The Sphinx and the egg: Evolutionary enigmas of the (glyco)sphingolipid biosynthetic pathway.

In eukaryotes, the de novo synthesis of sphingolipids (SLs) consists of multiple sequential steps which are compartmentalized between the endoplasmic reticulum and the Golgi apparatus. Studies over many decades have identified the enzymes in the pathway, their localization, topology and an array of regulatory mechanisms. However, little is known about the evolutionary forces that underly the generation of this complex pathway or of its anteome, i.e., the metabolic pathways that converge on the SL biosynthetic pathway and are essential for its activity. After briefly describing the pathway, we discuss the mechanisms by which the enzymes of the SL biosynthetic pathway are targeted to their different subcellular locations, how the pathway per se may have evolved, including its compartmentalization, and the relationship of the pathway to eukaryogenesis. We discuss the circular interdependence of the evolution of the SL pathway, and comment on whether current Darwinian evolutionary models are able to provide genuine mechanistic insight into how the pathway came into being.

Computational design and molecular dynamics simulations suggest the mode of substrate binding in ceramide synthases.

Until now, membrane-protein stabilization has relied on iterations of mutations and screening. We now validate a one-step algorithm, mPROSS, for stabilizing membrane proteins directly from an AlphaFold2 model structure. Applied to the lipid-generating enzyme, ceramide synthase, 37 designed mutations lead to a more stable form of human CerS2. Together with molecular dynamics simulations, we propose a pathway by which substrates might be delivered to the ceramide synthases.

Inhibiting a promiscuous GPCR: iterative discovery of bitter taste receptor ligands.

The human GPCR family comprises circa 800 members, activated by hundreds of thousands of compounds. Bitter taste receptors, TAS2Rs, constitute a large and distinct subfamily, expressed orally and extra-orally and involved in physiological and pathological conditions. TAS2R14 is the most promiscuous member, with over 150 agonists and 3 antagonists known prior to this study. Due to the scarcity of inhibitors and to the importance of chemical probes for exploring TAS2R14 functions, we aimed to discover new ligands for this receptor, with emphasis on antagonists. To cope with the lack of experimental structure of the receptor, we used a mixed experimental/computational methodology which iteratively improved the performance of the predicted structure. The increasing number of active compounds, obtained here through experimental screening of FDA-approved drug library, and through chemically synthesized flufenamic acid derivatives, enabled the refinement of the binding pocket, which in turn improved the structure-based virtual screening reliability. This mixed approach led to the identification of 10 new antagonists and 200 new agonists of TAS2R14, illustrating the untapped potential of rigorous medicinal chemistry for TAS2Rs. 9% of the ~ 1800 pharmaceutical drugs here tested activate TAS2R14, nine of them at sub-micromolar concentrations. The iterative framework suggested residues involved in the activation process, is suitable for expanding bitter and bitter-masking chemical space, and is applicable to other promiscuous GPCRs lacking experimental structures.

Elevation of gangliosides in four brain regions from Parkinson's disease patients with a GBA mutation.

A number of genetic risk factors have been identified over the past decade for Parkinson's Disease (PD), with variants in GBA prominent among them. GBA encodes the lysosomal enzyme that degrades the glycosphingolipid, glucosylceramide (GlcCer), with the activity of this enzyme defective in Gaucher disease. Based on the ill-defined relationship between glycosphingolipid metabolism and PD, we now analyze levels of various lipids by liquid chromatography/electrospray ionization-tandem mass spectrometry in four brain regions from age- and sex-matched patient samples, including idiopathic PD, PD patients with a GBA mutation and compare both to control brains (n = 21 for each group) obtained from individuals who died from a cause unrelated to PD. Of all the glycerolipids, sterols, and (glyco)sphingolipids (251 lipids in total), the only lipid class which showed significant differences were the gangliosides (sialic acid-containing complex glycosphingolipids), which were elevated in 3 of the 4 PD-GBA brain regions. There was no clear correlation between levels of individual gangliosides and the genetic variant in Gaucher disease [9 samples of severe (neuronopathic), 4 samples of mild (non-neuronopathic) GBA variants, and 8 samples with low pathogenicity variants which have a higher risk for development of PD]. Most brain regions, i.e. occipital cortex, cingulate gyrus, and striatum, did not show a statistically significant elevation of GlcCer in PD-GBA. Only one region, the middle temporal gyrus, showed a small, but significant elevation in GlcCer concentration in PD-GBA. We conclude that changes in ganglioside, but not in GlcCer levels, may contribute to the association between PD and GBA mutations.

The sphingolipid anteome: implications for evolution of the sphingolipid metabolic pathway.

Modern cell membranes contain a bewildering complexity of lipids, among them sphingolipids (SLs). Advances in mass spectrometry have led to the realization that the number and combinatorial complexity of lipids, including SLs, is much greater than previously appreciated. SLs are generated de novo by four enzymes, namely serine palmitoyltransferase, 3-ketodihydrosphingosine reductase, ceramide synthase and dihydroceramide Δ4-desaturase 1. Some of these enzymes depend on the availability of specific substrates and cofactors, which are themselves supplied by other complex metabolic pathways. The evolution of these four enzymes is poorly understood and likely depends on the co-evolution of the metabolic pathways that supply the other essential reaction components. Here, we introduce the concept of the 'anteome', from the Latin ante ('before') to describe the network of metabolic ('omic') pathways that must have converged in order for these pathways to co-evolve and permit SL synthesis. We also suggest that the current origin of life and evolutionary models lack appropriate experimental support to explain the appearance of this complex metabolic pathway and its anteome.

Combined presentation and immunogenicity analysis reveals a recurrent RAS.Q61K neoantigen in melanoma.

Neoantigens are now recognized drivers of the antitumor immune response. Recurrent neoantigens, shared among groups of patients, have thus become increasingly coveted therapeutic targets. Here, we report on the data-driven identification of a robustly presented, immunogenic neoantigen that is derived from the combination of HLA-A*01:01 and RAS.Q61K. Analysis of large patient cohorts indicated that this combination applies to 3% of patients with melanoma. Using HLA peptidomics, we were able to demonstrate robust endogenous presentation of the neoantigen in 10 tumor samples. We detected specific reactivity to the mutated peptide within tumor-infiltrating lymphocytes (TILs) from 2 unrelated patients, thus confirming its natural immunogenicity. We further investigated the neoantigen-specific clones and their T cell receptors (TCRs) via a combination of TCR sequencing, TCR overexpression, functional assays, and single-cell transcriptomics. Our analysis revealed a diverse repertoire of neoantigen-specific clones with both intra- and interpatient TCR similarities. Moreover, 1 dominant clone proved to cross-react with the highly prevalent RAS.Q61R variant. Transcriptome analysis revealed a high association of TCR clones with specific T cell phenotypes in response to cognate melanoma, with neoantigen-specific cells showing an activated and dysfunctional phenotype. Identification of recurrent neoantigens and their reactive TCRs can promote "off-the-shelf" precision immunotherapies, alleviating limitations of personalized treatments.

'Dual-reference' method for high-precision infrared measurement of leaf surface temperature under field conditions.

Temperature is a key control over biological activities from the cellular to the ecosystem scales. However, direct, high-precision measurements of surface temperature of small objects, such as leaves, under field conditions with large variations in ambient conditions remain rare. Contact methods, such as thermocouples, are prone to large errors. The use of noncontact remote-sensing methods, such as thermal infrared measurements, provides an ideal solution, but their accuracy has been low (c. 2°C) owing to the necessity for corrections for material emissivity and fluctuations in background radiation Lbg . A novel 'dual-reference' method was developed to increase the accuracy of infrared needle-leaf surface temperature measurements in the field. It accounts for variations in Lbg and corrects for the systematic camera offset using two reference plates. We accurately captured surface temperature and leaf-to-air temperature differences of needle-leaves in a forest ecosystem with large diurnal and seasonal temperature fluctuations with an uncertainty of ± 0.23°C and ± 0.28°C, respectively. Routine high-precision leaf temperature measurements even under harsh field conditions, such as demonstrated here, opens the way for investigating a wide range of leaf-scale processes and their dynamics.

The role of the 'sphingoid motif' in shaping the molecular interactions of sphingolipids in biomembranes.

Sphingolipids can be differentiated from other membrane lipids by the distinctive chemistry of the sphingoid long chain base (LCB), which is generated by the condensation of an amino acid (normally but not always serine) and a fatty acyl CoA (normally palmitoyl CoA) by the pyridoxal phosphate-dependent enzyme, serine palmitoyl transferase (SPT). The first five carbon atoms of the sphingoid LCB, herein defined as the 'sphingoid motif', are largely responsible for the unique chemical and biophysical properties of sphingolipids since they can undergo a relatively large number (compared to other lipid species) of molecular interactions with other membrane lipids, via hydrogen-bonding, charge-pairing, hydrophobic and van der Waals interactions. These interactions are responsible, for instance, for the association of sphingolipids with cholesterol in the membrane lipid bilayer. Here, we discuss some of the unique properties of this sphingoid motif, and in addition to outlining how this structural motif drives intra-bilayer interactions, discuss the atomic details of the interactions with two critical players in the biosynthetic pathway, namely SPT, and the ceramide transport protein, CERT. In the former, the selectivity of sphingolipid synthesis relies on a hydrogen bond interaction between Lys379 of SPTLC2 and the l-serine sidechain hydroxyl moiety. In the latter, the entire sphingoid motif is stereoselectively recognized by a hydrogen-bonding network involving all three sphingoid motif heteroatoms. The remarkable selectivity of these interactions, and the subtle means by which these interactions are modified and regulated in eukaryotic cells raises a number of challenging questions about the generation of these proteins, and of their interactions with the sphingoid motif in evolutionary history.

The fine-tuning of cell membrane lipid bilayers accentuates their compositional complexity.

Cell membranes are now emerging as finely tuned molecular systems, signifying that re-evaluation of our understanding of their structure is essential. Although the idea that cell membrane lipid bilayers do little more than give shape and form to cells and limit diffusion between cells and their environment is totally passé, the structural, compositional, and functional complexity of lipid bilayers often catches cell and molecular biologists by surprise. Models of lipid bilayer structure have developed considerably since the heyday of the fluid mosaic model, principally by the discovery of the restricted diffusion of membrane proteins and lipids within the plane of the bilayer. In reviewing this field, we now suggest that further refinement of current models is necessary and propose that describing lipid bilayers as "finely-tuned molecular assemblies" best portrays their complexity and function. Also see the video abstract here: https://www.youtube.com/watch?v=ddkP-QRZTl8.

Bitter-Tasting Amino Acids l-Arginine and l-Isoleucine Differentially Regulate Proton Secretion via T2R1 Signaling in Human Parietal Cells in Culture.

This study aimed at identifying whether the bitter-tasting amino acids l-arginine (l-ARG) and l-isoleucine (l-ILE) differentially regulate mechanisms of gastric acid secretion in human parietal cells (HGT-1 cells) via activation of bitter taste sensing receptors (T2Rs). In a first set of experiments, involvement of T2Rs in l-ARG and l-ILE-modulated proton secretion was demonstrated by co-treatment of HGT-1 cells with T2R antagonists. Subsequent whole genome screenings by means of cDNA arrays revealed T2R1 as a prominent target for both amino acids. Next, the functional role of T2R1 was verified by means of a T2R1 CRISPR-Cas9 knock-out approach. Here, the effect of l-ARG on proton secretion decreased by 65.7 ± 21.9% and the effect of l-ILE increased by 93.2 ± 24.1% in HGT-1 T2R1 ko versus HGT-1 wt cells (p < 0.05). Overall, our results indicate differential effects of l-ARG and l-ILE on proton secretion in HGT-1 cells and our molecular docking studies predict distinct binding for these amino acids in the binding site of T2R1. Further studies will elucidate whether the mechanism of differential effects involves structure-specific ligand-biased signaling of T2R1 or additional cellular targets.

3,3'-Disubstituted 5,5'-Bi(1,2,4-triazine) Derivatives with Potent in Vitro and in Vivo Antimalarial Activity.

A series of 3,3'-disubstituted 5,5'-bi(1,2,4-triazine) derivatives was synthesized and screened against the erythrocytic stage of Plasmodium falciparum 3D7 line. The most potent dimer, 6k, with an IC50 (50% inhibitory concentration) of 0.008 µM, had high in vitro potency against P. falciparum lines resistant to chloroquine (W2, IC50 = 0.0047 ± 0.0011 µM) and artemisinin (MRA1240, IC50 = 0.0086 ± 0.0010 µM). Excellent ex vivo potency of 6k was shown against clinical field isolates of both P. falciparum (IC50 = 0.022-0.034 µM) and Plasmodium vivax (IC50 = 0.0093-0.031 µM) from the blood of outpatients with uncomplicated malaria. Despite 6k being cleared relatively rapidly in mice, it suppressed parasitemia in the Peters 4-day test, with a mean ED50 value (50% effective dose) of 1.47 mg kg-1 day-1 following oral administration. The disubstituted triazine dimer 6k represents a new class of orally available antimalarial compounds of considerable interest for further development.

Virtual Screening Against Carbohydrate-Binding Proteins: Evaluation and Application to Bacterial Burkholderia ambifaria Lectin.

Bacterial adhesion to human epithelia via lectins constitutes a therapeutic opportunity to prevent infection. Specifically, BambL (the lectin from Burkholderia ambifaria) is implicated in cystic fibrosis, where lectin-mediated bacterial adhesion to fucosylated lung epithelia is suspected to play an important role. We employed structure-based virtual screening to identify inhibitors of BambL-saccharide interaction with potential therapeutic value. To enable such discovery, a virtual screening protocol was iteratively developed via 194 retrospective screening protocols against 4 bacterial lectins (BambL, BC2L-A, FimH, and LecA) with known ligands. Specific attention was given to the rigorous evaluation of retrospective screening, including calculation of analytical errors for enrichment metrics. The developed virtual screening workflow used crystallographic constraints, pharmacophore filters, and a final manual selection step. The protocol was applied to BambL, predicting 15 active compounds from virtual libraries of approximately 7 million compounds. Experimental validation using fluorescence polarization confirmed micromolar inhibitory activity for two compounds, which were further characterized by isothermal titration calorimetry and surface plasmon resonance. Subsequent testing against LecB from Pseudomonas aeruginosa demonstrated binding specificity of one of the hit compounds. This report demonstrates the utility of virtual screening protocols, integrating ligand-based pharmacophore filtering and structure-based constraints, in the search for bacterial lectin inhibitors.

Molecular Simulations of Carbohydrates with a Fucose-Binding Burkholderia ambifaria Lectin Suggest Modulation by Surface Residues Outside the Fucose-Binding Pocket.

Burkholderia ambifaria is an opportunistic respiratory pathogen belonging to the Burkholderia cepacia complex, a collection of species responsible for the rapidly fatal cepacia syndrome in cystic fibrosis patients. A fucose-binding lectin identified in the B. ambifaria genome, BambL, is able to adhere to lung tissue, and may play a role in respiratory infection. X-ray crystallography has revealed the bound complex structures for four fucosylated human blood group epitopes (blood group B, H type 1, H type 2, and Lex determinants). The present study employed computational approaches, including docking and molecular dynamics (MD), to extend the structural analysis of BambL-oligosaccharide complexes to include four additional blood group saccharides (A, Lea, Leb, and Ley) and a library of blood-group-related carbohydrates. Carbohydrate recognition is dominated by interactions with fucose via a hydrogen-bonding network involving Arg15, Glu26, Ala38, and Trp79 and a stacking interaction with Trp74. Additional hydrogen bonds to non-fucose residues are formed with Asp30, Tyr35, Thr36, and Trp74. BambL recognition is dominated by interactions with fucose, but also features interactions with other parts of the ligands that may modulate specificity or affinity. The detailed computational characterization of the BambL carbohydrate-binding site provides guidelines for the future design of lectin inhibitors.

Structure and Characterisation of a Key Epitope in the Conserved C-Terminal Domain of the Malaria Vaccine Candidate MSP2.

Merozoite surface protein 2 (MSP2) is an intrinsically disordered antigen that is abundant on the surface of the malaria parasite Plasmodium falciparum. The two allelic families of MSP2, 3D7 and FC27, differ in their central variable regions, which are flanked by highly conserved C-terminal and N-terminal regions. In a vaccine trial, full-length 3D7 MSP2 induced a strain-specific protective immune response despite the detectable presence of conserved region antibodies. This work focuses on the conserved C-terminal region of MSP2, which includes the only disulphide bond in the protein and encompasses key epitopes recognised by the mouse monoclonal antibodies 4D11 and 9H4. Although the 4D11 and 9H4 epitopes are overlapping, immunofluorescence assays have shown that the mouse monoclonal antibody 4D11 binds to MSP2 on the merozoite surface with a much stronger signal than 9H4. Understanding the structural basis for this antigenic difference between these antibodies will help direct the design of a broad-spectrum and MSP2-based malaria vaccine. 4D11 and 9H4 were reengineered into antibody fragments [variable region fragment (Fv) and single-chain Fv (scFv)] and were validated as suitable models for their full-sized IgG counterparts by surface plasmon resonance and isothermal titration calorimetry. An alanine scan of the 13-residue epitope 3D7-MSP2207-222 identified the minimal binding epitope of 4D11 and the key residues involved in binding. A 2.2-Å crystal structure of 4D11 Fv bound to the eight-residue epitope NKENCGAA provided valuable insight into the possible conformation of the C-terminal region of MSP2 on the parasite. This work underpins continued efforts to optimise recombinant MSP2 constructs for evaluation as potential vaccine candidates.

Antibody-Carbohydrate Recognition from Docked Ensembles Using the AutoMap Procedure.

Carbohydrate-protein recognition is vital to many processes in health and disease. In particular, elucidation of the structural basis of carbohydrate binding is important to the development of oligosaccharides and oligosaccharide mimetics as vaccines for infectious diseases and cancer. Computational structural techniques are valuable for the study of carbohydrate-protein recognition due to the challenges associated with experimental determination of carbohydrate-protein complexes. AutoMap is a computer program that we have developed to study protein-ligand recognition. AutoMap determines the interactions taking place in a set of highly ranked poses obtained from molecular docking and processes these to identify the protein residues most likely to be involved in interactions. In this protocol, we describe the use of AutoMap and illustrate its suitability for studying antibody recognition of the Lewis Y tetrasaccharide, which is a potential cancer vaccine antigen.

Structural biology of antibody recognition of carbohydrate epitopes and potential uses for targeted cancer immunotherapies.

Monoclonal antibodies represent the most successful class of biopharmaceuticals for the treatment of cancer. Mechanisms of action of therapeutic antibodies are very diverse and reflect their ability to engage in antibody-dependent effector mechanisms, internalize to deliver cytotoxic payloads, and display direct effects on cells by lysis or by modulating the biological pathways of their target antigens. Importantly, one of the universal changes in cancer is glycosylation and carbohydrate-binding antibodies can be produced to selectively recognize tumor cells over normal tissues. A promising group of cell surface antibody targets consists of carbohydrates presented as glycolipids or glycoproteins. In this review, we outline the basic principles of antibody-based targeting of carbohydrate antigens in cancer. We also present a detailed structural view of antibody recognition and the conformational properties of a series of related tissue-blood group (Lewis) carbohydrates that are being pursued as potential targets of cancer immunotherapy.

The carbohydrate-binding promiscuity of Euonymus europaeus lectin is predicted to involve a single binding site.

Euonymus europaeus lectin (EEL) is a carbohydrate-binding protein derived from the fruit of the European spindle tree. EEL was first identified for its erythrocyte agglutinating properties and specificity for B and H blood groups. However, a detailed molecular picture of the structural basis of carbohydrate recognition by EEL remains to be developed. In this study, we performed fluorescence titrations of a range of carbohydrates against EEL. Binding of EEL to a wide range of carbohydrates was observed, including a series of blood group-related carbohydrates, mannosides, chitotriose and sialic acid. Affinity was strongest for carbohydrates with H-related structures and the B trisaccharide. A homology model of EEL was produced from templates identified using the HHPred server, which employs hidden Markov models (HMMs) to identify templates. The HMM approach identified that the best templates for EEL were proteins featuring a ricin B-like (R-type) fold. Separate templates were used to model the core and binding site regions of the lectin. Through the use of constrained docking and spatial comparison with a template ligand, binding modes for the carbohydrate ligands were predicted. A relationship between the experimental binding energies and the computed binding energies of the selected docked poses was determined and optimized. Collectively, our results suggest that EEL utilizes a single site for recognition of carbohydrates terminating in a variety of monosaccharides.