Directed evolution of Tau class glutathione transferases reveals a site that regulates catalytic efficiency and masks co-operativity.

A library of Tau class GSTs (glutathione transferases) was constructed by DNA shuffling using the DNA encoding the Glycine max GSTs GmGSTU2-2, GmGSTU4-4 and GmGSTU10-10. The parental GSTs are >88% identical at the sequence level; however, their specificity varies towards different substrates. The DNA library contained chimaeric structures of alternated segments of the parental sequences and point mutations. Chimaeric GST sequences were expressed in Escherichia coli and their enzymatic activities towards CDNB (1-chloro-2,4-dinitrobenzene) and the herbicide fluorodifen (4-nitrophenyl α,α,α-trifluoro-2-nitro-p-tolyl ether) were determined. A chimaeric clone (Sh14) with enhanced CDNB- and fluorodifen-detoxifying activities, and unusual co-operative kinetics towards CDNB and fluorodifen, but not towards GSH, was identified. The structure of Sh14 was determined at 1.75 Å (1 Å=0.1 nm) resolution in complex with S-(p-nitrobenzyl)-glutathione. Analysis of the Sh14 structure showed that a W114C point mutation is responsible for the altered kinetic properties. This was confirmed by the kinetic properties of the Sh14 C114W mutant. It is suggested that the replacement of the bulky tryptophan residue by a smaller amino acid (cysteine) results in conformational changes of the active-site cavity, leading to enhanced catalytic activity of Sh14. Moreover, the structural changes allow the strengthening of the two salt bridges between Glu(66) and Lys(104) at the dimer interface that triggers an allosteric effect and the communication between the hydrophobic sites.

Functional characterization of two novel germline mutations of the KCNJ5 gene in hypertensive patients without primary aldosteronism but with ACTH-dependent aldosterone hypersecretion.

BACKGROUND: Germline mutations of the KCNJ5 gene encoding Kir3·4, a member of the inwardly rectifying K+ channel, have been identified in 'normal' adrenal glands, patients with familial hyperaldosteronism (FH) type III, aldosterone-producing adenomas (APAs) and sporadic cases of primary aldosteronism (PA). OBJECTIVE: To present two novel KCNJ5 gene mutations in hypertensive patients without PA, but with Adrenocorticotropic hormone (ACTH)-dependent aldosterone hypersecretion. DESIGN AND PATIENTS: Two hypertensive patients without PA, who exhibited enhanced ACTH-dependent response of aldosterone secretion, underwent genetic testing for the presence of the CYP11B1/CYP11B2 chimeric gene and KCNJ5 gene mutations. Genomic DNA was isolated from peripheral white blood cells, and the exons of the entire coding regions of the above genes were amplified and sequenced. Electrophysiological studies were performed to determine the effect of identified mutation(s) on the membrane reversal potentials. Structural biology studies were also carried out. RESULTS: Two novel germline heterozygous KCNJ5 mutations, p.V259M and p.Y348N, were detected in the two subjects. Electrophysiological studies showed that the Y348N mutation resulted in significantly less negative reversal potentials, suggesting loss of ion selectivity, while the V259M mutation did not affect the Kir3.4 current. In the mutated structural biology model, the N348 mutant resulted in significant loss of the ability for hydrogen bonding, while the M259 mutant was capable of establishing weaker interactions. The CYP11B1/CYP11B2 chimeric gene was not detected. CONCLUSIONS: These findings expand on the clinical spectrum of phenotypes associated with KCNJ5 mutations and implicate these mutations in the pathogenesis of hypertension associated with increased aldosterone response to ACTH stimulation.

Functional characterization of the hGRαT556I causing Chrousos syndrome.

BACKGROUND: Chrousos syndrome is a rare pathologic condition characterized by generalized, partial resistance of target tissues to glucocorticoids and caused by inactivating mutations of the human glucocorticoid receptor (hGR) gene. A novel case of Chrousos syndrome has been reported in a patient with adrenal incidentaloma, who harboured a heterozygous point mutation in the hGR gene, which resulted in threonine (T) to isoleucine (I) substitution at amino acid position 556 in the ligand-binding domain of the receptor. OBJECTIVE: To delineate the molecular mechanisms through which the mutant receptor hGRαT556I causes Chrousos syndrome. DESIGN AND RESULTS: Compared with the wild-type receptor, the mutant receptor hGRαT556I demonstrated 50% reduction in its ability to transactivate glucocorticoid-responsive genes and in the affinity for the ligand, 30% increase in the ability to transrepress the nuclear factor-κB-target genes and a 3,4-fold delay in the cytoplasmic-to-nuclear translocation. The mutant receptor hGRαT556I did not exert a dominant negative effect upon the hGRα-mediated transcriptional activity; it preserved its ability to bind to DNA and interacted with the glucocorticoid receptor-interacting protein 1 coactivator mostly through its activation function-1 domain. Structural biology studies revealed that the T556I mutation caused disruption of the hydrogen bond formed by the T556 with the =O group of P637 backbone, which resulted in a significant relocation of the P637-bearing loop. This conformational alteration affected the local 3D arrangement of the receptor and hence the electrostatic surface of the region. CONCLUSIONS: The hGRαT556I causes Chrousos syndrome by impairing multiple steps of the glucocorticoid signal transduction pathway.

State-of-the-art technology in modern computer-aided drug design.

The quest for small drug-like compounds that selectively inhibit the function of biological targets has always been a major focus in the pharmaceutical industry and in academia as well. High-throughput screening of compound libraries requires time, cost and resources. Therefore, the use of alternative methods is necessary for facilitating lead discovery. Computational techniques that dock small molecules into macromolecular targets and predict the affinity and activity of the small molecule are widely used in drug design and discovery, and have become an integral part of the industrial and academic research. In this review, we present an overview of some state-of-the-art technologies in modern drug design that have been developed for expediting the search for novel drug candidates.

Deciphering the protein interaction in adhesion of Francisella tularensis subsp. holarctica to the endothelial cells.

Extracellular form of Francisella is able to cross various cell barriers and invade multiple organs, such as skin, liver, lung and central nervous system. Transient adhesion of Francisella to endothelial cells may trigger the process of translocation. In this report, we showed that Francisella tularensis subsp. holarctica (Fth) is able to adhere to the endothelial cells, while ICAM-1 may serve as an adhesion molecule for Fth. Pull down and affinity ligand binding assays indicated that the PilE4 could be the probable ligand for ICAM-1. Further deciphering of this ligand:receptor interaction revealed that PilE4 interacts with Ig-like C2-type 1 domain of ICAM-1. To corroborate the role of PilE4 and ICAM-1 interaction in adhesion of extracellular form of Fth to endothelial cells, ICAM-1 was blocked with monoclonal anti-ICAM-1 antibody prior to the incubation with Fth and numbers of adherent bacteria were counted. Blocking of the ICAM-1 significantly reduced (500-fold, P < 0.05) number of adherent Fth compared to unblocked cells. PilE4:ICAM-1 interaction unfolded here may provide a new perspective on molecules involved in the adhesion of extracellular form of Francisella to endothelial cells and probably its translocation across endothelial barriers.

A novel mutation of the hGR gene causing Chrousos syndrome.

BACKGROUND: Natural mutations in the human glucocorticoid receptor (hGR, NR3C1) gene cause Chrousos syndrome, a rare condition characterized by generalized, partial, target-tissue insensitivity to glucocorticoids. OBJECTIVE: To present a new case of Chrousos syndrome caused by a novel mutation in the hGR gene, and to elucidate the molecular mechanisms through which the natural mutant receptor affects glucocorticoid signal transduction. DESIGN AND RESULTS: The index case presented with hirsutism, acne, alopecia, anxiety, fatigue and irregular menstrual cycles, but no clinical manifestations suggestive of Cushing's syndrome. Endocrinologic evaluation revealed elevated 08:00 h plasma adrenocorticotropic hormone, serum cortisol and androstenedione concentrations and increased urinary free cortisol excretion. The patient harbored a novel A > G transition at nucleotide position 2177, which resulted in histidine (H) to arginine (R) substitution at amino acid position 726 of the receptor (c.2177A > G, p.H726R). Compared with the wild-type receptor, the mutant receptor hGRαH726R demonstrated decreased ability to transactivate glucocorticoid-responsive genes and to transrepress the nuclear factor-κB signalling pathway, displayed 55% lower affinity for the ligand and a four-fold delay in nuclear translocation, and interacted with the glucocorticoid receptor-interacting protein 1 coactivator mostly through its activation function-1 domain. Finally, a 3-dimensional molecular modelling study of the H726R mutation revealed a significant structural shift in the rigidity of helix 10 of the receptor, which resulted in reduced flexibility and decreased affinity of the mutant receptor for binding to the ligand. CONCLUSIONS: The natural mutant receptor hGRαH726R impairs multiple steps of glucocorticoid signal transduction, thereby decreasing tissue sensitivity to glucocorticoids.

Evolution of the F0F1 ATP synthase complex in light of the patchy distribution of different bioenergetic pathways across prokaryotes.

Bacteria and archaea are characterized by an amazing metabolic diversity, which allows them to persist in diverse and often extreme habitats. Apart from oxygenic photosynthesis and oxidative phosphorylation, well-studied processes from chloroplasts and mitochondria of plants and animals, prokaryotes utilize various chemo- or lithotrophic modes, such as anoxygenic photosynthesis, iron oxidation and reduction, sulfate reduction, and methanogenesis. Most bioenergetic pathways have a similar general structure, with an electron transport chain composed of protein complexes acting as electron donors and acceptors, as well as a central cytochrome complex, mobile electron carriers, and an ATP synthase. While each pathway has been studied in considerable detail in isolation, not much is known about their relative evolutionary relationships. Wanting to address how this metabolic diversity evolved, we mapped the distribution of nine bioenergetic modes on a phylogenetic tree based on 16S rRNA sequences from 272 species representing the full diversity of prokaryotic lineages. This highlights the patchy distribution of many pathways across different lineages, and suggests either up to 26 independent origins or 17 horizontal gene transfer events. Next, we used comparative genomics and phylogenetic analysis of all subunits of the F0F1 ATP synthase, common to most bacterial lineages regardless of their bioenergetic mode. Our results indicate an ancient origin of this protein complex, and no clustering based on bioenergetic mode, which suggests that no special modifications are needed for the ATP synthase to work with different electron transport chains. Moreover, examination of the ATP synthase genetic locus indicates various gene rearrangements in the different bacterial lineages, ancient duplications of atpI and of the beta subunit of the F0 subcomplex, as well as more recent stochastic lineage-specific and species-specific duplications of all subunits. We discuss the implications of the overall pattern of conservation and flexibility of the F0F1 ATP synthase genetic locus.

Notch signaling and ageing.

Notch signaling is a master controller of the neural stem cell and neural development maintaining a significant role in the normal brain function. Notch genes are involved in embryogenesis, nervous system, and cardiovascular and endocrine function. On the other side, there are studies representing the involvement of Notch mutations in sporadic Alzheimer disease, other neurodegenerative diseases such as Down syndrome, Pick's and Prion's disease, and CADASIL. This manuscript attempts to present a holistic view of the positive or negative contribution of Notch signaling in the adult brain, and at the same time to present and promote the promising research fields of study.

Frequent gene fissions associated with human pathogenic bacteria.

Gene fusion and fission events are important for evolutionary studies and for predicting protein-protein interactions. Previous studies have shown that fusion events always predominate over fission events and, in their majority, they represent singular events throughout evolution. In this project, the role of fusion and fission events in the genome evolution of 104 human bacterial pathogens was studied. 141 protein pairs were identified to be involved in gene fusion or fission events. Surprisingly, we find that, in the species analyzed, gene fissions prevail over fusions. Moreover, while most events appear to have occurred only once in evolution, 23% of the gene fusion and fission events identified are deduced to have occurred independently multiple times. Comparison of the analyzed bacteria with non-pathogenic close relatives indicates that this impressive result is associated with the recent evolutionary history of the human bacterial pathogens, and thus is probably caused by their pathogenic lifestyle.

3D molecular modeling and evolutionary study of the Trypanosoma brucei DNA Topoisomerase IB, as a new emerging pharmacological target.

In the present study, an outline is proposed that may lead to specific drug design targeting of the Trypanosoma brucei DNA Topoisomerase IB. In this direction, an unequivocally specific platform was designed for the development of selective modulators. The designed platform is focused on the unique structural and catalytic features of the enzyme. Extensive phylogenetic analysis based on all available published genomes indicated a broad distribution of DNA topoisomerases across eukaryotic species and revealed structurally important amino acids which could be assigned as potentially strong contributors to the regulation of the mechanism of the T. brucei DNA Topoisomerase IB. Based on the above, we propose a comprehensive in silico 3D model for the structure of the T. brucei DNA Topoisomerase IB. Our approach provides an efficient intergraded platform with both evolutionary and structural insights for the rational design of pharmacophore models as well as novel modulators as the anti-T. brucei DNA Topoisomerase IB agents with therapeutic potential.

Design, synthesis and application of benzyl-sulfonate biomimetic affinity adsorbents for monoclonal antibody purification from transgenic corn.

The human anti-human immunodeficiency virus (HIV) antibody 2G12 (mAb 2G12) is one of the most broadly neutralizing antibodies against HIV that recognizes a unique epitope on the surface glycoprotein gp120. In the present work, a limited affinity-ligand library was synthesized and evaluated for its ability to bind and purify recombinant mAb 2G12 expressed in transgenic corn. The affinity ligands were structural fragments of polysulfonate triazine dye Cibacron Blue 3GA (CB3GA) and represent novel lead scaffolds for designing synthetic affinity ligands. Solid phase chemistry was used to synthesize variants of CB3GA lead ligand. One immobilized ligand, bearing 4-aminobenzyl sulfonic acid (4ABS) linked on two chlorine atoms of the triazine ring (4ABS-Trz-4ABS), displayed high affinity for mAb 2G12. Absorption equilibrium, 3D molecular modelling and molecular dynamics simulation studies were carried out to provide a detailed picture of the 4ABS-Trz-4ABS interaction with mAb 2G12. This biomimetic affinity ligand was exploited for the development of a facile two-step purification protocol for mAb 2G12. In the first step of the procedure, mAb 2G12 was purified on an S-Sepharose FF cation exchanger, and in the second step, mAb 2G12 was purified using affinity chromatography on 4ABS-Trz-4ABS affinity adsorbent. Analysis of the antibody preparation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and enzyme-linked immunosorbent assay showed that the mAb 2G12 was fully active and of sufficient purity suitable for analytical applications.

Introducing Drugster: a comprehensive and fully integrated drug design, lead and structure optimization toolkit.

SUMMARY: Drugster is a fully interactive pipeline designed to break the command line barrier and introduce a new user-friendly environment to perform drug design, lead and structure optimization experiments through an efficient combination of the PDB2PQR, Ligbuilder, Gromacs and Dock suites. Our platform features a novel workflow that guides the user through each logical step of the iterative 3D structural optimization setup and drug design process, by providing a seamless interface to all incorporated packages. AVAILABILITY: Drugster can be freely downloaded via our dedicated server system at http://www.bioacademy.gr/bioinformatics/drugster/.

Transcriptome map of mouse isochores in embryonic and neonatal cortex.

Several studies on adult tissues agree on the presence of a positive effect of the genomic and genic base composition on mammalian gene expression. Recent literature supports the idea that during developmental processes GC-poor genomic regions are preferentially implicated. We investigate the relationship between the compositional properties of the isochores and of the genes with their respective expression activity during developmental processes. Using RNA-seq data from two distinct developmental stages of the mouse cortex, embryonic day 18 (E18) and postnatal day 7 (P7), we established for the first time a developmental-related transcriptome map of the mouse isochores. Additionally, for each stage we estimated the correlation between isochores' GC level and their expression activity, and the genes' expression patterns for each isochore family. Our analyses add evidence supporting the idea that during development GC-poor isochores are preferentially implicated, and confirm the positive effect of genes' GC level on their expression activity.

Unraveling Desmin's Head Domain Structure and Function.

Understanding the structure and function of intermediate filaments (IFs) is necessary in order to explain why more than 70 related IF genes have evolved in vertebrates while maintaining such dramatically tissue-specific expression. Desmin is a member of the large multigene family of IF proteins and is specifically expressed in myocytes. In an effort to elucidate its muscle-specific behavior, we have used a yeast two-hybrid system in order to identify desmin's head binding partners. We described a mitochondrial and a lysosomal protein, NADH ubiquinone oxidoreductase core subunit S2 (NDUFS2), and saposin D, respectively, as direct desmin binding partners. In silico analysis indicated that both interactions at the atomic level occur in a very similar way, by the formation of a three-helix bundle with hydrophobic interactions in the interdomain space and hydrogen bonds at R16 and S32 of the desmin head domain. The interactions, confirmed also by GST pull-down assays, indicating the necessity of the desmin head domain and, furthermore, point out its role in function of mitochondria and lysosomes, organelles which are disrupted in myopathies due to desmin head domain mutations.

IMGT/Collier-de-Perles: a two-dimensional visualization tool for amino acid domain sequences.

IMGT/Collier-de-Perles is a tool that allows the user to analyze and draw two-dimensional graphical representations (or IMGT Collier de Perles) of protein domains (e.g., hydropathy plots). The IMGT/Collier-de-Perles specializes in the area of immunoglobulins (IG) or antibodies, T cell receptors (TR) and major histocompatibility (MH) of human and other vertebrate species as well as other proteins of the immunoglobulin superfamily (IgSF) and of the major histocompatibility superfamily (MhSF) and related proteins of the immune system of vertebrates and invertebrates.

Towards the elucidation of the regulatory network guiding the insulin producing cells' differentiation.

This study pertains to the regulatory network of neurogenin3 (NGN3, approved symbol: NEUROG3), the main regulator of insulin producing cells' formation. In silico regulatory region analyses of known and novel targets of NGN3 revealed the presence of two variants of a regulatory module that appeared conserved at the most phylogenetically distant species with pancreas. Both variants of this module contained binding sites of six transcription factors implicated in pancreas development. Nevertheless, an additional factor was found only into the module of the down-regulated by NGN3 genes. Whole genome analyses confirmed the statistical significance of these regulatory modules. Investigation of protein-protein interactions among the factors bound into these sequences indicated the formation of alternative protein complexes resulting into the up- or down-regulation of the respective genes. Subsequently, an NGN3-guided regulatory network, was modeled, describing the interactions among the analyzed genes with their transcriptional regulators, leading into the differentiation of cells capable of producing insulin.

Molecular fusion events in carcinogenic organisms: a bioinformatics study for the detection of fused proteins between viruses, bacteria and eukaryotes.

Molecular fusion events have a prominent role in the initial steps of carcinogenesis. In this study, a bioinformatics analysis was performed between four organisms that are known to induce cancer development in humans: two viruses, Human Herpesvirus 4, and Human T-cell leukaemia virus, one bacterium, Helicobacter Pylori, and one trematode, Schistosoma mansoni. The annotated proteomes from these organisms were analysed using the SAFE software to identify protein fusion events, which may provide insight into protein function similarities and possible merging events during the course of evolution. Based on the results, five fused proteins with very similar functions were detected, whereas proteins with different functions that might act in the same molecular complex or biochemical pathway were not found. Thus, this study analysed the above four well-known cancer-related organisms with de novo bioinformatics programs and provided useful information on protein fusion events, hopefully leading to deeper understanding of carcinogenenesis.

Noise reduction in protein-protein interaction graphs by the implementation of a novel weighting scheme.

BACKGROUND: Recent technological advances applied to biology such as yeast-two-hybrid, phage display and mass spectrometry have enabled us to create a detailed map of protein interaction networks. These interaction networks represent a rich, yet noisy, source of data that could be used to extract meaningful information, such as protein complexes. Several interaction network weighting schemes have been proposed so far in the literature in order to eliminate the noise inherent in interactome data. In this paper, we propose a novel weighting scheme and apply it to the S. cerevisiae interactome. Complex prediction rates are improved by up to 39%, depending on the clustering algorithm applied. RESULTS: We adopt a two step procedure. During the first step, by applying both novel and well established protein-protein interaction (PPI) weighting methods, weights are introduced to the original interactome graph based on the confidence level that a given interaction is a true-positive one. The second step applies clustering using established algorithms in the field of graph theory, as well as two variations of Spectral clustering. The clustered interactome networks are also cross-validated against the confirmed protein complexes present in the MIPS database. CONCLUSIONS: The results of our experimental work demonstrate that interactome graph weighting methods clearly improve the clustering results of several clustering algorithms. Moreover, our proposed weighting scheme outperforms other approaches of PPI graph weighting.

Protein functional links in Trypanosoma brucei, identified by gene fusion analysis.

BACKGROUND: Domain or gene fusion analysis is a bioinformatics method for detecting gene fusions in one organism by comparing its genome to that of other organisms. The occurrence of gene fusions suggests that the two original genes that participated in the fusion are functionally linked, i.e. their gene products interact either as part of a multi-subunit protein complex, or in a metabolic pathway. Gene fusion analysis has been used to identify protein functional links in prokaryotes as well as in eukaryotic model organisms, such as yeast and Drosophila. RESULTS: In this study we have extended this approach to include a number of recently sequenced protists, four of which are pathogenic, to identify fusion linked proteins in Trypanosoma brucei, the causative agent of African sleeping sickness. We have also examined the evolution of the gene fusion events identified, to determine whether they can be attributed to fusion or fission, by looking at the conservation of the fused genes and of the individual component genes across the major eukaryotic and prokaryotic lineages. We find relatively limited occurrence of gene fusions/fissions within the protist lineages examined. Our results point to two trypanosome-specific gene fissions, which have recently been experimentally confirmed, one fusion involving proteins involved in the same metabolic pathway, as well as two novel putative functional links between fusion-linked protein pairs. CONCLUSIONS: This is the first study of protein functional links in T. brucei identified by gene fusion analysis. We have used strict thresholds and only discuss results which are highly likely to be genuine and which either have already been or can be experimentally verified. We discuss the possible impact of the identification of these novel putative protein-protein interactions, to the development of new trypanosome therapeutic drugs.

Transcriptome map of mouse isochores.

BACKGROUND: The availability of fully sequenced genomes and the implementation of transcriptome technologies have increased the studies investigating the expression profiles for a variety of tissues, conditions, and species. In this study, using RNA-seq data for three distinct tissues (brain, liver, and muscle), we investigate how base composition affects mammalian gene expression, an issue of prime practical and evolutionary interest. RESULTS: We present the transcriptome map of the mouse isochores (DNA segments with a fairly homogeneous base composition) for the three different tissues and the effects of isochores' base composition on their expression activity. Our analyses also cover the relations between the genes' expression activity and their localization in the isochore families. CONCLUSIONS: This study is the first where next-generation sequencing data are used to associate the effects of both genomic and genic compositional properties to their corresponding expression activity. Our findings confirm previous results, and further support the existence of a relationship between isochores and gene expression. This relationship corroborates that isochores are primarily a product of evolutionary adaptation rather than a simple by-product of neutral evolutionary processes.