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Various single nucleotide polymorphisms (SNPs) in the oxytocin receptor (OXTR) gene have been associated with behavioral traits, autism spectrum disorder (ASD) and other diseases. The non-synonymous SNP rs4686302 results in the OXTR variant A218T and has been linked to core characteristics of ASD, trait empathy and preterm birth. However, the molecular and intracellular mechanisms underlying those associations are still elusive. Here, we uncovered the molecular and intracellular consequences of this mutation that may affect the psychological or behavioral outcome of oxytocin (OXT)-treatment regimens in clinical studies, and provide a mechanistic explanation for an altered receptor function. We created two monoclonal HEK293 cell lines, stably expressing either the wild-type or A218T OXTR. We detected an increased OXTR protein stability, accompanied by a shift in Ca2+ dynamics and reduced MAPK pathway activation in the A218T cells. Combined whole-genome and RNA sequencing analyses in OXT-treated cells revealed 7823 differentially regulated genes in A218T compared to wild-type cells, including 429 genes being associated with ASD. Furthermore, computational modeling provided a molecular basis for the observed change in OXTR stability suggesting that the OXTR mutation affects downstream events by altering receptor activation and signaling, in agreement with our in vitro results. In summary, our study provides the cellular mechanism that links the OXTR rs4686302 SNP with genetic dysregulations associated with aspects of ASD.

Bitter molecules in humans are detected by ∼25 G protein-coupled receptors (GPCRs). The lack of atomic resolution structure for any of them is complicating an in depth understanding of the molecular mechanisms underlying bitter taste perception. Here, we investigate the molecular determinants of the interaction of the TAS2R38 bitter taste receptor with its agonists phenylthiocarbamide (PTC) and propylthiouracil (PROP). We use the recently developed hybrid Molecular Mechanics/Coarse Grained (MM/CG) method tailored specifically for GPCRs. The method, through an extensive exploration of the conformational space in the binding pocket, allows the identification of several residues important for agonist binding that would have been very difficult to capture from the standard bioinformatics/docking approach. Our calculations suggest that both agonists bind to Asn103, Phe197, Phe264 and Trp201, whilst they do not interact with the so-called extra cellular loop 2, involved in cis-retinal binding in the GPCR rhodopsin. These predictions are consistent with data sets based on more than 20 site-directed mutagenesis and functional calcium imaging experiments of TAS2R38. The method could be readily used for other GPCRs for which experimental information is currently lacking.

Advances in coarse-grained molecular dynamics (CGMD) simulations have extended the use of computational studies on biological macromolecules and their complexes, as well as the interactions of membrane protein and lipid complexes at a reduced level of representation, allowing longer and larger molecular dynamics simulations. Here, we present a computational platform dedicated to the preparation, running, and analysis of CGMD simulations. The platform is built on a completely revisited version of our Martini coarsE gRained MembrAne proteIn Dynamics (MERMAID) web server, and it integrates this with other three dedicated services. In its current version, the platform expands the existing implementation of the Martini force field for membrane proteins to also allow the simulation of soluble proteins using the Martini and the SIRAH force fields. Moreover, it offers an automated protocol for carrying out the backmapping of the coarse-grained description of the system into an atomistic one.

Human G-protein coupled receptors (GPCRs) are important targets for pharmaceutical intervention against neurological diseases. Here, we use molecular simulation to investigate the key step in ligand recognition governed by the extracellular domains in the neuronal adenosine receptor type 2A (hA2AR), a target for neuroprotective compounds. The ligand is the high-affinity antagonist (4-(2-(7-amino-2-(furan-2-yl)-[1,2,4]triazolo[1,5-a][1,3,5]triazin-5-ylamino)ethyl)phenol), embedded in a neuronal membrane mimic environment. Free energy calculations, based on well-tempered metadynamics, reproduce the experimentally measured binding affinity. The results are consistent with the available mutagenesis studies. The calculations identify a vestibular binding site, where lipids molecules can actively participate to stabilize ligand binding. Bioinformatic analyses suggest that such vestibular binding site and, in particular, the second extracellular loop, might drive the ligand toward the orthosteric binding pocket, possibly by allosteric modulation. Taken together, these findings point to a fundamental role of the interaction between extracellular loops and membrane lipids for ligands’ molecular recognition and ligand design in hA2AR.

Background Strongyloidiasis is a neglected tropical disease affecting an estimated 600 million people, particularly in resource-limited settings. The infection can persist lifelong due to unusual auto-infective cycle of Strongyloides stercoralis . The lack of a diagnostic gold standard and limited knowledge of the mechanisms underpinning this chronic infection are key issues in disease management. To date, only a few proteomics studies have been conducted to elucidate the molecular mechanisms associated with Strongyloides parasitism or to highlight novel immunological markers, with the result that our knowledge of S. stercoralis proteome remains limited. This study aims at expanding the characterization of S. stercoralis infective larvae (iL3) in order to further explore the mechanisms of parasitism and to highlight possible novel targets for serodiagnosis. Methods iL3 obtained from an infected subject were analysed by high-throughput tandem mass spectrometry. To achieve a more comprehensive characterization of the iL3 proteome we analysed the experimental dataset using an automatic search strategy combined with manual annotation, which included gene ontology (GO) analysis, InterPro annotation, assessment of the homology with Homo sapiens and other pathogens of clinical importance and B-cell epitope prediction. Results Our pipeline identified 430 S. stercoralis proteins, 187 (43%) of which were uncharacterized. Oxidoreductases and peptidases were amongst the most represented protein categories, as highlighted by molecular function GO analyses, while membrane and mitochondrial proteins were the most represented cellular component GO categories. A high proportion of proteins bearing the CAP, SCP or thioredoxin domain or belonging to cysteine-rich secretory, transthyretin-like or peptidase protein families were also identified. Additionally, we highlighted nine proteins displaying low homology with H. sapiens or other related pathogens and bearing amino acid sequences with immunogenic properties. Conclusions Our comprehensive description and annotation of the S. stercoralis iL3 proteome contribute to expanding the ‘omics characterization of this parasite and provide experimental evidence on the most represented proteins associated with S. stercoralis parasitism, as inferred from genomic and transcriptomic data. Moreover, novel candidate immunogenic proteins to be evaluated as novel serological diagnostic markers are highlighted. Graphical Abstract

The translocator protein (TSPO) is a transmembrane protein present across the three domains of life. Its functional quaternary structure consists of one or more subunits. In mice, the dimer-to-monomer equilibrium is shifted in vitro towards the monomer by adding cholesterol, a natural component of mammalian membranes. Here, we present a coarse-grained molecular dynamics study on the mouse protein in the presence of a physiological content and of an excess of cholesterol. The latter turns out to weaken the interfaces of the dimer by clusterizing mostly at the inter-monomeric space and pushing the contact residues apart. It also increases the compactness and the rigidity of the monomer. These two factors might play a role for the experimentally observed incremented stability of the monomeric form with increased content of cholesterol. Comparison with simulations on bacterial proteins suggests that the effect of cholesterol is much less pronounced for the latter than for the mouse protein.

Clinical and experimental studies indicate that muscarinic acetylcholine receptors are potential pharmacological targets for the treatment of neurological diseases. Although these receptors have been described in human, bovine and rat cerebral microvascular tissue, a subtype functional characterization in mouse brain endothelium is lacking. Here, we show that all muscarinic acetylcholine receptors (M 1 -M 5 ) are expressed in mouse brain microvascular endothelial cells. The mRNA expression of M 2 , M 3 , and M 5 correlates with their respective protein abundance, but a mismatch exists for M 1 and M 4 mRNA versus protein levels. Acetylcholine activates calcium transients in brain endothelium via muscarinic, but not nicotinic, receptors. Moreover, although M 1 and M 3 are the most abundant receptors, only a small fraction of M 1 is present in the plasma membrane and functions in ACh-induced Ca 2+ signaling. Bioinformatic analyses performed on eukaryotic muscarinic receptors demonstrate a high degree of conservation of the orthosteric binding site and a great variability of the allosteric site. In line with previous studies, this result indicates muscarinic acetylcholine receptors as potential pharmacological targets in future translational studies. We argue that research on drug development should especially focus on the allosteric binding sites of the M 1 and M 3 receptors.

Understanding how ligands bind to G-protein coupled receptors (GPCRs) provides insights into a myriad of cell processes and is crucial for drug development. Here we extend a hybrid molecular mechanics/coarse-grained (MM/CG) approach applied previously to enzymes to GPCR/ligand complexes. The accuracy of this method for structural predictions is established by comparison with recent atomistic molecular dynamics simulations on the human β2 adrenergic receptor, a member of the GPCRs superfamily. The results obtained with the MM/CG methodology show a good agreement with previous all-atom classical dynamics simulations, in particular in the structural description of the ligand binding site. This approach could be used for high-throughput predictions of ligand poses in a variety of GPCRs.

The translocator protein (TSPO) is a 18kDa transmembrane protein, ubiquitously present in human mitochondria. It is overexpressed in tumor cells and at the sites of neuroinflammation, thus representing an important biomarker, as well as a promising drug target. In mammalian TSPO, there are cholesterol–binding motifs, as well as a binding cavity able to accommodate different chemical compounds. Given the lack of structural information for the human protein, we built a model of human (h) TSPO in the apo state and in complex with PK11195, a molecule routinely used in positron emission tomography (PET) for imaging of neuroinflammatory sites. To better understand the interactions of PK11195 and cholesterol with this pharmacologically relevant protein, we ran molecular dynamics simulations of the apo and holo proteins embedded in a model membrane. We found that: (i) PK11195 stabilizes hTSPO structural fold; (ii) PK11195 might enter in the binding site through transmembrane helices I and II of hTSPO; (iii) PK11195 reduces the frequency of cholesterol binding to the lower, N–terminal part of hTSPO in the inner membrane leaflet, while this impact is less pronounced for the upper, C–terminal part in the outer membrane leaflet, where the ligand binding site is located; (iv) very interestingly, cholesterol most frequently binds simultaneously to the so-called CRAC and CARC regions in TM V in the free form (residues L150–X–Y152–X(3)–R156 and R135–X(2)–Y138–X(2)–L141, respectively). However, when the protein is in complex with PK11195, cholesterol binds equally frequently to the CRAC–resembling motif that we observed in TM I (residues L17–X(2)–F20–X(3)–R24) and to CRAC in TM V. We expect that the CRAC–like motif in TM I will be of interest in future experimental investigations. Thus, our MD simulations provide insight into the structural features of hTSPO and the previously unknown interplay between PK11195 and cholesterol interactions with this pharmacologically relevant protein.

Humans' bitter taste perception is mediated by the hTAS2R subfamily of the G protein-coupled membrane receptors (GPCRs). Structural information on these receptors is currently limited. Here we identify residues involved in the binding of phenylthiocarbamide (PTC) and in receptor activation in one of the most widely studied hTAS2Rs (hTAS2R38) by means of structural bioinformatics and molecular docking. The predictions are validated by site-directed mutagenesis experiments that involve specific residues located in the putative binding site and trans-membrane (TM) helices 6 and 7 putatively involved in receptor activation. Based on our measurements, we suggest that (i) residue N103 participates actively in PTC binding, in line with previous computational studies. (ii) W99, M100 and S259 contribute to define the size and shape of the binding cavity. (iii) W99 and M100, along with F255 and V296, play a key role for receptor activation, providing insights on bitter taste receptor activation not emerging from the previously reported computational models.