specific compartments or conditions. This review discusses the different roles proteolysis plays in regulating G protein-coupled receptors (GPCRs). At the cell-surface, certain GPCRs are regulated by the hydrolytic inactivation of bioactive peptides by membrane-anchored peptidases, which prevents signaling. Conversely, cell-surface peptidases can also generate bioactive peptides that directly activate GPCRs. Alternatively, cell-surface peptidases activated by GPCRs, can generate bioactive peptides to cause transactivation of receptor tyrosine kinases, thereby promoting signaling. Certain peptidases can signals directly to cells, by cleaving GPCR to initiate intracellular signaling cascades. Intracellular peptidases also regulate GPCRs; lysosomal peptidases destroy GPCRs in lysosomes to permanently terminate signaling and mediate downregulation; endosomal peptidases cleave internalized peptide agonists to regulate GPCR recycling, resensitization and signaling; and soluble intracellular peptidases also participate in GPCR function by regulating the ubiquitination state of GPCRs, thereby altering GPCR signaling and fate. Although the use of peptidase inhibitors has already brought success in the treatment of diseases such as hypertension, the discovery of new regulatory mechanisms involving proteolysis that control GPCRs may provide additional targets to modulate dysregulated GPCR signaling in disease.

Transcription profiling in human platelets reveals LRRFIP1 as a novel protein regulating platelet function

Within the healthy population, there is substantial, heritable, and interindividual variability in the platelet response. We explored whether a proportion of this variability could be accounted for by interindividual variation in gene expression. Through a correlative analysis of genome-wide platelet RNA expression data from 37 subjects representing the normal range of platelet responsiveness within a cohort of 500 subjects, we identified 63 genes in which transcript levels correlated with variation in the platelet response to adenosine diphosphate and/or the collagen-mimetic peptide, cross-linked collagen-related peptide. Many of these encode proteins with no reported function in platelets. An association study of 6 of the 63 genes in 4235 cases and 6379 controls showed a putative association with myocardial infarction for COMMD7 (COMM domain-containing protein 7) and a major deviation from the null hypo thesis for LRRFIP1 [leucine-rich repeat (in FLII) interacting protein 1]. Morpholino-based silencing in Danio rerio identified a modest role for commd7 and a significant effect for lrrfip1 as positive regulators of thrombus formation. Proteomic analysis of human platelet LRRFIP1-interacting proteins indicated that LRRFIP1 functions as a component of the platelet cytoskeleton, where it interacts with the actin-remodeling proteins Flightless-1 and Drebrin. Taken together, these data reveal novel proteins regulating the platelet response.