Nikita V. Ivanisenko

ANDSystem: an Associative Network Discovery System for automated literature mining in the field of biology

BackgroundSufficient knowledge of molecular and genetic interactions, which comprise the entire basis of the functioning of living systems, is one of the necessary requirements for successfully answering almost any research question in the field of biology and medicine. To date, more than 24 million scientific papers can be found in PubMed, with many of them containing descriptions of a wide range of biological processes. The analysis of such tremendous amounts of data requires the use of automated text-mining approaches. Although a handful of tools have recently been developed to meet this need, none of them provide error-free extraction of highly detailed information.ResultsThe ANDSystem package was developed for the reconstruction and analysis of molecular genetic networks based on an automated text-mining technique. It provides a detailed description of the various types of interactions between genes, proteins, microRNAs, metabolites, cellular components, pathways and diseases, taking into account the specificity of cell lines and organisms. Although the accuracy of ANDSystem is comparable to other well known text-mining tools, such as Pathway Studio and STRING, it outperforms them in having the ability to identify an increased number of interaction types.ConclusionThe use of ANDSystem, in combination with Pathway Studio and STRING, can improve the quality of the automated reconstruction of molecular and genetic networks. ANDSystem should provide a useful tool for researchers working in a number of different fields, including biology, biotechnology, pharmacology and medicine.

The substitutions G245C and G245D in the Zn(2+)-binding pocket of the p53 protein result in differences of conformational flexibility of the DNA-binding domain.

Transcription activation of the proapoptotic target genes is a means by which the p53 protein implements its function of tumor suppression. Zn2+ is a known regulator of p53 binding to the target genes. We have previously obtained an evidence that amino acid substitutions in the p53 Zn2+-binding pocket can presumably exert an influence on Zn2+ position in the Zn2+-p53 complex and thereby affect p53 binding to DNA. With these background considerations, our aim was to estimate the effect of the putative changes in the Zn2+ position in its binding pocket due to the G245C and G245D substitutions on the conformation of the p53 DNA-binding motif. Statistical analysis of the molecular dynamics (MD) trajectories of the mutant p53-Zn2+ complexes was used to detect significant deviations in conformation of the mutant p53 forms. MD simulations demonstrated that (1) the two substitutions in the Zn2+-binding pocket caused changes in the conformation of the p53 DNA-binding motif, as compared with the wild-type (WT) p53; (2) binding of Zn2+ to the p53 mutant forms reduced the effect of the substitutions on conformational change; and (3) Zn2+ binding in the normal position compensated the effect of the mutations on the conformation in comparison to the altered Zn2+ position.

Systemic lupus erythematosus: molecular cloning and analysis of recombinant monoclonal kappa light chain NGTA2-Me-pro-ChTr possessing two different activities—trypsin-like and metalloprotease

Polyclonal antibodies hydrolyzing myelin basic protein (MBP) can play an important role in the pathogenesis of multiple sclerosis and systemic lupus erythematosus (SLE). An immunoglobulin light chain phagemid library derived from peripheral blood lymphocytes of patients with SLE was used. The small pools of phage particles displaying light chains with different affinity for MBP were isolated by affinity chromatography on MBP-Sepharose. The fraction eluted with 0.5M NaCl was used for preparation of individual monoclonal light chains (MLChs, 26-27kDa). The clones were expressed in Escherichia coli in a soluble form; MLChs were purified by metal-chelating chromatography followed by gel filtration. In mammalians, there are serine proteases and metalloproteases. These and many other enzymes usually have only one active site and catalyze only one chemical reaction. In contrast to canonical proteases, one MLCh (NGTA2-Me-pro-ChTr) efficiently hydrolyzed MBP (but not other proteins) and four different oligopeptides corresponding to four immunodominant sequences containing cleavage sites of MBP. The proteolytic activity of MLCh was efficiently inhibited only by specific inhibitors of serine-like (phenylmethanesulfonylfluoride, PMSF) and metalloproteases (EDTA). It was shown that MLCh possess independent serine-like and metal-dependent activities. The principal existence of monoclonal antibodies with two different proteolytic activities is unexpected but very important for the further understanding of at present unknown biological functions of human antibodies.

A New Stochastic Model for Subgenomic Hepatitis C Virus Replication Considers Drug Resistant Mutants

As an RNA virus, hepatitis C virus (HCV) is able to rapidly acquire drug resistance, and for this reason the design of effective anti-HCV drugs is a real challenge. The HCV subgenomic replicon-containing cells are widely used for experimental studies of the HCV genome replication mechanisms, for drug testing in vitro and in studies of HCV drug resistance. The NS3/4A protease is essential for virus replication and, therefore, it is one of the most attractive targets for developing specific antiviral agents against HCV. We have developed a stochastic model of subgenomic HCV replicon replication, in which the emergence and selection of drug resistant mutant viral RNAs in replicon cells is taken into account. Incorporation into the model of key NS3 protease mutations leading to resistance to BILN-2061 (A156T, D168V, R155Q), VX-950 (A156S, A156T, T54A) and SCH 503034 (A156T, A156S, T54A) inhibitors allows us to describe the long term dynamics of the viral RNA suppression for various inhibitor concentrations. We theoretically showed that the observable difference between the viral RNA kinetics for different inhibitor concentrations can be explained by differences in the replication rate and inhibitor sensitivity of the mutant RNAs. The pre-existing mutants of the NS3 protease contribute more significantly to appearance of new resistant mutants during treatment with inhibitors than wild-type replicon. The model can be used to interpret the results of anti-HCV drug testing on replicon systems, as well as to estimate the efficacy of potential drugs and predict optimal schemes of their usage.

Computational screening for new inhibitors of M. tuberculosis mycolyltransferases antigen 85 group of proteins as potential drug targets.

The group of antigen 85 proteins of Mycobacterium tuberculosis is responsible for converting trehalose monomycolate to trehalose dimycolate, which contributes to cell wall stability. Here, we have used a serial enrichment approach to identify new potential inhibitors by searching the libraries of compounds using both 2D atom pair descriptors and binary fingerprints followed by molecular docking. Three different docking softwares AutoDock, GOLD, and LigandFit were used for docking calculations. In addition, we applied the criteria of selecting compounds with binding efficiency close to the starting known inhibitor and showing potential to form hydrogen bonds with the active site amino acid residues. The starting inhibitor was ethyl-3-phenoxybenzyl-butylphosphonate, which had IC50 value of 2.0 μM in mycolyltransferase inhibition assay. Our search from more than 34 million compounds from public libraries yielded 49 compounds. Subsequently, selection was restricted to compounds conforming to the Lipinski rule of five and exhibiting hydrogen bonding to any of the amino acid residues in the active site pocket of all three proteins of antigen 85A, 85B, and 85C. Finally, we selected those ligands which were ranked top in the table with other known decoys in all the docking results. The compound NIH415032 from tuberculosis antimicrobial acquisition and coordinating facility was further examined using molecular dynamics simulations for 10 ns. These results showed that the binding is stable, although some of the hydrogen bond atom pairs varied through the course of simulation. The NIH415032 has antitubercular properties with IC90 at 20 μg/ml (53.023 μM). These results will be helpful to the medicinal chemists for developing new antitubercular molecules for testing.

Exploring Interaction of TNF and Orthopoxviral CrmB Protein by Surface Plasmon Resonance and Free Energy Calculation

Inhibition of the activity of the tumor necrosis factor (TNF) has become the main strategy for treating inflammatory diseases. The orthopoxvirus TNF-binding proteins can bind and efficiently neutralize TNF. To analyze the mechanisms of the interaction between human (hTNF) or mouse (mTNF) TNF and the cowpox virus N-terminal binding domain (TNFBD-CPXV), also the variola virus N-terminal binding domain (TNFBD-VARV) and to define the amino acids most importantly involved in the formation of complexes, computer models, derived from the X-ray structure of a homologous hTNF/TNFRII complex, were used together with experiments. The hTNF/TNFBD-CPXV, hTNF/TNFBD-VARV, mTNF/TNFBD-CPXV, and mTNF/TNFBD-VARV complexes were used in the molecular dynamics (MD) simulations and MM/GBSA free energy calculations. The complexes were ordered as hTNF/TNFBD-CPXV, hTNF/TNFBD-VARV, mTNF/TNFBD-CPXV and mTNF/TNFBD-VARV according to increase in the binding affinity. The calculations were in agreement with surface plasmon resonance (SPR) measurements of the binding constants. Key residues involved in complex formation were identified.

Enhanced Differential Evolution Entirely Parallel Method for Biomedical Applications

A considerable enhancement is proposed for the Differential Evolution Entirely Parallel DEEP method developed recently. A new selection rule was implemented in order to increase the robustness of DEEP. To simplify the approach a population is not divided now into branches, instead of it, several oldest individuals are substituted with the same number of the best ones after the predefined number of iterations. The individuals are selected on the basis of the number of generations, in which they survived without any change. We demonstrate how the enhanced DEEP provides new solutions to problems with several objective functions.

Antibodies to H1 histone from the sera of HIV‐infected patients recognize and catalyze site‐specific degradation of this histone

Histones and their posttranslational modifications have key roles in chromatin remodeling and gene transcription. Besides intranuclear functions, histones act as damage‐associated molecules when they are released into the extracellular space. Administration of histones to animals leads to systemic inflammatory and toxic responses. Autoantibodies with enzymatic activities (abzymes) are distinctive feature of some autoimmune and viral diseases. Electrophoretically and immunologically homogeneous IgGs containing no canonical enzymes were isolated from sera of human immunodeficiency virus–infected patients by chromatography on several affinity sorbents. In contrast to canonical proteases (trypsin, chymotrypsin, and proteinase K), IgGs from human immunodeficiency virus–infected patients purified by affinity chromatography on Sepharose containing immobilized histones specifically recognized and hydrolyzed only histones but not many other tested globular proteins. Using matrix‐assisted laser desorption/ionization mass spectrometry, the sites of H1 histone (193 amino acids [AAs]) cleavage by anti‐H1 histone IgGs were determined for the first time. It was shown that 1 cluster of 2 major and 4 moderate sites of cleavage is located at the beginning (106‐112 AAs) of the known antigenic determinants disposed at the long C‐terminal sequence of H1. Two clusters of minor and very weak sites of the protein cleavage correspond to middle (8 sites, 138‐158 AAs) and terminal (5 sites, 166‐176 AAs) parts of the antigenic determinants. It was shown that in contrast to canonical proteases, N‐terminal part of H1 histone (1‐136 AAs) containing no antigenic determinants is an unpredictably very resistant against hydrolysis by abzymes, while it can be easily cleavage by canonical proteases. Because histones act as damage‐associated molecules, abzymes against H1 and other histones can play important role in pathogenesis of acquired immune deficiency syndrome and probably other different diseases.

Dynamic properties of SOD1 mutants can predict survival time of patients carrying familial amyotrophic lateral sclerosis.

One of the reasons for the death of motor neurons of the brain and spinal cord in patients with amyotrophic lateral sclerosis is known to be formation of subcellular protein aggregates that are caused by mutations in the SOD1 gene. Patient survival time was earlier shown to have limiting correlation with thermostability change of SOD1 mutant forms of patients’ carriers. We hypothesized that aggregation of mutant SOD1 may occur not only due to the protein destabilization, but through formation of novel interatomic bonds which stabilize “pathogenic” conformations of the mutant as well. To estimate these effects in the present paper, we performed statistical analysis of occupancy of intramolecular hydrogen bonds, hydrogen bonds between the protein and water molecules, and water bridges with use of molecular dynamics simulation for 38 mutant SOD1 forms. Multiple regression model based on these kinds of bonds demonstrated correlation with patient survival time significantly better (R = .9, p-value < 10−11) than the thermostability of SOD1 mutants only. It was shown that the occupancy of intramolecular hydrogen bonds between amino acid residues is a key determinant (R = .89, p-value < 10−10) in predicting patients’ survival time.

How human serum albumin recognizes DNA and RNA

Abstract We show here for the first time that HSA possesses two nucleic acid-(NA) binding sites and we estimated the relative contributions of the nucleotide links of (pN)n to their total affinity for these binding sites with higher and lower affinity for NAs. The minimal ligands of these binding sites are orthophosphate (Kd=3.0 and 20.0 mm), various dNMPs (5.6–400 μm and 0.063–18 mm) and different rNMPs (4.9–30 μm and 14–250 μm). Maximal contribution to the total affinity of all NAs to the first and second sites was observed for one nucleotide and was remarkably lower for three additional nucleotide units of (pN)n (n=1–4) with a significant decrease in the contribution at n=5–6, and at n≥7–8 all dependencies reached plateaus. For d(pA)n and r(pA)n a relatively gradual decrease in the contribution to the affinity at n=1–6 was observed, while several d(pN)n, demonstrated a sharp increase in the contribution at n=2–4. Finally, all (pN)n>10 demonstrated high affinity for the first (1.4–150 nm) and the second (80–2400 nm) sites of HSA. Double-stranded NAs showed significantly lower affinity comparing with single-stranded ligands. The thermodynamic parameters characterizing the specific contribution of every nucleotide link of all (pN)1−9 (ΔG°) to their total affinity for HSA were estimated.