Advances in GPCR modeling evaluated by the GPCR Dock 2013 assessment: Meeting new challenges

© 2014 Elsevier Ltd All rights reserved. Despite tremendous successes of GPCR crystallography, the receptors with available structures represent only a small fraction of human GPCRs. An important role of the modeling community is to maximize structural insights for the remaining receptors and complexes. The community-wide GPCR Dock assessment was established to stimulate and monitor the progress in molecular modeling and ligand docking for GPCRs. The four targets in the present third assessment round presented new and diverse challenges for modelers, including prediction of allosteric ligand interaction and activation states in 5-hydroxytryptamine receptors 1B and 2B, and modeling by extremely distant homology for smoothened receptor. Forty-four modeling groups participated in the assessment. State-of-the-art modeling approaches achieved close-to-experimental accuracy for small rigid orthosteric ligands and models built by close homology, and they correctly predicted protein fold for distant homology targets. Predictions of long loops and GPCR activation states remain unsolved problems

Identification de nouvelles protéines effectrices dans la signalisation des récepteurs Eph

La réponse cellulaire aux stimuli extracellulaires est souvent médiée par des voies de signalisation qui agissent en aval des récepteurs transmembranaires, comme les récepteurs tyrosine kinases (RTK). Avec quatorze membres, la famille des récepteurs Eph représente la plus grande famille de RTK chez l'humain. Contrairement aux autres RTK, les ligands des récepteurs Eph, les éphrines, sont des protéines associées à la membrane cellulaire. La signalisation Eph-éprhines est donc principalement impliquée dans des événements de communication qui impliquent des contacts cellule-cellule comme la migration cellulaire, la répulsion et l'adhésion cellule-cellule. Ces événements sont cruciaux pour certains processus biologiques tels le guidage axonal et l'organisation tissulaire dans l'organisme en développement et chez l'adulte. Les récepteurs Eph sont fréquemment surexprimés ou dérégulés dans divers cancers, en particulier dans les plus agressifs et mortels. Récemment, la signalisation Eph-éphrines est devenue une nouvelle cible émergente pour le traitement du cancer. Bien que les fonctions biologiques des récepteurs Eph aient été largement étudiées, notre compréhension des mécanismes moléculaires grâce auxquels les récepteurs Eph régulent des phénotypes cellulaires précis demeure incomplète. Pour mieux comprendre le système de signalisation impliquant les Eph, mes travaux ont porté sur l'identification de nouvelles protéines effectrices en aval des récepteurs Eph et sur l'étude de leurs implications dans les fonctions régulées par les récepteurs Eph. Pour mieux comprendre les complexes de signalisation associés aux récepteurs Eph dans des conditions natives, j'ai appliqué une approche basée sur la spectrométrie de masse (MS), le marquage de proximité BioID. Cela m'a permis de surmonter les limites de l'utilisation d'approches conventionnelles de purification par affinité pour cartographier les interactions protéine-protéine liées aux récepteurs transmembranaires. J'ai obtenu un réseau de signalisation dépendant des récepteurs EphA4, - B2, -B3 et -B4, qui comprend 395 protéines, dont la plupart n'avaient jamais été liées à la signalisation Eph-dépendante. Pour tester la pertinence biologique des partenaires identifiés, j'ai examiné la contribution de 17 candidats en utilisant une approche de perte de fonction dans une expérience de tri cellulaire dépendante des récepteurs Eph. J'ai pu montrer que la déplétion de quelques candidats, incluant la protéine Par3, bloque le tri des cellules. En utilisant la purification par affinité combinée à la MS, j'ai aussi identifié un complexe de signalisation impliquant la kinase C-terminal SRC (CSK), dont le recrutement aux complexes Par3 dépend des signaux des récepteurs Eph. Pour mieux comprendre les interactions protéiques suivant la liaison Eph-éphrine, j'ai effectué des expériences de TurboID. Ces études m'ont permis d'identifier des complexes protéiques associés au récepteur EphA4 lorsqu'il est lié à l'éphrine-B2. J'ai également étudié les interactions protéine-protéine dépendantes de la liaison du récepteur EphB2 aux éphrines-B1 et -B2. Pour explorer si l'interaction d'EphB2 avec ces deux ligands peut mener à une réponse de signalisation inverse différente, j'ai identifié les partenaires de des ephrin-B1/-B2 lorsque stimulés par EphB2. Enfin, j'ai cartographié les réseaux de signalisation dépendants des récepteurs EphA4 et EphB2 sauvages ou kinase-inactifs, ce qui m'a permis de conclure que la perte de leur activité catalytique a conduit à des changements majeurs dans les interactomes dépendants de ces récepteurs. L'ensemble de mes résultats a permis de mieux définir les complexes protéiques dépendants des récepteurs Eph. Mes études ont mené à une meilleure compréhension des mécanismes moléculaires sous-jacents aux récepteurs Eph et de leur contribution dans le processus de délimitation des tissus, un processus souvent perturbé dans des maladies comme le cancer.The cellular response to extracellular stimuli is often mediated by signaling pathways that act downstream of transmembrane receptors, such as receptor tyrosine kinases (RTKs). With fourteen members, the Eph family of RTKs is the largest in humans. In contrast to other RTKs, Eph receptor cognate ligands, ephrins, are tethered to the cell surface. This results in Eph receptor-ephrin signaling being mainly involved in short-range cell-cell communication events that regulate cell migration, repulsion and cell-cell adhesion. These events are crucial in biological processes such as axon guidance and tissue boundary formation in the developing and adult organisms. Eph receptors are frequently overexpressed or deregulated in a variety of human tumors, especially in the more aggressive and lethal ones. In recent years, the Eph-ephrin signaling system became an emerging new target for cancer treatment. Although a plethora of Eph receptor biological functions have been extensively studied, we still have a vague idea on the molecular mechanisms of Eph receptor signal transduction, underlying how Eph receptors regulate precise cellular phenotypes. To better understand the Eph receptor signaling system, my studies focused on the identification of novel Eph receptor downstream effector proteins and the determination of their requirement for Eph receptor-regulated functions. To unravel Eph receptor-associated signaling complexes under native conditions, I applied a mass spectrometry (MS)-based approach, namely BioID proximity labeling. This allowed me to overcome the limitations of conventional affinity purification approaches for mapping protein-protein interactions of transmembrane receptors. I obtained a composite signaling network from EphA4, -B2, -B3 and -B4 receptors that comprises 395 proteins, most of which not previously linked to Eph signaling. To test the biological relevance of the identified Eph receptor proximity interactors, I examined the contribution of 17 candidates using a loss-of-function approach in an Eph receptor-dependent cell sorting assay. I showed that depletion of a few candidates, including the signaling scaffold Par3, blocks Eph receptordependent cell sorting. Using affinity purification combined with MS, I further delineated a signaling complex involving C-terminal SRC kinase (CSK), whose recruitment to Par3 complexes is dependent on Eph receptor signals. To further elucidate Eph receptor-centric signaling complexes that are formed upon ephrin binding and are affected by Eph receptor catalytic activity I performed TurboID experiments. I systematically mapped ligand stimulation-dependent signaling networks downstream of EphA4 and EphB2 receptors. I dissected the impact of ephrin-B2 stimulation on the formation of EphA4- nucleated proximal protein complexes. Moreover, I showed the differential recruitment of EphB2 partners upon receptor binding to the same subclass of ligands, ephrin-B1 and ephrin-B2. To explore whether the EphB2 interactions with these two ephrin-B ligands elicit different reverse signaling responses, I delineated ephrin-B1/-B2 proximity partners recruited upon EphB2 stimulation. I also determined that the kinase domain of EphA4/-B2 plays a major role in determining the composition of signaling networks around the receptors, as a loss of catalytic activity led to a drastic decrease in a number of interactors with the receptors. Collectively, my definition of Eph receptor signaling networks sheds light on physiologically relevant Eph receptor-centered protein complexes that occur in living cells. These studies will lead to a better understanding of the mechanisms by which Eph receptors transmit signals at the membrane and give insight into how Eph receptor-mediated signaling pathways contribute to boundary formation, a process often disrupted in diseases like cancer

Development of an affinity pair “Tag-Receptor” for recombinant protein expression and purification

Dissertação apresentada para a obtenção do Grau de Mestre em Biotecnologia, pela Universidade Nova de Lisboa, Faculdade de Ciências e TecnologiaThe main objective of this work was the development of an affinity pair for the purification of recombinant proteins. In this work, ligands based on the Ugi Reaction and the 1,3,5-Triazine scaffold were synthesised in solid-phase and screened for binding to an affinity tag, an hexapeptide constituted by asparagine aminoacid (N). The ligands were tested against pure solutions of the hexapeptide and Green Fluorescence Protein (GFP), used as a model protein. The ligands that had the highest affinity for the hexapeptide and lowest affinity for the protein were selected for further studies with cellular extracts. The cellular extracts were produced in HEK 293T cells transfected with two designed vectors: one containing the GFP tagged with the affinity tag, and the other containing GFP without tag. The efficient expression of a recombinant GFP fused with the designed affinity tag was demonstrated. The cellular extracts were then loaded onto chromatographic columns containing the lead ligands immobilised onto agarose, and the amount of total protein and GFP bound and eluted noted. The results demonstrated that the Ugi ligands were less selective than the Triazine ligands for the hexapeptide. The triazine ligand 7,4 has been considered as the most selective for the designed affinity tag. In addition, preselected lead ligands for another hexapeptide (RW) of interest were studied. Mammalian cells HEK 293T were transfected with a vector expressing for GFP tagged with this peptide. The ligands immobilized onto agarose were loaded with cellular extracts, being noted that the lead A6C3 showed a high selectivity for the tag tested

The interplay of descriptor-based computational analysis with pharmacophore modeling builds the basis for a novel classification scheme for feruloyl esterases

One of the most intriguing groups of enzymes, the feruloyl esterases (FAEs), is ubiquitous in both simple and complex organisms. FAEs have gained importance in biofuel, medicine and food industries due to their capability of acting on a large range of substrates for cleaving ester bonds and synthesizing high-added value molecules through esterification and transesterification reactions. During the past two decades extensive studies have been carried out on the production and partial characterization of FAEs from fungi, while much less is known about FAEs of bacterial or plant origin. Initial classification studies on FAEs were restricted on sequence similarity and substrate specificity on just four model substrates and considered only a handful of FAEs belonging to the fungal kingdom. This study centers on the descriptor-based classification and structural analysis of experimentally verified and putative FAEs; nevertheless, the framework presented here is applicable to every poorly characterized enzyme family. 365 FAE-related sequences of fungal, bacterial and plantae origin were collected and they were clustered using Self Organizing Maps followed by k-means clustering into distinct groups based on amino acid composition and physico-chemical composition descriptors derived from the respective amino acid sequence. A Support Vector Machine model was subsequently constructed for the classification of new FAEs into the pre-assigned clusters. The model successfully recognized 98.2% of the training sequences and all the sequences of the blind test. The underlying functionality of the 12 proposed FAE families was validated against a combination of prediction tools and published experimental data. Another important aspect of the present work involves the development of pharmacophore models for the new FAE families, for which sufficient information on known substrates existed. Knowing the pharmacophoric features of a small molecule that are essential for binding to the members of a certain family opens a window of opportunities for tailored applications of FAEs

The Use of Computational Methods in the Toxicological Assessment of Chemicals in Food: Current Status and Future Prospects

A wide range of chemicals are intentionally added to, or unintentially found in, food products, often in very small amounts. Depending on the situation, the experimental data needed to complete a dietary risk assessment, which is the scientific basis for protecting human health, may not be available or obtainable, for reasons of cost, time and animal welfare. For example, toxicity data are often lacking for the metabolites and degradation products of pesticide active ingredients. There is therefore an interest in the development and application of efficient and effective non-animal methods for assessing chemical toxicity, including Quantitative Structure-Activity Relationship (QSAR) models and related computational methods. This report gives an overview of how computational methods are currently used in the field of food safety by national regulatory bodies, international advisory organisations and the food industry. On the basis of an international survey, a comprehensive literature review and a detailed QSAR analysis, a range of recommendations are made with the long-term aim of promoting the judicious use of suitable QSAR methods. The current status of QSAR methods is reviewed not only for toxicological endpoints relevant to dietary risk assessment, but also for Absorption, Distribution, Metabolism and Excretion (ADME) properties, which are often important in discriminating between the toxicological profiles of parent compounds and their reaction products. By referring to the concept of the Threshold of Toxicological Concern (TTC), the risk assessment context in which QSAR methods can be expected to be used is also discussed. This Joint Research Centre (JRC) Reference Report provides a summary and update of the findings obtained in a study carried out by the JRC under the terms of a contract awarded by the European Food Safety Authority (EFSA).JRC.DG.I.6-Systems toxicolog

Establishing the Role of Vitamin D signalling in Immunity and Melanoma Specific Survival

1α,25-dihydroxyvitamin D3 signals via its canonical nuclear receptor: Vitamin D Receptor (VDR). While higher levels of serum vitamin D have been reported to be associated with thinner primary melanomas and better outcome, increased VDR expression has been associated with decreased tumour progression and improved prognosis in melanoma primaries. However, the genomic basis of this effect remains to be explored and a causal mechanism is yet to be established. To address this question, I have used microarray data from a cohort of 703 treatment-naïve primary melanomas from the Leeds Melanoma Cohort (LMC) and corresponding clinical data. In the LMC primary melanomas, serum vitamin D was not significantly associated with melanoma survival. However, tumour VDR expression was significantly (and independently) protective for melanoma death in both the LMC and the TCGA metastatic melanoma datasets. Tumour VDR expression was found to be significantly positively correlated with genes enriched for ECM organization, TNF signalling, IFNg signalling, IL12-mediated signalling and NFkB signalling, which are predominantly immune-related. Concordantly, VDR expression was lower in tumours graded by the pathologist as having no immune infiltrate, compared to tumours with brisk and nonbrisk immune infiltrate. Additionally, VDR correlated positively with imputed immune cells scores. Conversely, the negatively correlated genes were enriched for Mitotic Prophase, Wnt signalling pathway, Mitochondrial translation, citric acid cycle and oxidative phosphorylation, which are predominantly proliferation-related. Of particular interest among the negatively correlated pathways was the Wnt/b-catenin signalling pathway. Functional validation using an in vivo tail-vein metastasis assay revealed that murine melanoma cells stably expressing VDR produced significantly fewer pulmonary metastases compared to control cells with null VDR expression. VDR-expressing cells also had significantly lower expression of Wnt/b-catenin signalling genes compared to control cells. These findings indicate that vitamin D-VDR signalling contributes to control of pro-proliferative and immunosuppressive Wnt/b-catenin signalling in melanoma and that this is associated with less proliferative, less metastatic disease and stronger host immune responses

The architecture of polyketide synthases

Since the discovery of penicillin over a century ago, secondary metabolites from all kingdoms of life have proven to be of high medical value. One class of proteins prevalent in the production of secondary metabolites are polyketide synthases (PKSs). Their polyketide products are complex organic compounds based on carbon chains assembled from carboxylic acid precursors. Many polyketides are produced by their hosts with the primary purpose of gaining an advantage in their ecological niche. To contribute to such an advantage, a significant proportion of polyketides are active against pro- and eukaryotic microorganisms. Type I PKSs are giant multienzyme proteins employing an assembly line logic for the synthesis of the most complex polyketides. They are composed of one or more functional and structural modules, each capable of carrying out one step of precursor elongation during the formation of an extended polyketide product. In this thesis, I address two fundamental and open questions in the biosynthesis of polyketides: First, what is the unique architecture underlying the assembly line logic of multimodular PKS assembly lines; and second, how is atomic accuracy achieved in cyclization and aromatic ring formation in the final step of PKS action. The first aim is addressed in chapter two, which provides for the first time detailed structural insights into the organization of type I PKS multimodules. This is achieved by cryo-electron microscopic analysis of filamentous and non-filamentous forms of K3DAK4, a bimodular trans-acyltransferase (AT) PKS fragment from Brevibacillus brevis. Overall reconstructions are provided at an intermediate resolution of 7 Å, with detailed insights into individual domains at sub-3Å resolution from cryo-electron microscopy and X-ray crystallography. The bimodule core displays a vertical stacking of its two modules along the central dimer axis of all three enzymatic domains involved. Additionally, K3DAK4 oligomerizes into filaments horizontally via small scaffolding domains in a trans-AT PKS-specific manner. In chapter three the second aim is tackled, as I visualize an intermediate of the enigmatic targeted cyclization and aromatic ring formation in the product template domain (PT) of the aflatoxin-producing PksA at 2.7 Å resolution using X-ray crystallography. To this end a substrate-analogue mimicking the transient intermediate after the first of two cyclization steps facilitated by the enzyme is covalently crosslinked to the active site. The positioning of the ligand relative to previously known ligands representing the pre-and post-cyclization states indicate an outward movement of the substrate throughout the process and a substantial effect of progressing cyclization on the meticulous positioning of the intermediates. The work provides detailed insights into core aspects of PKS biology from the atomistic picture of guided product modification to the giant overall assembly line architecture. In chapter four, both of these levels are put into context with current advances in the analysis of modular structure and dynamics of PKSs, such as recent structural models of cis-AT PKS modules and iterative PKSs. Furthermore, it addresses currently open questions, such as the interaction of trans-AT PKS with their cognate trans-acting enzymes. Altogether, the current progress in mechanistic understanding of PKS systems makes systematic and structure-guided efforts to unleash the full potential of PKS bioengineering ever more achievable

IN SILICO METHODS FOR DRUG DESIGN AND DISCOVERY

Computer-aided drug design (CADD) methodologies are playing an ever-increasing role in drug discovery that are critical in the cost-effective identification of promising drug candidates. These computational methods are relevant in limiting the use of animal models in pharmacological research, for aiding the rational design of novel and safe drug candidates, and for repositioning marketed drugs, supporting medicinal chemists and pharmacologists during the drug discovery trajectory.Within this field of research, we launched a Research Topic in Frontiers in Chemistry in March 2019 entitled “In silico Methods for Drug Design and Discovery,” which involved two sections of the journal: Medicinal and Pharmaceutical Chemistry and Theoretical and Computational Chemistry. For the reasons mentioned, this Research Topic attracted the attention of scientists and received a large number of submitted manuscripts. Among them 27 Original Research articles, five Review articles, and two Perspective articles have been published within the Research Topic. The Original Research articles cover most of the topics in CADD, reporting advanced in silico methods in drug discovery, while the Review articles offer a point of view of some computer-driven techniques applied to drug research. Finally, the Perspective articles provide a vision of specific computational approaches with an outlook in the modern era of CADD

Conformational and functional analysis of molecular dynamics trajectories by Self-Organising Maps

<p>Abstract</p> <p>Background</p> <p>Molecular dynamics (MD) simulations are powerful tools to investigate the conformational dynamics of proteins that is often a critical element of their function. Identification of functionally relevant conformations is generally done clustering the large ensemble of structures that are generated. Recently, Self-Organising Maps (SOMs) were reported performing more accurately and providing more consistent results than traditional clustering algorithms in various data mining problems. We present a novel strategy to analyse and compare conformational ensembles of protein domains using a two-level approach that combines SOMs and hierarchical clustering.</p> <p>Results</p> <p>The conformational dynamics of the α-spectrin SH3 protein domain and six single mutants were analysed by MD simulations. The Cα's Cartesian coordinates of conformations sampled in the essential space were used as input data vectors for SOM training, then complete linkage clustering was performed on the SOM prototype vectors. A specific protocol to optimize a SOM for structural ensembles was proposed: the optimal SOM was selected by means of a Taguchi experimental design plan applied to different data sets, and the optimal sampling rate of the MD trajectory was selected. The proposed two-level approach was applied to single trajectories of the SH3 domain independently as well as to groups of them at the same time. The results demonstrated the potential of this approach in the analysis of large ensembles of molecular structures: the possibility of producing a topological mapping of the conformational space in a simple 2D visualisation, as well as of effectively highlighting differences in the conformational dynamics directly related to biological functions.</p> <p>Conclusions</p> <p>The use of a two-level approach combining SOMs and hierarchical clustering for conformational analysis of structural ensembles of proteins was proposed. It can easily be extended to other study cases and to conformational ensembles from other sources.</p