Computational Prediction and Experimental Verification of New MAP Kinase Docking Sites and Substrates Including Gli Transcription Factors

In order to fully understand protein kinase networks, new methods are needed to identify regulators and substrates of kinases, especially for weakly expressed proteins. Here we have developed a hybrid computational search algorithm that combines machine learning and expert knowledge to identify kinase docking sites, and used this algorithm to search the human genome for novel MAP kinase substrates and regulators focused on the JNK family of MAP kinases. Predictions were tested by peptide array followed by rigorous biochemical verification with in vitro binding and kinase assays on wild-type and mutant proteins. Using this procedure, we found new ‘D-site’ class docking sites in previously known JNK substrates (hnRNP-K, PPM1J/PP2Czeta), as well as new JNK-interacting proteins (MLL4, NEIL1). Finally, we identified new D-site-dependent MAPK substrates, including the hedgehog-regulated transcription factors Gli1 and Gli3, suggesting that a direct connection between MAP kinase and hedgehog signaling may occur at the level of these key regulators. These results demonstrate that a genome-wide search for MAP kinase docking sites can be used to find new docking sites and substrates

Prediction of protein binding sites in protein structures using hidden Markov support vector machine

<p>Abstract</p> <p>Background</p> <p>Predicting the binding sites between two interacting proteins provides important clues to the function of a protein. Recent research on protein binding site prediction has been mainly based on widely known machine learning techniques, such as artificial neural networks, support vector machines, conditional random field, etc. However, the prediction performance is still too low to be used in practice. It is necessary to explore new algorithms, theories and features to further improve the performance.</p> <p>Results</p> <p>In this study, we introduce a novel machine learning model hidden Markov support vector machine for protein binding site prediction. The model treats the protein binding site prediction as a sequential labelling task based on the maximum margin criterion. Common features derived from protein sequences and structures, including protein sequence profile and residue accessible surface area, are used to train hidden Markov support vector machine. When tested on six data sets, the method based on hidden Markov support vector machine shows better performance than some state-of-the-art methods, including artificial neural networks, support vector machines and conditional random field. Furthermore, its running time is several orders of magnitude shorter than that of the compared methods.</p> <p>Conclusion</p> <p>The improved prediction performance and computational efficiency of the method based on hidden Markov support vector machine can be attributed to the following three factors. Firstly, the relation between labels of neighbouring residues is useful for protein binding site prediction. Secondly, the kernel trick is very advantageous to this field. Thirdly, the complexity of the training step for hidden Markov support vector machine is linear with the number of training samples by using the cutting-plane algorithm.</p

Fast learning optimized prediction methodology for protein secondary structure prediction, relative solvent accessibility prediction and phosphorylation prediction

Computational methods are rapidly gaining importance in the field of structural biology, mostly due to the explosive progress in genome sequencing projects and the large disparity between the number of sequences and the number of structures. There has been an exponential growth in the number of available protein sequences and a slower growth in the number of structures. There is therefore an urgent need to develop computed structures and identify the functions of these sequences. Developing methods that will satisfy these needs both efficiently and accurately is of paramount importance for advances in many biomedical fields, for a better basic understanding of aberrant states of stress and disease, including drug discovery and discovery of biomarkers. Several aspects of secondary structure predictions and other protein structure-related predictions are investigated using different types of information such as data obtained from knowledge-based potentials derived from amino acids in protein sequences, physicochemical properties of amino acids and propensities of amino acids to appear at the ends of secondary structures. Investigating the performance of these secondary structure predictions by type of amino acid highlights some interesting aspects relating to the influences of the individual amino acid types on formation of secondary structures and points toward ways to make further gains. Other research areas include Relative Solvent Accessibility (RSA) predictions and predictions of phosphorylation sites, which is one of the Post-Translational Modification (PTM) sites in proteins. Protein secondary structures and other features of proteins are predicted efficiently, reliably, less expensively and more accurately. A novel method called Fast Learning Optimized PREDiction (FLOPRED) Methodology is proposed for predicting protein secondary structures and other features, using knowledge-based potentials, a Neural Network based Extreme Learning Machine (ELM) and advanced Particle Swarm Optimization (PSO) techniques that yield better and faster convergence to produce more accurate results. These techniques yield superior classification of secondary structures, with a training accuracy of 93.33% and a testing accuracy of 92.24% with a standard deviation of 0.48% obtained for a small group of 84 proteins. We have a Matthew\u27s correlation-coefficient ranging between 80.58% and 84.30% for these secondary structures. Accuracies for individual amino acids range between 83% and 92% with an average standard deviation between 0.3% and 2.9% for the 20 amino acids. On a larger set of 415 proteins, we obtain a testing accuracy of 86.5% with a standard deviation of 1.38%. These results are significantly higher than those found in the literature. Prediction of protein secondary structure based on amino acid sequence is a common technique used to predict its 3-D structure. Additional information such as the biophysical properties of the amino acids can help improve the results of secondary structure prediction. A database of protein physicochemical properties is used as features to encode protein sequences and this data is used for secondary structure prediction using FLOPRED. Preliminary studies using a Genetic Algorithm (GA) for feature selection, Principal Component Analysis (PCA) for feature reduction and FLOPRED for classification give promising results. Some amino acids appear more often at the ends of secondary structures than others. A preliminary study has indicated that secondary structure accuracy can be improved as much as 6% by including these effects for those residues present at the ends of alpha-helix, beta-strand and coil. A study on RSA prediction using ELM shows large gains in processing speed compared to using support vector machines for classification. This indicates that ELM yields a distinct advantage in terms of processing speed and performance for RSA. Additional gains in accuracies are possible when the more advanced FLOPRED algorithm and PSO optimization are implemented. Phosphorylation is a post-translational modification on proteins often controls and regulates their activities. It is an important mechanism for regulation. Phosphorylated sites are known to be present often in intrinsically disordered regions of proteins lacking unique tertiary structures, and thus less information is available about the structures of phosphorylated sites. It is important to be able to computationally predict phosphorylation sites in protein sequences obtained from mass-scale sequencing of genomes. Phosphorylation sites may aid in the determination of the functions of a protein and to better understanding the mechanisms of protein functions in healthy and diseased states. FLOPRED is used to model and predict experimentally determined phosphorylation sites in protein sequences. Our new PSO optimization included in FLOPRED enable the prediction of phosphorylation sites with higher accuracy and with better generalization. Our preliminary studies on 984 sequences demonstrate that this model can predict phosphorylation sites with a training accuracy of 92.53% , a testing accuracy 91.42% and Matthew\u27s correlation coefficient of 83.9%. In summary, secondary structure prediction, Relative Solvent Accessibility and phosphorylation site prediction have been carried out on multiple sets of data, encoded with a variety of information drawn from proteins and the physicochemical properties of their constituent amino acids. Improved and efficient algorithms called S-ELM and FLOPRED, which are based on Neural Networks and Particle Swarm Optimization are used for classifying and predicting protein sequences. Analysis of the results of these studies provide new and interesting insights into the influence of amino acids on secondary structure prediction. S-ELM and FLOPRED have also proven to be robust and efficient for predicting relative solvent accessibility of proteins and phosphorylation sites. These studies show that our method is robust and resilient and can be applied for a variety of purposes. It can be expected to yield higher classification accuracy and better generalization performance compared to previous methods

PlantPhos: using maximal dependence decomposition to identify plant phosphorylation sites with substrate site specificity

<p>Abstract</p> <p>Background</p> <p>Protein phosphorylation catalyzed by kinases plays crucial regulatory roles in intracellular signal transduction. Due to the difficulty in performing high-throughput mass spectrometry-based experiment, there is a desire to predict phosphorylation sites using computational methods. However, previous studies regarding <it>in silico </it>prediction of plant phosphorylation sites lack the consideration of kinase-specific phosphorylation data. Thus, we are motivated to propose a new method that investigates different substrate specificities in plant phosphorylation sites.</p> <p>Results</p> <p>Experimentally verified phosphorylation data were extracted from TAIR9-a protein database containing 3006 phosphorylation data from the plant species <it>Arabidopsis thaliana</it>. In an attempt to investigate the various substrate motifs in plant phosphorylation, maximal dependence decomposition (MDD) is employed to cluster a large set of phosphorylation data into subgroups containing significantly conserved motifs. Profile hidden Markov model (HMM) is then applied to learn a predictive model for each subgroup. Cross-validation evaluation on the MDD-clustered HMMs yields an average accuracy of 82.4% for serine, 78.6% for threonine, and 89.0% for tyrosine models. Moreover, independent test results using <it>Arabidopsis thaliana </it>phosphorylation data from UniProtKB/Swiss-Prot show that the proposed models are able to correctly predict 81.4% phosphoserine, 77.1% phosphothreonine, and 83.7% phosphotyrosine sites. Interestingly, several MDD-clustered subgroups are observed to have similar amino acid conservation with the substrate motifs of well-known kinases from Phospho.ELM-a database containing kinase-specific phosphorylation data from multiple organisms.</p> <p>Conclusions</p> <p>This work presents a novel method for identifying plant phosphorylation sites with various substrate motifs. Based on cross-validation and independent testing, results show that the MDD-clustered models outperform models trained without using MDD. The proposed method has been implemented as a web-based plant phosphorylation prediction tool, PlantPhos <url>http://csb.cse.yzu.edu.tw/PlantPhos/</url>. Additionally, two case studies have been demonstrated to further evaluate the effectiveness of PlantPhos.</p

Allosteric Regulation at the Crossroads of New Technologies: Multiscale Modeling, Networks, and Machine Learning

Allosteric regulation is a common mechanism employed by complex biomolecular systems for regulation of activity and adaptability in the cellular environment, serving as an effective molecular tool for cellular communication. As an intrinsic but elusive property, allostery is a ubiquitous phenomenon where binding or disturbing of a distal site in a protein can functionally control its activity and is considered as the “second secret of life.” The fundamental biological importance and complexity of these processes require a multi-faceted platform of synergistically integrated approaches for prediction and characterization of allosteric functional states, atomistic reconstruction of allosteric regulatory mechanisms and discovery of allosteric modulators. The unifying theme and overarching goal of allosteric regulation studies in recent years have been integration between emerging experiment and computational approaches and technologies to advance quantitative characterization of allosteric mechanisms in proteins. Despite significant advances, the quantitative characterization and reliable prediction of functional allosteric states, interactions, and mechanisms continue to present highly challenging problems in the field. In this review, we discuss simulation-based multiscale approaches, experiment-informed Markovian models, and network modeling of allostery and information-theoretical approaches that can describe the thermodynamics and hierarchy allosteric states and the molecular basis of allosteric mechanisms. The wealth of structural and functional information along with diversity and complexity of allosteric mechanisms in therapeutically important protein families have provided a well-suited platform for development of data-driven research strategies. Data-centric integration of chemistry, biology and computer science using artificial intelligence technologies has gained a significant momentum and at the forefront of many cross-disciplinary efforts. We discuss new developments in the machine learning field and the emergence of deep learning and deep reinforcement learning applications in modeling of molecular mechanisms and allosteric proteins. The experiment-guided integrated approaches empowered by recent advances in multiscale modeling, network science, and machine learning can lead to more reliable prediction of allosteric regulatory mechanisms and discovery of allosteric modulators for therapeutically important protein targets

KinasePhos: a web tool for identifying protein kinase-specific phosphorylation sites

KinasePhos is a novel web server for computationally identifying catalytic kinase-specific phosphorylation sites. The known phosphorylation sites from public domain data sources are categorized by their annotated protein kinases. Based on the profile hidden Markov model, computational models are learned from the kinase-specific groups of the phosphorylation sites. After evaluating the learned models, the model with highest accuracy was selected from each kinase-specific group, for use in a web-based prediction tool for identifying protein phosphorylation sites. Therefore, this work developed a kinase-specific phosphorylation site prediction tool with both high sensitivity and specificity. The prediction tool is freely available at

Predicting domain-domain interaction based on domain profiles with feature selection and support vector machines

<p>Abstract</p> <p>Background</p> <p>Protein-protein interaction (PPI) plays essential roles in cellular functions. The cost, time and other limitations associated with the current experimental methods have motivated the development of computational methods for predicting PPIs. As protein interactions generally occur via domains instead of the whole molecules, predicting domain-domain interaction (DDI) is an important step toward PPI prediction. Computational methods developed so far have utilized information from various sources at different levels, from primary sequences, to molecular structures, to evolutionary profiles.</p> <p>Results</p> <p>In this paper, we propose a computational method to predict DDI using support vector machines (SVMs), based on domains represented as interaction profile hidden Markov models (ipHMM) where interacting residues in domains are explicitly modeled according to the three dimensional structural information available at the Protein Data Bank (PDB). Features about the domains are extracted first as the Fisher scores derived from the ipHMM and then selected using singular value decomposition (SVD). Domain pairs are represented by concatenating their selected feature vectors, and classified by a support vector machine trained on these feature vectors. The method is tested by leave-one-out cross validation experiments with a set of interacting protein pairs adopted from the 3DID database. The prediction accuracy has shown significant improvement as compared to <it>InterPreTS </it>(Interaction Prediction through Tertiary Structure), an existing method for PPI prediction that also uses the sequences and complexes of known 3D structure.</p> <p>Conclusions</p> <p>We show that domain-domain interaction prediction can be significantly enhanced by exploiting information inherent in the domain profiles via feature selection based on Fisher scores, singular value decomposition and supervised learning based on support vector machines. Datasets and source code are freely available on the web at <url>http://liao.cis.udel.edu/pub/svdsvm</url>. Implemented in Matlab and supported on Linux and MS Windows.</p

Nuclear export signals (NESs) in Arabidopsis thaliana : development and experimental validation of a prediction tool

Rubiano Castellanos CC. Nuclear export signals (NESs) in Arabidopsis thaliana : development and experimental validation of a prediction tool. Bielefeld (Germany): Bielefeld University; 2010.It is well established that nucleo-cytoplasmic shuttling regulates not only the localization but also the activity of many proteins like transcription factors, cell cycle regulators and tumor suppressor proteins just to mention some. Also in plants the nucleo-cytoplasmic partitioning of proteins emerges as an important regulation mechanism for many plant-specific processes. One requirement for a protein to shuttle between nucleus and cytoplasm lies in its nuclear export activity. The widely used mechanism for export of proteins from the nucleus involves the receptor Exportin 1 and the presence of a nuclear export signal (NES) in the cargo protein. Given the big amount of sequence data available nowadays the possibility to use a computational tool to predict the proteins potentially containing an NES would help to facilitate the screening and experimental characterization of NES-containing proteins. However, the computational prediction of NESs is a challenging task. Currently there is only one NES prediction tool and that is unfortunately not accurate for predicting these signals in proteins of plants. In that direction, this study aimed mainly at developing a prediction method for identifying NESs in proteins from Arabidopsis and to validate its usefulness experimentally. It included also the definition of the influence of the NES protein context in the nuclear export activity of specific proteins of Arabidopsis. Three machine-learning algorithms (i.e. k-NN, SVM and Random Forests) were trained with experimentally validated NES sequences from proteins of Arabidopsis and other organisms. Two kinds of features were included, the sequence of the NESs expressed as the score obtained from an HMM profile constructed with the NES sequences of proteins from Arabidopsis, and physicochemical properties of the amino acid residues expressed as amino acid index values. The Random Forest classifier was selected among the three classifiers after evaluation of the performance by different methods. It showed to be highly accurate (accuracy values over 85 percent, classification error around 10 percent, MCC around 0.7 and area under the ROC curve around 0.90) and performed better than the other two trained classifiers. Using the Random Forest classifier around 5000 proteins from the total of protein sequences from Arabidopsis were predicted as containing NESs. A group of these proteins was selected by using Gene Ontologies (GO) and from this last group, 13 proteins were experimentally tested for nuclear export activity. 11 out of those 13 proteins showed positive interaction with the receptor Exportin 1 (XPO1a) from Arabidopsis in yeast two-hybrid assays. The proteins showing nuclear export activity include 9 transcription factors and 2 DNA metabolism-related proteins. Furthermore, it was established that the amino acid residues located between the hydrophobic residues in the NES as well as the protein structure of the regions around the NES could modify the nuclear export activity of some proteins. In conclusion, this work presents a new prediction tool for NESs in proteins of Arabidopsis based on a Random Forest classifier. The experimental validation of the nuclear export activity in a selected group of proteins is an indicative of the usefulness of the tool. From the biological point of view, the nuclear export activity observed in those proteins strongly suggest that nucleo-cytoplasmic partitioning could be involved in the regulation of their functions. For the follow up research the further characterization of the proteins showing positive nuclear export activity as well as the validation of additional predicted NES-containing proteins is envisioned. In the near future, the developed tool is going to be available as a web application to facilitate and promote its further usage