Pawel Siedlecki

Establishment and functional validation of a structural homology model for human DNA methyltransferase 1

Changes in DNA methylation patterns play an important role in tumorigenesis. The DNA methyltransferase 1 (DNMT1) protein represents a major DNA methyltransferase activity in human cells and is therefore a prominent target for experimental cancer therapies. However, there are only few available inhibitors and their high toxicity and low specificity have so far precluded their broad use in chemotherapy. Based on the strong conservation of catalytic DNA methyltransferase domains we have used a homology modeling approach to determine the three-dimensional structure of the DNMT1 catalytic domain. Our results suggest an overall structural conservation with other DNA methyltransferases but also indicate local conformational differences. To prove the validity of our model we used it as a template to design a novel derivative of the known DNA methyltransferase inhibitor 5-azacytidine. The resulting compound (N4-fluoroacetyl-5-azacytidine) functioned as an efficient inhibitor of DNA methylation in human tumor cell lines and also provides novel opportunities for pharmacological applications.

Nicotiana tabacum Osmotic Stress-activated Kinase Is Regulated by Phosphorylation on Ser-154 and Ser-158 in the Kinase Activation Loop

NtOSAK (Nicotiana tabacum osmotic stress-activated protein kinase), a member of the SnRK2 subfamily, is activated rapidly in response to hyperosmotic stress. Our previous results as well as data presented by others indicate that phosphorylation is involved in activation of SnRK2 kinases. Here, we have mapped the regulatory phosphorylation sites of NtOSAK by mass spectrometry with collision-induced peptide fragmentation. We show that active NtOSAK, isolated from NaCl-treated tobacco BY-2 cells, is phosphorylated on Ser-154 and Ser-158 in the kinase activation loop. Prediction of the NtOSAK three-dimensional structure indicates that phosphorylation of Ser-154 and Ser-158 triggers changes in enzyme conformation resulting in its activation. The involvement of Ser-154 and Ser-158 phosphorylation in regulation of NtOSAK activity was confirmed by site-directed mutagenesis of NtOSAK expressed in bacteria and in maize protoplasts. Our data reveal that phosphorylation of Ser-158 is essential for NtOSAK activation, whereas phosphorylation of Ser-154 most probably facilitates Ser-158 phosphorylation. The time course of NtOSAK phosphorylation on Ser-154 and Ser-158 in BY-2 cells subjected to osmotic stress correlates with NtOSAK activity, indicating that NtOSAK is regulated by reversible phosphorylation of these residues in vivo. Importantly, Ser-154 and Ser-158 are conserved in all SnRK2 subfamily members, suggesting that phosphorylation at these sites may be a general mechanism for SnRK2 activation.

A Specialized Histone H1 Variant Is Required for Adaptive Responses to Complex Abiotic Stress and Related DNA Methylation in Arabidopsis

Stress-inducible linker histone variant is required for adaptive response of Arabidopsis to complex environmental stress. Linker (H1) histones play critical roles in chromatin compaction in higher eukaryotes. They are also the most variable of the histones, with numerous nonallelic variants cooccurring in the same cell. Plants contain a distinct subclass of minor H1 variants that are induced by drought and abscisic acid and have been implicated in mediating adaptive responses to stress. However, how these variants facilitate adaptation remains poorly understood. Here, we show that the single Arabidopsis (Arabidopsis thaliana) stress-inducible variant H1.3 occurs in plants in two separate and most likely autonomous pools: a constitutive guard cell-specific pool and a facultative environmentally controlled pool localized in other tissues. Physiological and transcriptomic analyses of h1.3 null mutants demonstrate that H1.3 is required for both proper stomatal functioning under normal growth conditions and adaptive developmental responses to combined light and water deficiency. Using fluorescence recovery after photobleaching analysis, we show that H1.3 has superfast chromatin dynamics, and in contrast to the main Arabidopsis H1 variants H1.1 and H1.2, it has no stable bound fraction. The results of global occupancy studies demonstrate that, while H1.3 has the same overall binding properties as the main H1 variants, including predominant heterochromatin localization, it differs from them in its preferences for chromatin regions with epigenetic signatures of active and repressed transcription. We also show that H1.3 is required for a substantial part of DNA methylation associated with environmental stress, suggesting that the likely mechanism underlying H1.3 function may be the facilitation of chromatin accessibility by direct competition with the main H1 variants.

Performance of machine-learning scoring functions in structure-based virtual screening

Classical scoring functions have reached a plateau in their performance in virtual screening and binding affinity prediction. Recently, machine-learning scoring functions trained on protein-ligand complexes have shown great promise in small tailored studies. They have also raised controversy, specifically concerning model overfitting and applicability to novel targets. Here we provide a new ready-to-use scoring function (RF-Score-VS) trained on 15 426 active and 893 897 inactive molecules docked to a set of 102 targets. We use the full DUD-E data sets along with three docking tools, five classical and three machine-learning scoring functions for model building and performance assessment. Our results show RF-Score-VS can substantially improve virtual screening performance: RF-Score-VS top 1% provides 55.6% hit rate, whereas that of Vina only 16.2% (for smaller percent the difference is even more encouraging: RF-Score-VS top 0.1% achieves 88.6% hit rate for 27.5% using Vina). In addition, RF-Score-VS provides much better prediction of measured binding affinity than Vina (Pearson correlation of 0.56 and −0.18, respectively). Lastly, we test RF-Score-VS on an independent test set from the DEKOIS benchmark and observed comparable results. We provide full data sets to facilitate further research in this area (http://github.com/oddt/rfscorevs) as well as ready-to-use RF-Score-VS (http://github.com/oddt/rfscorevs_binary).

Sequence diversity and potential recombination events in the coat protein gene of Apple stem pitting virus.

The variability of the Apple stem pitting virus (ASPV) coat protein (CP) gene was investigated. The CP gene of ten virus isolates from apple and pear trees was sequenced. Comparison of all sequenced virus isolates revealed high diversity of the CP gene (70.7-93.5% at the nucleotide level and 77.8-98.7% at the amino acid level). Additionally, one or two deletions in the N-terminal part of the coat protein gene of the studied virus isolates were identified. The ratio of nonsynonymous to synonymous polymorphic sites indicated that purifying selection has acted to eliminate deleterious mutations in coding sites. Moreover, the evidences for recombination in analyzed sequences were provided. It is likely that recombination, along with selection, enhances the speed of elimination of deleterious mutations in ASPV, following the mutational deterministic hypothesis of Kondrashov.

Genetic diversity of hemagglutinin gene of A(H1N1)pdm09 influenza strains isolated in Taiwan and its potential impact on HA-neutralizing epitope interaction.

Pandemic influenza A(H1N1)pdm09 virus is a global health threat and between 2009–2011 it became the predominant influenza virus subtype circulating in the world. The research describes the MSSCP (Multitemperature Single Strand Conformation Polymorphism) analysis of the hemagglutinin (HA) region encompassing major neutralizing epitope in pandemic influenza isolates from Taiwan. Several genetically distinct changes appeared in isolates obtained in 2010 and 2011. The majority of changes in HA protein did not result in significant modifications, however three modifications were localized in epitope E of H1 and one was part of the interface binding antibodies BH151 and HC45 possibly making the current vaccine less effective.-Taking into account the possibility of the emergence of influenza A with antibody evading potential, the MSSCP method provides an alternative approach for detection of minor variants which escape detection by conventional Sanger sequencing.

Genetic Diversity of SCN5A Gene and Its Possible Association with the Concealed Form of Brugada Syndrome Development in Polish Group of Patients

Brugada Syndrome (BS) is an inherited channelopathy associated with a high incidence of sudden cardiac death. The paper presents the discovery of new genetic variants of SCN5A gene which might be associated with the development of a concealed form of Brugada Syndrome. The study involved a group of 59 patients (37 men) with suspected concealed form of Brugada Syndrome. Pharmacological provocation with intravenous ajmaline administration was performed. Six patients with positive test results were subjected to molecular analysis of SCN5A gene with MSSCP method. Additionally, MSSCP genotyping was performed for samples obtained from the family members with Brugada Syndrome, despite the fact that they had negative ajmaline challenge test results. Genetic examinations of the SCN5A gene at 6 positive patients showed 6 known polymorphisms, 8 new single nucleotide point (SNP) variants located at exons, and 12 new single nucleotide point variants located at introns. Among new SNPs localized in SCN5A gene exons three SNPs affected the protein sequence.

DeCAF—Discrimination, Comparison, Alignment Tool for 2D PHarmacophores

Comparison of small molecules is a common component of many cheminformatics workflows, including the design of new compounds and libraries as well as side-effect predictions and drug repurposing. Currently, large-scale comparison methods rely mostly on simple fingerprint representation of molecules, which take into account the structural similarities of compounds. Methods that utilize 3D information depend on multiple conformer generation steps, which are computationally expensive and can greatly influence their results. The aim of this study was to augment molecule representation with spatial and physicochemical properties while simultaneously avoiding conformer generation. To achieve this goal, we describe a molecule as an undirected graph in which the nodes correspond to atoms with pharmacophoric properties and the edges of the graph represent the distances between features. This approach combines the benefits of a conformation-free representation of a molecule with additional spatial information. We implemented our approach as an open-source Python module called DeCAF (Discrimination, Comparison, Alignment tool for 2D PHarmacophores), freely available at http://bitbucket.org/marta-sd/decaf. We show DeCAF’s strengths and weaknesses with usage examples and thorough statistical evaluation. Additionally, we show that our method can be manually tweaked to further improve the results for specific tasks. The full dataset on which DeCAF was evaluated and all scripts used to calculate and analyze the results are also provided.

Characterization of mAb6-9-1 monoclonal antibody against hemagglutinin of avian influenza virus H5N1 and its engineered derivative, single-chain variable fragment antibody.

Hemagglutinin (HA), as a major surface antigen of influenza virus, is widely used as a target for production of neutralizing antibodies. Monoclonal antibody, mAb6-9-1, directed against HA of highly pathogenic avian influenza virus A/swan/Poland/305-135V08/2006(H5N1) was purified from mouse hybridoma cells culture and characterized. The antigenic specificity of mAb6-9-1 was verified by testing its cross-reactivity with several variants of HA. The mimotopes recognized by mAb6-9-1 were selected from two types of phage display peptide libraries. The comparative structural model of the HA variant used for antibody generation was developed to further facilitate epitope mapping. Based on the sequences of the affinity- selected polypeptides and the structural model of HA the epitope was located to the region near the receptor binding site (RBS). Such localization of the epitope recognized by mAb6-9-1 is in concordance with its moderate hemagglutination inhibiting activity and its antigenic specificity. Additionally, total RNA isolated from the hybridoma cell line secreting mAb6-9-1 was used for obtaining two variants of cDNA encoding recombinant single-chain variable fragment (scFv) antibody. To ensure high production level and solubility in bacterial expression system, the scFv fragments were produced as chimeric proteins in fusion with thioredoxin or displayed on a phage surface after cloning into the phagemid vector. Specificity and affinity of the recombinant soluble and phage-bound scFv were assayed by suitable variants of ELISA test. The observed differences in specificity were discussed.