Synthesis and unexpected binding of monofluorinated N,N'-diacetylchitobiose and LacdiNAc to wheat germ agglutinin.

Fluorination of carbohydrate ligands of lectins is a useful approach to examine their binding profile, improve their metabolic stability and lipophilicity, and convert them into 19F NMR-active probes. However, monofluorination of monovalent carbohydrate ligands often leads to a decreased or completely lost affinity. By chemical glycosylation, we synthesized the full series of methyl ß-glycosides of N,N'-diacetylchitobiose (GlcNAcß(1-4)GlcNAcß1-OMe) and LacdiNAc (GalNAcß(1-4)GlcNAcß1-OMe) systematically monofluorinated at all hydroxyl positions. A competitive enzyme-linked lectin assay revealed that the fluorination at the 6'-position of chitobioside resulted in an unprecedented increase in affinity to wheat germ agglutinin (WGA) by one order of magnitude. For the first time, we have characterized the binding profile of a previously underexplored WGA ligand LacdiNAc. Surprisingly, 4'-fluoro-LacdiNAc bound WGA even stronger than unmodified LacdiNAc. These observations were interpreted using molecular dynamic calculations along with STD and transferred NOESY NMR techniques, which gave evidence for the strengthening of CH/π interactions after deoxyfluorination of the side chain of the non-reducing GlcNAc. These results highlight the potential of fluorinated glycomimetics as high-affinity ligands of lectins and 19F NMR-active probes.

Uncertainties of predictions from temperature replica exchange simulations.

Parallel tempering molecular dynamics simulation, also known as temperature replica exchange simulation, is a popular enhanced sampling method used to study biomolecular systems. This method makes it possible to calculate the free energy differences between states of the system for a series of temperatures. We developed a method to easily calculate the errors (standard errors or confidence intervals) of these predictions using a modified version of our recently introduced JumpCount method. The number of transitions between states (e.g., protein folding events) is counted for each temperature. This number of transitions, together with the temperature, fully determines the value of the standard error or the confidence interval of the free energy difference. We also address the issue of convergence in the situation where all replicas start from one state by developing an estimator of the equilibrium constant from simulations that are not fully equilibrated. The prerequisite of the method is the Markovianity of the process studied.

Deep learning in systems medicine.

Systems medicine (SM) has emerged as a powerful tool for studying the human body at the systems level with the aim of improving our understanding, prevention and treatment of complex diseases. Being able to automatically extract relevant features needed for a given task from high-dimensional, heterogeneous data, deep learning (DL) holds great promise in this endeavour. This review paper addresses the main developments of DL algorithms and a set of general topics where DL is decisive, namely, within the SM landscape. It discusses how DL can be applied to SM with an emphasis on the applications to predictive, preventive and precision medicine. Several key challenges have been highlighted including delivering clinical impact and improving interpretability. We used some prototypical examples to highlight the relevance and significance of the adoption of DL in SM, one of them is involving the creation of a model for personalized Parkinson's disease. The review offers valuable insights and informs the research in DL and SM.

Property Map Collective Variable as a Useful Tool for a Force Field Correction.

The accuracy of biomolecular simulations depends on the accuracy of an empirical molecular mechanics potential known as a force field: a set of parameters and expressions to estimate the potential from atomic coordinates. Accurate parametrization of force fields for small organic molecules is a challenge due to their high diversity. One of the possible approaches is to apply a correction to the existing force fields. Here, we propose an approach to estimate the density functional theory (DFT)-derived force field correction which is calculated during the run of molecular dynamics without significantly affecting its speed. Using the formula known as a property map collective variable, we approximate the force field correction by a weighted average of this force field correction calculated only for a small series of reference structures. To validate this method, we used seven AMBER force fields, and we show how it is possible to convert one force field to behave like the other one. We also present the force field correction for the important anticancer drug Imatinib as a use case example. Our method appears to be suitable for adjusting the force field for general drug-like molecules. We provide a pipeline that generates the correction; this pipeline is available at https://pmcvff-correction.cerit-sc.cz/.

A framework to assess the quality and impact of bioinformatics training across ELIXIR.

ELIXIR is a pan-European intergovernmental organisation for life science that aims to coordinate bioinformatics resources in a single infrastructure across Europe; bioinformatics training is central to its strategy, which aims to develop a training community that spans all ELIXIR member states. In an evidence-based approach for strengthening bioinformatics training programmes across Europe, the ELIXIR Training Platform, led by the ELIXIR EXCELERATE Quality and Impact Assessment Subtask in collaboration with the ELIXIR Training Coordinators Group, has implemented an assessment strategy to measure quality and impact of its entire training portfolio. Here, we present ELIXIR's framework for assessing training quality and impact, which includes the following: specifying assessment aims, determining what data to collect in order to address these aims, and our strategy for centralised data collection to allow for ELIXIR-wide analyses. In addition, we present an overview of the ELIXIR training data collected over the past 4 years. We highlight the importance of a coordinated and consistent data collection approach and the relevance of defining specific metrics and answer scales for consortium-wide analyses as well as for comparison of data across iterations of the same course.

Arabinogalactan-like Glycoproteins from Ulva lactuca (Chlorophyta) Show Unique Features Compared to Land Plants AGPs.

Arabinogalactan proteins (AGPs) encompass a diverse group of plant cell wall proteoglycans, which play an essential role in plant development, signaling, plant-microbe interactions, and many others. Although they are widely distributed throughout the plant kingdom and extensively studied, they remain largely unexplored in the lower plants, especially in seaweeds. Ulva species have high economic potential since various applications were previously described including bioremediation, biofuel production, and as a source of bioactive compounds. This article presents the first experimental confirmation of AGP-like glycoproteins in Ulva species and provides a simple extraction protocol of Ulva lactuca AGP-like glycoproteins, their partial characterization and unique comparison to scarcely described Solanum lycopersicum AGPs. The reactivity with primary anti-AGP antibodies as well as Yariv reagent showed a great variety between Ulva lactuca and Solanum lycopersicum AGP-like glycoproteins. While the amino acid analysis of the AGP-like glycoproteins purified by the ß-d-glucosyl Yariv reagent showed a similarity between algal and land plant AGP-like glycoproteins, neutral saccharide analysis revealed unique glycosylation of the Ulva lactuca AGP-like glycoproteins. Surprisingly, arabinose and galactose were not the most prevalent monosaccharides and the most outstanding was the presence of 3-O-methyl-hexose, which has never been described in the AGPs. The exceptional structure of the Ulva lactuca AGP-like glycoproteins implies a specialized adaptation to the marine environment and might bring new insight into the evolution of the plant cell wall.

Na+/K+-ATPase Revisited: On Its Mechanism of Action, Role in Cancer, and Activity Modulation.

Maintenance of Na+ and K+ gradients across the cell plasma membrane is an essential process for mammalian cell survival. An enzyme responsible for this process, sodium-potassium ATPase (NKA), has been currently extensively studied as a potential anticancer target, especially in lung cancer and glioblastoma. To date, many NKA inhibitors, mainly of natural origin from the family of cardiac steroids (CSs), have been reported and extensively studied. Interestingly, upon CS binding to NKA at nontoxic doses, the role of NKA as a receptor is activated and intracellular signaling is triggered, upon which cancer cell death occurs, which lies in the expression of different NKA isoforms than in healthy cells. Two major CSs, digoxin and digitoxin, originally used for the treatment of cardiac arrhythmias, are also being tested for another indication-cancer. Such drug repositioning has a big advantage in smoother approval processes. Besides this, novel CS derivatives with improved performance are being developed and evaluated in combination therapy. This article deals with the NKA structure, mechanism of action, activity modulation, and its most important inhibitors, some of which could serve not only as a powerful tool to combat cancer, but also help to decipher the so-far poorly understood NKA regulation.

Regioselective 3-O-Substitution of Unprotected Thiodigalactosides: Direct Route to Galectin Inhibitors.

The synthesis of tailored bioactive carbohydrates usually comprises challenging (de)protection steps, which lowers synthetic yields and increases time demands. We present here a regioselective single-step introduction of benzylic substituents at 3-hydroxy groups of ß-d-galactopyranosyl-(1→1)-thio-ß-d-galactopyranoside (TDG) employing dibutyltin oxide in good yields. These glycomimetics act as inhibitors of galectins-human lectins, which are biomedically attractive targets for therapeutic inhibition in, for example, cancerogenesis. The affinity of the prepared glycomimetics to galectin-1 and galectin-3 was studied in enzyme-linked immunosorbent (ELISA)-type assays and their potential to inhibit galectin binding on the cell surface was shown. We used our original in vivo biotinylated galectin constructs for easy detection by flow cytometry. The results of the biological experiments were compared with data from molecular modeling with both galectins. The present work reveals a facile and elegant synthetic route for the preparation of TDG-derived glycomimetics that exhibit differing selectivity and affinity to galectins depending on the choice of 3-O-substitution.

Active site complementation and hexameric arrangement in the GH family 29; a structure-function study of α-l-fucosidase isoenzyme 1 from Paenibacillus thiaminolyticus.

α-l-Fucosidase isoenzyme 1 from bacterium Paenibacillus thiaminolyticus is a member of the glycoside hydrolase family GH29 capable of cleaving l-fucose from nonreducing termini of oligosaccharides and glycoconjugates. Here we present the first crystal structure of this protein revealing a novel quaternary state within this family. The protein is in a unique hexameric assembly revealing the first observed case of active site complementation by a residue from an adjacent monomer in this family. Mutation of the complementing tryptophan residue caused changes in the catalytic properties including a shift of the pH optimum, a change of affinity to an artificial chromogenic substrate and a decreased reaction rate for a natural substrate. The wild-type enzyme was active on most of the tested naturally occurring oligosaccharides and capable of transglycosylation on a variety of acceptor molecules, including saccharides, alcohols or chromogenic substrates. Mutation of the complementing residue changed neither substrate specificity nor the preference for the type of transglycosylation acceptor molecule; however, the yields of the reactions were lower in both cases. Maltose molecules bound to the enzyme in the crystal structure identified surface carbohydrate-binding sites, possibly participating in binding of larger oligosaccharides.

PEGylated Purpurin 18 with Improved Solubility: Potent Compounds for Photodynamic Therapy of Cancer.

Purpurin 18 derivatives with a polyethylene glycol (PEG) linker were synthesized as novel photosensitizers (PSs) with the goal of using them in photodynamic therapy (PDT) for cancer. These compounds, derived from a second-generation PS, exhibit absorption at long wavelengths; considerable singlet oxygen generation and, in contrast to purpurin 18, have higher hydrophilicity due to decreased logP. Together, these properties make them potentially ideal PSs. To verify this, we screened the developed compounds for cell uptake, intracellular localization, antitumor activity and induced cell death type. All of the tested compounds were taken up into cancer cells of various origin and localized in organelles known to be important PDT targets, specifically, mitochondria and the endoplasmic reticulum. The incorporation of a zinc ion and PEGylation significantly enhanced the photosensitizing efficacy, decreasing IC50 (half maximal inhibitory compound concentration) in HeLa cells by up to 170 times compared with the parental purpurin 18. At effective PDT concentrations, the predominant type of induced cell death was apoptosis. Overall, our results show that the PEGylated derivatives presented have significant potential as novel PSs with substantially augmented phototoxicity for application in the PDT of cervical, prostate, pancreatic and breast cancer.

Archangelolide: A sesquiterpene lactone with immunobiological potential from Laserpitium archangelica.

Sesquiterpene lactones are secondary plant metabolites with sundry biological effects. In plants, they are synthesized, among others, for pesticidal and antimicrobial effects. Two such compounds, archangelolide and trilobolide of the guaianolide type, are structurally similar to the well-known and clinically tested lactone thapsigargin. While trilobolide has already been studied by us and others, there are only scarce reports on the biological activity of archangelolide. Here we present the preparation of its fluorescent derivative based on a dansyl moiety using azide-alkyne Huisgen cycloaddition having obtained the two sesquiterpene lactones from the seeds of Laserpitium archangelica Wulfen using supercritical CO2 extraction. We show that dansyl-archangelolide localizes in the endoplasmic reticulum of living cells similarly to trilobolide; localization in mitochondria was also detected. This led us to a more detailed study of the anticancer potential of archangelolide. Interestingly, we found that neither archangelolide nor its dansyl conjugate did exhibit cytotoxic effects in contrast to the structurally closely related counterparts trilobolide and thapsigargin. We explain this observation by a molecular dynamics simulation, in which, in contrast to trilobolide, archangelolide did not bind into the sarco/endoplasmic reticular calcium ATPase cavity utilized by thapsigargin. Last, but not least, archangelolide exhibited anti-inflammatory activity, which makes it promising compound for medicinal purposes.

The dehydration stress of couch grass is associated with its lipid metabolism, the induction of transporters and the re-programming of development coordinated by ABA.

BACKGROUND: The wild relatives of crop species represent a potentially valuable source of novel genetic variation, particularly in the context of improving the crop's level of tolerance to abiotic stress. The mechanistic basis of these tolerances remains largely unexplored. Here, the focus was to characterize the transcriptomic response of the nodes (meristematic tissue) of couch grass (a relative of barley) to dehydration stress, and to compare it to that of the barley crown formed by both a drought tolerant and a drought sensitive barley cultivar. RESULTS: Many of the genes up-regulated in the nodes by the stress were homologs of genes known to be mediated by abscisic acid during the response to drought, or were linked to either development or lipid metabolism. Transporters also featured prominently, as did genes acting on root architecture. The resilience of the couch grass node arise from both their capacity to develop an altered, more effective root architecture, but also from their formation of a lipid barrier on their outer surface and their ability to modify both their lipid metabolism and transporter activity when challenged by dehydration stress. CONCLUSIONS: Our analysis revealed the nature of dehydration stress response in couch grass. We suggested the tolerance is associated with lipid metabolism, the induction of transporters and the re-programming of development coordinated by ABA. We also proved the applicability of barley microarray for couch grass stress-response analysis.

Poly-N-Acetyllactosamine Neo-Glycoproteins as Nanomolar Ligands of Human Galectin-3: Binding Kinetics and Modeling.

Galectin-3 (Gal-3) is recognized as a prognostic marker in several cancer types. Its involvement in tumor development and proliferation makes this lectin a promising target for early cancer diagnosis and anti-cancer therapies. Gal-3 recognizes poly-N-acetyllactosamine (LacNAc)-based carbohydrate motifs of glycoproteins and glycolipids with a high specificity for internal LacNAc epitopes. This study analyzes the mode and kinetics of binding of Gal-3 to a series of multivalent neo-glycoproteins presenting complex poly-LacNAc-based oligosaccharide ligands on a scaffold of bovine serum albumin. These neo-glycoproteins rank among the strongest Gal-3 ligands reported, with Kd reaching sub-nanomolar values as determined by surface plasmon resonance. Significant differences in the binding kinetics were observed within the ligand series, showing the tetrasaccharide capped with N,N'-diacetyllactosamine (LacdiNAc) as the strongest ligand of Gal-3 in this study. A molecular model of the Gal-3 carbohydrate recognition domain with docked oligosaccharide ligands is presented that shows the relations in the binding site at the molecular level. The neo-glycoproteins presented herein may be applied for selective recognition of Gal-3 both on the cell surface and in blood serum.

Reducing the number of mean-square deviation calculations with floating close structure in metadynamics.

Metadynamics is an important collective-coordinate-based enhanced sampling simulation method. Its performance depends significantly on the capability of collective coordinates to describe the studied molecular processes. Collective coordinates based on comparison with reference landmark structures can be used to enhance sampling in highly complex systems; however, they may slow down simulations due to high number of structure-structure distance (e.g., mean-square deviation) calculations. Here we introduce an approximation of root-mean-square or mean-square deviation that significantly reduces numbers of computationally expensive operations. We evaluate its accuracy and theoretical performance gain with metadynamics simulations on two molecular systems.

Multisystem altruistic metadynamics-Well-tempered variant.

Metadynamics method has been widely used to enhance sampling in molecular simulations. Its original form suffers two major drawbacks, poor convergence in complex (especially biomolecular) systems and its serial nature. The first drawback has been addressed by introduction of a convergent variant known as well-tempered metadynamics. The second was addressed by introduction of a parallel multisystem metadynamics referred to as altruistic metadynamics. Here, we combine both approaches into well-tempered altruistic metadynamics. We provide mathematical arguments and trial simulations to show that it accurately predicts free energy surfaces.

CH/π Interactions in Carbohydrate Recognition.

Many carbohydrate-binding proteins contain aromatic amino acid residues in their binding sites. These residues interact with carbohydrates in a stacking geometry via CH/π interactions. These interactions can be found in carbohydrate-binding proteins, including lectins, enzymes and carbohydrate transporters. Besides this, many non-protein aromatic molecules (natural as well as artificial) can bind saccharides using these interactions. Recent computational and experimental studies have shown that carbohydrate-aromatic CH/π interactions are dispersion interactions, tuned by electrostatics and partially stabilized by a hydrophobic effect in solvated systems.

Biocatalyzed synthesis of difuranosides and their ability to trigger production of TNF-α.

Transglycosylation reactions biocatalyzed by the native arabinofuranosidase Araf51 and using d-galactosyl, d-fucosyl and 6-deoxy-6-fluoro-D-galactosyl derivatives as donors and acceptors provided di-to pentahexofuranosides. The immunostimulatory potency of these compounds, and more especially their ability to induce production of TNF-α, was evaluated on the murine macrophage cell line, Raw 264.7. The results obtained showed concentration-dependent and most importantly, structure-dependent responses. Interestingly, oligoarabinofuranosides belonging to the oligopentafuranoside family displayed concentration-, chain length and aglycon-dependent bioactivities irrespective of their fine chemical variations. Thus, neo-oligofuranosides in D-Galf series, as well as their D-Fucf and 6-fluorinated counterparts are indeed potential sources of immunostimulating agents.

Alpha-L-fucosidase isoenzyme iso2 from Paenibacillus thiaminolyticus.

BACKGROUND: α-L-Fucosidases are enzymes involved in metabolism of α-L-fucosylated molecules, compounds with a fundamental role in different life essential processes including immune response, fertilization and development, but also in some serious pathological events. According to the CAZy database, these enzymes belong to families 29 and 95. Some of them are also reported to be able to catalyze transglycosylation reactions, during which α-L-fucosylated molecules, representing compounds of interest especially for pharmaceutical industry, are formed. METHODS: Activity-based screening of a genomic library was used to isolate the gene encoding a novel α-L-fucosidase. The enzyme was expressed in E.coli and affinity chromatography was used for purification of His-tagged α-L-fucosidase. Standard activity assay was used for enzyme characterization. Thin layer chromatography and mass spectrometry were used for transglycosylation reactions evaluation. RESULTS: Using a genomic library of Paenibacillus thiaminolyticus, constructed in E.coli DH5α cells, nucleotide sequence of a new α-L-fucosidase isoenzyme was determined and submitted to the EMBL database (HE654122). However, no similarity with enzymes from CAZy database families 29 and 95 was detected. This enzyme was produced in form of histidine-tagged protein in E.coli BL21 (DE3) cells and purified by metaloaffinity chromatography. Hydrolytic and transglycosylation abilities of α-L-fucosidase iso2 were tested using different acceptor molecules. CONCLUSIONS: In this study, new enzyme α-L-fucosidase iso2 originating from Paenibacillus thiaminolyticus was described and prepared in recombinant form and its hydrolytic and transglycosylation properties were characterized. As a very low amino acid sequence similarity with known α-L-fucosidases was found, following study could be important for different biochemical disciplines involving molecular modelling.

Role of Mason-Pfizer monkey virus CA-NC spacer peptide-like domain in assembly of immature particles.

UNLABELLED: The hexameric lattice of an immature retroviral particle consists of Gag polyprotein, which is the precursor of all viral structural proteins. Lentiviral and alpharetroviral Gag proteins contain a peptide sequence called the spacer peptide (SP), which is localized between the capsid (CA) and nucleocapsid (NC) domains. SP plays a critical role in intermolecular interactions during the assembly of immature particles of several retroviruses. Published models of supramolecular structures of immature particles suggest that in lentiviruses and alpharetroviruses, SP adopts a rod-like six-helix bundle organization. In contrast, Mason-Pfizer monkey virus (M-PMV), a betaretrovirus that assembles in the cytoplasm, does not contain a distinct SP sequence, and the CA-NC connecting region is not organized into a clear rod-like structure. Nevertheless, the CA-NC junction comprises a sequence critical for assembly of immature M-PMV particles. In the present work, we characterized this region, called the SP-like domain, in detail. We provide biochemical data confirming the critical role of the M-PMV SP-like domain in immature particle assembly, release, processing, and infectivity. Circular dichroism spectroscopy revealed that, in contrast to the SP regions of other retroviruses, a short SP-like domain-derived peptide (SPLP) does not form a purely helical structure in aqueous or helix-promoting solution. Using 8-Å cryo-electron microscopy density maps of immature M-PMV particles, we prepared computational models of the SP-like domain and indicate the structural features required for M-PMV immature particle assembly. IMPORTANCE: Retroviruses such as HIV-1 are of great medical importance. Using Mason-Pfizer monkey virus (M-PMV) as a model retrovirus, we provide biochemical and structural data confirming the general relevance of a short segment of the structural polyprotein Gag for retrovirus assembly and infectivity. Although this segment is critical for assembly of immature particles of lentiviruses, alpharetroviruses, and betaretroviruses, the organization of this domain is strikingly different. A previously published electron microscopic structure of an immature M-PMV particle allowed us to model this important region into the electron density map. The data presented here help explain the different packing of the Gag segments of various retroviruses, such as HIV, Rous sarcoma virus (RSV), and M-PMV. Such knowledge contributes to understanding the importance of this region and its structural flexibility among retroviral species. The region might play a key role in Gag-Gag interactions, leading to different morphological pathways of immature particle assembly.

Nonlinear vs. linear biasing in Trp-cage folding simulations.

Biased simulations have great potential for the study of slow processes, including protein folding. Atomic motions in molecules are nonlinear, which suggests that simulations with enhanced sampling of collective motions traced by nonlinear dimensionality reduction methods may perform better than linear ones. In this study, we compare an unbiased folding simulation of the Trp-cage miniprotein with metadynamics simulations using both linear (principle component analysis) and nonlinear (Isomap) low dimensional embeddings as collective variables. Folding of the mini-protein was successfully simulated in 200 ns simulation with linear biasing and non-linear motion biasing. The folded state was correctly predicted as the free energy minimum in both simulations. We found that the advantage of linear motion biasing is that it can sample a larger conformational space, whereas the advantage of nonlinear motion biasing lies in slightly better resolution of the resulting free energy surface. In terms of sampling efficiency, both methods are comparable.