Importance of RNA secondary structure information for yeast donor and acceptor splice site predictions by neural networks

Previously, Patterson et al. showed that mRNA structure information aids splice site prediction in human genes [Patterson, D.J., Yasuhara, K., Ruzzo, W.L., 2002. Pre-mRNA secondary structure prediction aids splice site prediction. Pac. Symp. Biocomput. 7, 223-234]. Here, we have attempted to predict splice sites in selected genes of Saccharomyces cerevisiae using the information obtained from the secondary structures of corresponding mRNAs. From Ares database, 154 genes were selected and their structures were predicted by Mfold. We selected a 20-nucleotide window around each site, each containing 4 nucleotides in the exon region. Based on whether the nucleotide is in a stem or not, the conventional four-letter nucleotide alphabet was translated into an eight-letter alphabet. Two different three-layer-based perceptron neural networks were devised to predict the 5' and 3' splice sites. In case of 5' site determination, a network with 3 neurons at the hidden layer was chosen, while in case of 3' site 20 neurons acted more efficiently. Both neural nets were trained applying Levenberg-Marquardt backpropagation method, using half of the available genes as training inputs and the other half for testing and cross-validations. Sequences with GUs and AGs non-sites were used as negative controls. The correlation coefficients in the predictions of 5' and 3' splice sites using eight-letter alphabet were 98.0% and 69.6%, respectively, while these values were 89.3% and 57.1% when four-letter alphabet is applied. Our results suggest that considering the secondary structure of mRNA molecules positively affects both donor and acceptor site predictions by increasing the capacity of neural networks in learning the patterns.     

Virtual screening of the SAMPL4 blinded HIV integrase inhibitors dataset

Several combinations of docking software and scoring functions were evaluated for their ability to predict the binding of a dataset of potential HIV integrase inhibitors. We found that different docking software were appropriate for each one of the three binding sites considered (LEDGF, Y3 and fragment sites), and the most suitable two docking protocols, involving Glide SP and Gold ChemScore, were selected using a training set of compounds identified from the structural data available. These protocols could successfully predict respectively 20.0 and 23.6 % of the HIV integrase binders, all of them being present in the LEDGF site. When a different analysis of the results was carried out by removing all alternate isomers of binders from the set, our predictions were dramatically improved, with an overall ROC AUC of 0.73 and enrichment factor at 10 % of 2.89 for the prediction obtained using Gold ChemScore. This study highlighted the ability of the selected docking protocols to correctly position in most cases the ortho-alkoxy-carboxylate core functional group of the ligands in the corresponding binding site, but also their difficulties to correctly rank the docking poses.     

Expansion of the lysine acylation landscape

Leaving marks: the number of known posttranslational modifications for lysine has been expanded considerably. In addition to acetylation of side-chain amino functionalities of lysine residues in proteins, crotonylation, succinylation, and malonylation have now been identified as posttranslational modifications in histone and in non-histone proteins.     

Structure and binding of the H4 histone tail and the effects of lysine 16 acetylation

The H4 histone tail plays a critical role in chromatin folding and regulation--it mediates strong interactions with the acidic patch of proximal nucleosomes and its acetylation at lysine 16 (K16) leads to partial unfolding of chromatin. The molecular mechanism associated with the H4 tail/acidic patch interactions and its modulation via K16 acetylation remains unknown. Here we employ a combination of molecular dynamics simulations, molecular docking calculations, and free energy computations to investigate the structure of the H4 tail in solution, the binding of the H4 tail with the acidic patch, and the effects of K16 acetylation. The H4 tail exhibits a disordered configuration except in the region Ala15-Lys20, where it exhibits a strong propensity for an α-helical structure. This α-helical region is found to dock very favorably into the acidic patch groove of a nucleosome with a binding free energy of approximately -7 kcal mol(-1). We have identified the specific interactions that stabilize this binding as well as the associated energetics. The acetylation of K16 is found to reduce the α-helix forming propensity of the H4 tail and K16's accessibility for mediating external interactions. More importantly, K16 acetylation destabilizes the binding of the H4 tail at the acidic patch by mitigating specific salt bridges and longer-ranged electrostatic interactions mediated by K16. Our study thus provides new microscopic insights into the compaction of chromatin and its regulation via posttranslational modifications of histone tails, which could be of interest to chromatin biology, cancer, epigenetics, and drug design.     

Acetylation of the HIV-1 Tat protein: an in vitro study

In the last few years, the understanding of lysine acetylation as a regulatory post-translational modification of proteins in cell signalling cascades has increased. It is now known that not only histones but also non-histone factors can serve as substrates of different acetyltransferase enzymes. Acetylated lysine residues in non-histone factors are often identified using radioactive labelling experiments and immunochemical analysis of synthetic peptides. In this study of the human immunodeficiency virus 1 (HIV-1) Tat protein, we demonstrate the benefits of matrix-assisted laser desorption/ionisation mass spectrometry, proteolytic digestion and Edman sequencing for the mapping of acetylation sites. We confirmed that the HIV-1 Tat protein is acetylated in vitro by the acetyltransferase p300 at a specific lysine residue at position 50 in its RNA binding region. Furthermore, we showed that the Tat cysteine-rich region is acetylated at multiple cysteine residues in the absence of enzyme. Since this non-enzymatic cysteine acetylation occurs independently from the surrounding peptide sequence, we consider the presence of cysteine residues in acetylated peptides an important factor for the interpretation of in vitro acetylation assays in general.Abbreviations aa Amino acid - AcCoA Acetyl coenzyme A - acm Acetamidomethyl - ARM Arginine-rich motif - CRR Cysteine-rich region - HAT Histone acetyltransferaseThis article is dedicated to Harald zur Hausen on the occasion of his retirement as head of the German Cancer Research Center (Deutsches Krebsforschungszentrum) with gratitude and appreciation for 20 years of leadership     

Interrogating Substrate Selectivity and Composition of Endogenous Histone Deacetylase Complexes with Chemical Probes

Histone deacetylases (HDACs) regulate the function and activity of numerous cellular proteins by removing acetylation marks from regulatory lysine residues. We have developed peptide‐based HDAC probes that contain hydroxamate amino acids of various lengths to replace modified lysine residues in the context of known acetylation sites. The interaction profiles of all human HDACs were studied with three sets of probes, which derived from different acetylation sites, and sequence context was found to have a strong impact on substrate recognition and composition of HDAC complexes. By investigating K382 acetylation of the tumor suppressor p53 as an example, we further demonstrate that the interaction profiles reflect the catalytic activities of respective HDACs. These results underline the utility of the newly established probes for deciphering not only activity, but also substrate selectivity and composition of endogenous HDAC complexes, which can hardly be achieved otherwise.     

Mapping Post-translational Modifications of Histones H2A, H2B and H4 in Schizosaccharomyces pombe

Core histones are known to carry a variety of post-translational modifications (PTMs), including acetylation, phosphorylation, methylation and ubiquitination, which play important roles in the epigenetic control of gene expression. The nature and biological functions of these PTMs in histones from plants, animals and budding yeast have been extensively investigated. In contrast, the corresponding studies for fission yeast were mainly focused on histone H3. In the present study, we applied LC-nano-ESI-MS/MS, coupled with multiple protease digestion, to identify PTMs in histones H2A, H2B and H4 from Schizosaccharomyces pombe (S. pombe), the typical model organism of fission yeast. Various protease digestions provided high sequence coverage for PTM mapping, and accurate mass measurement of fragment ions allowed for unambiguous differentiation of acetylation from tri-methylation. Many modification sites conserved in other organisms were identified in S. pombe. In addition, some unique modification sites, including N-terminal acetylation in H2A and H2B as well as K123 acetylation in H2A.β, were observed. Our results provide a comprehensive picture of the PTMs of histones H2A, H2B and H4 in S. pombe, which serves as a foundation for future investigations on the regulation and functions of histone modifications in this important model organism.     

二进小波神经网络用于示波计时电位测定

提出了二进小波神经网络的结构及算法，并用于单组分和多组分示波计时电位信号的浓度计算。在二进小波神经网络中选用了Morlet母小波和修理的误差反传前向神经网络。探讨了二进小波神经网络中的中小波基个数，初始学习速率因子和动量因子等参数对网络预测结果的影响。结果表明：二进小波神经网络对双组分和单组分示波计时电位信号中去极剂浓度的预测均有很好效果。