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.

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.

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.

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.

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.

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.

DrugOn: a fully integrated pharmacophore modeling and structure optimization toolkit.

During the past few years, pharmacophore modeling has become one of the key components in computer-aided drug design and in modern drug discovery. DrugOn is a fully interactive pipeline designed to exploit the advantages of modern programming and overcome the command line barrier with two friendly environments for the user (either novice or experienced in the field of Computer Aided Drug Design) to perform pharmacophore modeling through an efficient combination of the PharmACOphore, Gromacs, Ligbuilder and PDB2PQR 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. For the pharmacophore modeling we are focusing on either the characteristics of the receptor or the full molecular system, including a set of selected ligands. DrugOn can be freely downloaded from our dedicated server system at www.bioacademy.gr/bioinformatics/drugon/.

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.

Computational Construction of Antibody-Drug Conjugates Using Surface Lysines as the Antibody Conjugation Site and a Non-cleavable Linker.

Antibody-drug conjugates (ADCs) constitute a category of anticancer targeted therapy that has gathered great interest during the last few years because of their potential to kill cancer cells while causing significantly fewer side effects than traditional chemotherapy. In this paper, a process of computational construction of ADCs is described, using the surface lysines of an antibody and a non-covalent linker molecule, as well as a cytotoxic substance, as files in Protein Data Bank format. Also, aspects related to the function, properties, and development of ADCs are discussed.

Structural models for the design of novel antiviral agents against Greek Goat Encephalitis.

The Greek Goat Encephalitis virus (GGE) belongs to the Flaviviridae family of the genus Flavivirus. The GGE virus constitutes an important pathogen of livestock that infects the goat's central nervous system. The viral enzymes of GGE, helicase and RNA-dependent RNA polymerase (RdRP), are ideal targets for inhibitor design, since those enzymes are crucial for the virus' survival, proliferation and transmission. In an effort to understand the molecular structure underlying the functions of those viral enzymes, the three dimensional structures of GGE NS3 helicase and NS5 RdRP have been modelled. The models were constructed in silico using conventional homology modelling techniques and the known 3D crystal structures of solved proteins from closely related species as templates. The established structural models of the GGE NS3 helicase and NS5 RdRP have been evaluated for their viability using a repertoire of in silico tools. The goal of this study is to present the 3D conformations of the GGE viral enzymes as reliable structural models that could provide the platform for the design of novel anti-GGE agents.

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.

AmalgamScope: merging annotations data across the human genome.

The past years have shown an enormous advancement in sequencing and array-based technologies, producing supplementary or alternative views of the genome stored in various formats and databases. Their sheer volume and different data scope pose a challenge to jointly visualize and integrate diverse data types. We present AmalgamScope a new interactive software tool focusing on assisting scientists with the annotation of the human genome and particularly the integration of the annotation files from multiple data types, using gene identifiers and genomic coordinates. Supported platforms include next-generation sequencing and microarray technologies. The available features of AmalgamScope range from the annotation of diverse data types across the human genome to integration of the data based on the annotational information and visualization of the merged files within chromosomal regions or the whole genome. Additionally, users can define custom transcriptome library files for any species and use the file exchanging distant server options of the tool.

On integrating multi-experiment microarray data.

With the extensive use of microarray technology as a potential prognostic and diagnostic tool, the comparison and reproducibility of results obtained from the use of different platforms is of interest. The integration of those datasets can yield more informative results corresponding to numerous datasets and microarray platforms. We developed a novel integration technique for microarray gene-expression data derived by different studies for the purpose of a two-way Bayesian partition modelling which estimates co-expression profiles under subsets of genes and between biological samples or experimental conditions. The suggested methodology transforms disparate gene-expression data on a common probability scale to obtain inter-study-validated gene signatures. We evaluated the performance of our model using artificial data. Finally, we applied our model to six publicly available cancer gene-expression datasets and compared our results with well-known integrative microarray data methods. Our study shows that the suggested framework can relieve the limited sample size problem while reporting high accuracies by integrating multi-experiment data.

Current state-of-the-art molecular dynamics methods and applications.

Molecular dynamics simulations are used to describe the patterns, strength, and properties of protein behavior, drug-receptor interactions, the solvation of molecules, the conformational changes that a protein or molecule may undergo under various conditions, and other events that require the systematic evaluation of molecular properties in dynamic molecular systems. Only few years ago proteins were considered to be rigid body structures with very limited conformational flexibility. However, it is now clear that proteins are highly dynamic structures, the internal organization of which is the key to their 3D spatial arrangement and hence biological function. The study of protein dynamics in the lab is a very complicated, expensive, and time-consuming process. Therefore, a lot of effort and hope lies with the computers and the in silico study of protein structure and molecular dynamics. Herein, an effort has been made to describe the ever-evolving field of molecular dynamics, the different algorithms, and force fields that are being used as well as to provide some insight on what the near future holds for this auspicious field of computational structural biology.

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.

Silencing motifs in the Clr2 protein from fission yeast, Schizosaccharomyces pombe.

The fission yeast, Schizosaccharomyces pombe, is a well-established model for heterochromatin formation, but the exact sequence of events for initiation remains to be elucidated. The essential factors involved include RNA transcribed from repeated sequences together with the methyltransferase Clr4. In addition, histone deacetylases, like Clr3, found in the SHREC complex are also necessary for transcriptional silencing. Clr2 is another crucial factor required for heterochromatin formation found in the SHREC complex. The function of Clr2 has been difficult to establish due to the lack of conserved domains or homology to proteins of known molecular function. Using a bioinformatics approach, three conserved motifs in Clr2 were identified, which contained amino acids important for transcriptional repression. Analysis of clr2 mutant strains revealed a major role for Clr2 in mating-type and rDNA silencing, and weaker effects on centromeric silencing. The effect on mating-type silencing showed variegation in several of the strains with mutated versions of Clr2 indicating an establishment or maintenance defect. Moreover, the critical amino acids in Clr2 were also necessary for transcriptional repression in a minimal system, by the tethering of Clr4 upstream of a reporter gene, inserted into the euchromatic part of the genome. Finally, in silico modeling suggested that the mutations in Clr2 cause disruption of secondary structures in the Clr2 protein. Identification of these critical amino acids in the protein provides a useful tool to explore the molecular mechanism behind the role of Clr2 in heterochromatin formation.

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.

3D structural analysis of proteins using electrostatic surfaces based on image segmentation.

Herein, we present a novel strategy to analyse and characterize proteins using protein molecular electro-static surfaces. Our approach starts by calculating a series of distinct molecular surfaces for each protein that are subsequently flattened out, thus reducing 3D information noise. RGB images are appropriately scaled by means of standard image processing techniques whilst retaining the weight information of each protein's molecular electrostatic surface. Then homogeneous areas in the protein surface are estimated based on unsupervised clustering of the 3D images, while performing similarity searches. This is a computationally fast approach, which efficiently highlights interesting structural areas among a group of proteins. Multiple protein electrostatic surfaces can be combined together and in conjunction with their processed images, they can provide the starting material for protein structural similarity and molecular docking experiments.

Molecular dynamics simulations through GPU video games technologies.

Bioinformatics is the scientific field that focuses on the application of computer technology to the management of biological information. Over the years, bioinformatics applications have been used to store, process and integrate biological and genetic information, using a wide range of methodologies. One of the most de novo techniques used to understand the physical movements of atoms and molecules is molecular dynamics (MD). MD is an in silico method to simulate the physical motions of atoms and molecules under certain conditions. This has become a state strategic technique and now plays a key role in many areas of exact sciences, such as chemistry, biology, physics and medicine. Due to their complexity, MD calculations could require enormous amounts of computer memory and time and therefore their execution has been a big problem. Despite the huge computational cost, molecular dynamics have been implemented using traditional computers with a central memory unit (CPU). A graphics processing unit (GPU) computing technology was first designed with the goal to improve video games, by rapidly creating and displaying images in a frame buffer such as screens. The hybrid GPU-CPU implementation, combined with parallel computing is a novel technology to perform a wide range of calculations. GPUs have been proposed and used to accelerate many scientific computations including MD simulations. Herein, we describe the new methodologies developed initially as video games and how they are now applied in MD simulations.