Single-cell and spatially resolved interactomics of tooth-associated keratinocytes in periodontitis.

Periodontitis affects billions of people worldwide. To address relationships of periodontal niche cell types and microbes in periodontitis, we generated an integrated single-cell RNA sequencing (scRNAseq) atlas of human periodontium (34-sample, 105918-cell), including sulcular and junctional keratinocytes (SK/JKs). SK/JKs displayed altered differentiation states and were enriched for effector cytokines in periodontitis. Single-cell metagenomics revealed 37 bacterial species with cell-specific tropism. Fluorescence in situ hybridization detected intracellular 16 S and mRNA signals of multiple species and correlated with SK/JK proinflammatory phenotypes in situ. Cell-cell communication analysis predicted keratinocyte-specific innate and adaptive immune interactions. Highly multiplexed immunofluorescence (33-antibody) revealed peri-epithelial immune foci, with innate cells often spatially constrained around JKs. Spatial phenotyping revealed immunosuppressed JK-microniches and SK-localized tertiary lymphoid structures in periodontitis. Here, we demonstrate impacts on and predicted interactomics of SK and JK cells in health and periodontitis, which requires further investigation to support precision periodontal interventions in states of chronic inflammation.

Evolutionary analyses reveal immune cell receptor GPR84 as a conserved receptor for bacteria-derived molecules.

The G protein-coupled receptor 84 (GPR84) is found in immune cells and its expression is increased under inflammatory conditions. Activation of GPR84 by medium-chain fatty acids results in pro-inflammatory responses. Here, we screened available vertebrate genome data and found that GPR84 is present in vertebrates for more than 500 million years but absent in birds and a pseudogene in bats. Cloning and functional characterization of several mammalian GPR84 orthologs in combination with evolutionary and model-based structural analyses revealed evidence for positive selection of bear GPR84 orthologs. Naturally occurring human GPR84 variants are most frequent in Asian populations causing a loss of function. Further, we identified cis- and trans-2-decenoic acid, both known to mediate bacterial communication, as evolutionary highly conserved ligands. Our integrated set of approaches contributes to a comprehensive understanding of GPR84 in terms of evolutionary and structural aspects, highlighting GPR84 as a conserved immune cell receptor for bacteria-derived molecules.

Pathogenic variants damage cell composition and single cell transcription in cardiomyopathies.

Pathogenic variants in genes that cause dilated cardiomyopathy (DCM) and arrhythmogenic cardiomyopathy (ACM) convey high risks for the development of heart failure through unknown mechanisms. Using single-nucleus RNA sequencing, we characterized the transcriptome of 880,000 nuclei from 18 control and 61 failing, nonischemic human hearts with pathogenic variants in DCM and ACM genes or idiopathic disease. We performed genotype-stratified analyses of the ventricular cell lineages and transcriptional states. The resultant DCM and ACM ventricular cell atlas demonstrated distinct right and left ventricular responses, highlighting genotype-associated pathways, intercellular interactions, and differential gene expression at single-cell resolution. Together, these data illuminate both shared and distinct cellular and molecular architectures of human heart failure and suggest candidate therapeutic targets.

Cells of the adult human heart.

Cardiovascular disease is the leading cause of death worldwide. Advanced insights into disease mechanisms and therapeutic strategies require a deeper understanding of the molecular processes involved in the healthy heart. Knowledge of the full repertoire of cardiac cells and their gene expression profiles is a fundamental first step in this endeavour. Here, using state-of-the-art analyses of large-scale single-cell and single-nucleus transcriptomes, we characterize six anatomical adult heart regions. Our results highlight the cellular heterogeneity of cardiomyocytes, pericytes and fibroblasts, and reveal distinct atrial and ventricular subsets of cells with diverse developmental origins and specialized properties. We define the complexity of the cardiac vasculature and its changes along the arterio-venous axis. In the immune compartment, we identify cardiac-resident macrophages with inflammatory and protective transcriptional signatures. Furthermore, analyses of cell-to-cell interactions highlight different networks of macrophages, fibroblasts and cardiomyocytes between atria and ventricles that are distinct from those of skeletal muscle. Our human cardiac cell atlas improves our understanding of the human heart and provides a valuable reference for future studies.

The Translational Landscape of the Human Heart.

Gene expression in human tissue has primarily been studied on the transcriptional level, largely neglecting translational regulation. Here, we analyze the translatomes of 80 human hearts to identify new translation events and quantify the effect of translational regulation. We show extensive translational control of cardiac gene expression, which is orchestrated in a process-specific manner. Translation downstream of predicted disease-causing protein-truncating variants appears to be frequent, suggesting inefficient translation termination. We identify hundreds of previously undetected microproteins, expressed from lncRNAs and circRNAs, for which we validate the protein products in vivo. The translation of microproteins is not restricted to the heart and prominent in the translatomes of human kidney and liver. We associate these microproteins with diverse cellular processes and compartments and find that many locate to the mitochondria. Importantly, dozens of microproteins are translated from lncRNAs with well-characterized noncoding functions, indicating previously unrecognized biology.

Structural changes of TasA in biofilm formation of Bacillus subtilis.

Microorganisms form surface-attached communities, termed biofilms, which can serve as protection against host immune reactions or antibiotics. Bacillus subtilis biofilms contain TasA as major proteinaceous component in addition to exopolysaccharides. In stark contrast to the initially unfolded biofilm proteins of other bacteria, TasA is a soluble, stably folded monomer, whose structure we have determined by X-ray crystallography. Subsequently, we characterized in vitro different oligomeric forms of TasA by NMR, EM, X-ray diffraction, and analytical ultracentrifugation (AUC) experiments. However, by magic-angle spinning (MAS) NMR on live biofilms, a swift structural change toward only one of these forms, consisting of homogeneous and protease-resistant, ß-sheet-rich fibrils, was observed in vivo. Thereby, we characterize a structural change from a globular state to a fibrillar form in a functional prokaryotic system on the molecular level.

Evidence of G-protein-coupled receptor and substrate transporter heteromerization at a single molecule level.

G-protein-coupled receptors (GPCRs) can constitute complexes with non-GPCR integral membrane proteins, while such interaction has not been demonstrated at a single molecule level so far. We here investigated the potential interaction between the thyrotropin receptor (TSHR) and the monocarboxylate transporter 8 (MCT8), a member of the major facilitator superfamily (MFS), using fluorescence cross-correlation spectroscopy (FCCS). Both the proteins are expressed endogenously on the basolateral plasma membrane of the thyrocytes and are involved in stimulation of thyroid hormone production and release. Indeed, we demonstrate strong interaction between both the proteins which causes a suppressed activation of Gq/11 by TSH-stimulated TSHR. Thus, we provide not only evidence for a novel interaction between the TSHR and MCT8, but could also prove this interaction on a single molecule level. Moreover, this interaction forces biased signaling at the TSHR. These results are of general interest for both the GPCR and the MFS research fields.

GPCR-SSFE 2.0-a fragment-based molecular modeling web tool for Class A G-protein coupled receptors.

G-protein coupled receptors (GPCRs) are key players in signal transduction and therefore a large proportion of pharmaceutical drugs target these receptors. Structural data of GPCRs are sparse yet important for elucidating the molecular basis of GPCR-related diseases and for performing structure-based drug design. To ameliorate this problem, GPCR-SSFE 2.0 (http://www.ssfa-7tmr.de/ssfe2/), an intuitive web server dedicated to providing three-dimensional Class A GPCR homology models has been developed. The updated web server includes 27 inactive template structures and incorporates various new functionalities. Uniquely, it uses a fingerprint correlation scoring strategy for identifying the optimal templates, which we demonstrate captures structural features that sequence similarity alone is unable to do. Template selection is carried out separately for each helix, allowing both single-template models and fragment-based models to be built. Additionally, GPCR-SSFE 2.0 stores a comprehensive set of pre-calculated and downloadable homology models and also incorporates interactive loop modeling using the tool SL2, allowing knowledge-based input by the user to guide the selection process. For visual analysis, the NGL viewer is embedded into the result pages. Finally, blind-testing using two recently published structures shows that GPCR-SSFE 2.0 performs comparably or better than other state-of-the art GPCR modeling web servers.

Structural-Functional Features of the Thyrotropin Receptor: A Class A G-Protein-Coupled Receptor at Work.

The thyroid-stimulating hormone receptor (TSHR) is a member of the glycoprotein hormone receptors, a sub-group of class A G-protein-coupled receptors (GPCRs). TSHR and its endogenous ligand thyrotropin (TSH) are of essential importance for growth and function of the thyroid gland and proper function of the TSH/TSHR system is pivotal for production and release of thyroid hormones. This receptor is also important with respect to pathophysiology, such as autoimmune (including ophthalmopathy) or non-autoimmune thyroid dysfunctions and cancer development. Pharmacological interventions directly targeting the TSHR should provide benefits to disease treatment compared to currently available therapies of dysfunctions associated with the TSHR or the thyroid gland. Upon TSHR activation, the molecular events conveying conformational changes from the extra- to the intracellular side of the cell across the membrane comprise reception, conversion, and amplification of the signal. These steps are highly dependent on structural features of this receptor and its intermolecular interaction partners, e.g., TSH, antibodies, small molecules, G-proteins, or arrestin. For better understanding of signal transduction, pathogenic mechanisms such as autoantibody action and mutational modifications or for developing new pharmacological strategies, it is essential to combine available structural data with functional information to generate homology models of the entire receptor. Although so far these insights are fragmental, in the past few decades essential contributions have been made to investigate in-depth the involved determinants, such as by structure determination via X-ray crystallography. This review summarizes available knowledge (as of December 2016) concerning the TSHR protein structure, associated functional aspects, and based on these insights we suggest several receptor complex models. Moreover, distinct TSHR properties will be highlighted in comparison to other class A GPCRs to understand the molecular activation mechanisms of this receptor comprehensively. Finally, limitations of current knowledge and lack of information are discussed highlighting the need for intensified efforts toward TSHR structure elucidation.

Molecular dissection of colorectal cancer in pre-clinical models identifies biomarkers predicting sensitivity to EGFR inhibitors.

Colorectal carcinoma represents a heterogeneous entity, with only a fraction of the tumours responding to available therapies, requiring a better molecular understanding of the disease in precision oncology. To address this challenge, the OncoTrack consortium recruited 106 CRC patients (stages I-IV) and developed a pre-clinical platform generating a compendium of drug sensitivity data totalling >4,000 assays testing 16 clinical drugs on patient-derived in vivo and in vitro models. This large biobank of 106 tumours, 35 organoids and 59 xenografts, with extensive omics data comparing donor tumours and derived models provides a resource for advancing our understanding of CRC. Models recapitulate many of the genetic and transcriptomic features of the donors, but defined less complex molecular sub-groups because of the loss of human stroma. Linking molecular profiles with drug sensitivity patterns identifies novel biomarkers, including a signature outperforming RAS/RAF mutations in predicting sensitivity to the EGFR inhibitor cetuximab.

Genomics and drug profiling of fatal TCF3-HLF-positive acute lymphoblastic leukemia identifies recurrent mutation patterns and therapeutic options.

TCF3-HLF-positive acute lymphoblastic leukemia (ALL) is currently incurable. Using an integrated approach, we uncovered distinct mutation, gene expression and drug response profiles in TCF3-HLF-positive and treatment-responsive TCF3-PBX1-positive ALL. We identified recurrent intragenic deletions of PAX5 or VPREB1 in constellation with the fusion of TCF3 and HLF. Moreover somatic mutations in the non-translocated allele of TCF3 and a reduction of PAX5 gene dosage in TCF3-HLF ALL suggest cooperation within a restricted genetic context. The enrichment for stem cell and myeloid features in the TCF3-HLF signature may reflect reprogramming by TCF3-HLF of a lymphoid-committed cell of origin toward a hybrid, drug-resistant hematopoietic state. Drug response profiling of matched patient-derived xenografts revealed a distinct profile for TCF3-HLF ALL with resistance to conventional chemotherapeutics but sensitivity to glucocorticoids, anthracyclines and agents in clinical development. Striking on-target sensitivity was achieved with the BCL2-specific inhibitor venetoclax (ABT-199). This integrated approach thus provides alternative treatment options for this deadly disease.

Recurrent somatic alterations of FGFR1 and NTRK2 in pilocytic astrocytoma.

Pilocytic astrocytoma, the most common childhood brain tumor, is typically associated with mitogen-activated protein kinase (MAPK) pathway alterations. Surgically inaccessible midline tumors are therapeutically challenging, showing sustained tendency for progression and often becoming a chronic disease with substantial morbidities. Here we describe whole-genome sequencing of 96 pilocytic astrocytomas, with matched RNA sequencing (n = 73), conducted by the International Cancer Genome Consortium (ICGC) PedBrain Tumor Project. We identified recurrent activating mutations in FGFR1 and PTPN11 and new NTRK2 fusion genes in non-cerebellar tumors. New BRAF-activating changes were also observed. MAPK pathway alterations affected all tumors analyzed, with no other significant mutations identified, indicating that pilocytic astrocytoma is predominantly a single-pathway disease. Notably, we identified the same FGFR1 mutations in a subset of H3F3A-mutated pediatric glioblastoma with additional alterations in the NF1 gene. Our findings thus identify new potential therapeutic targets in distinct subsets of pilocytic astrocytoma and childhood glioblastoma.

Residues of a proposed gate region in type I ATP-binding cassette import systems are crucial for function as revealed by mutational analysis.

The type I ATP-binding cassette (ABC) importer for positively charged amino acids of the thermophilic bacterium Geobacillus stearothermophilus consists of the extracellular solute binding protein, ArtJ, and a homodimer each of the transmembrane subunit, ArtM, and the nucleotide-binding and -hydrolyzing subunit, ArtP. We have investigated the functional consequences of mutations affecting conserved residues from two peptide regions in ArtM, recently proposed to form a 'gate' by which access of a substrate to the translocation path is controlled (Hollenstein et al., 2007 [14]). Transporter variants were reconstituted into proteoliposomes and assayed for ArtJ/arginine-stimulated ATPase activity. Replacement of residues from region 1 (Arg-63, Pro-66) caused no or only moderate reduction in ATPase activity. In contrast, mutating residues from gate region 2 (Lys-159, Leu-163) resulted in a substantial increase in ATPase activity which, however, as demonstrated for variants ArtM(K159I) and ArtM(K159E), is not coupled to transport. Replacing homologous residues in the closely related histidine transporter of Salmonella enterica serovar Typhimurium (HisJ-QMP2) caused different phenotypes. Mutation to isoleucine of HisQ(K163) or HisM(H172), both homologous to ArtM(K159), abolished ATPase activity. The mutations most likely caused a structural change as revealed by limited proteolysis. In contrast, substantial, albeit reduced, enzymatic activity was observed with variants of HisQ(L167→G) or HisM(L176→G), both homologous to ArtM(L163). Our study provides the first experimental evidence in favor of a crucial role of residues from the proposed gate region in type I ABC importer function.

Binding sites in membrane proteins--diversity, druggability and prospects.

The identification of novel drug targets is one of the major challenges in proteomics. Computational methods developed over the last decade have enhanced the process of drug design in both terms of time and quality. The main task is the design of selective compounds, which bind targets more specifically, dependent on the desired mode of action of the particular drug. This makes it necessary to create compounds, which either exhibit their functions on one single protein to exclude undesired cross-reactivity or to use the advantageous effect of less selective drugs that target numerous proteins and therefore exhibit their functions on whole protein classes. Main aspects in the assignment of interactions between ligands and putative targets involve the amino acid composition of the binding site, evolutionary conservation and similarity in sequence and structure of known targets. Similarities or differences within classified protein families can be the key to their function and give first hints to functional drug design. Hereby, binding site-based classification outnumbers sequence-based classifications since similar binding sites can also be found in more distant proteins. Membrane proteins are 'difficult targets', because of their special physicochemical characteristics and the general lack of structural information. Here, we describe recent advances in modeling methods dedicated to membrane proteins. Different descriptors of similarity between compounds and the similarity between binding sites are under development and elucidate important aspects like dynamics or entropy. The importance of computational drug design is undisputable. Nevertheless, the process of design is complicated by increasing complexity, which underlines the importance of accurate knowledge about the addressed target class(es) and particularly their binding sites. One main objective by considering named topics is to predict putative side effects and errant functions (off-target effects) of novel drugs, which requires a holistic (systems biology) view on drug-target-pathway relations. In the following, we give a brief summary about the recent discussion on drug-target interactions with emphasis on membrane proteins.

Development of immune-specific interaction potentials and their application in the multi-agent-system VaccImm.

Peptide vaccination in cancer therapy is a promising alternative to conventional methods. However, the parameters for this personalized treatment are difficult to access experimentally. In this respect, in silico models can help to narrow down the parameter space or to explain certain phenomena at a systems level. Herein, we develop two empirical interaction potentials specific to B-cell and T-cell receptor complexes and validate their applicability in comparison to a more general potential. The interaction potentials are applied to the model VaccImm which simulates the immune response against solid tumors under peptide vaccination therapy. This multi-agent system is derived from another immune system simulator (C-ImmSim) and now includes a module that enables the amino acid sequence of immune receptors and their ligands to be taken into account. The multi-agent approach is combined with approved methods for prediction of major histocompatibility complex (MHC)-binding peptides and the newly developed interaction potentials. In the analysis, we critically assess the impact of the different modules on the simulation with VaccImm and how they influence each other. In addition, we explore the reasons for failures in inducing an immune response by examining the activation states of the immune cell populations in detail.In summary, the present work introduces immune-specific interaction potentials and their application to the agent-based model VaccImm which simulates peptide vaccination in cancer therapy.

GPCR-SSFE: a comprehensive database of G-protein-coupled receptor template predictions and homology models.

BACKGROUND: G protein-coupled receptors (GPCRs) transduce a wide variety of extracellular signals to within the cell and therefore have a key role in regulating cell activity and physiological function. GPCR malfunction is responsible for a wide range of diseases including cancer, diabetes and hyperthyroidism and a large proportion of drugs on the market target these receptors. The three dimensional structure of GPCRs is important for elucidating the molecular mechanisms underlying these diseases and for performing structure-based drug design. Although structural data are restricted to only a handful of GPCRs, homology models can be used as a proxy for those receptors not having crystal structures. However, many researchers working on GPCRs are not experienced homology modellers and are therefore unable to benefit from the information that can be gleaned from such three-dimensional models. Here, we present a comprehensive database called the GPCR-SSFE, which provides initial homology models of the transmembrane helices for a large variety of family A GPCRs. DESCRIPTION: Extending on our previous theoretical work, we have developed an automated pipeline for GPCR homology modelling and applied it to a large set of family A GPCR sequences. Our pipeline is a fragment-based approach that exploits available family A crystal structures. The GPCR-SSFE database stores the template predictions, sequence alignments, identified sequence and structure motifs and homology models for 5025 family A GPCRs. Users are able to browse the GPCR dataset according to their pharmacological classification or search for results using a UniProt entry name. It is also possible for a user to submit a GPCR sequence that is not contained in the database for analysis and homology model building. The models can be viewed using a Jmol applet and are also available for download along with the alignments. CONCLUSIONS: The data provided by GPCR-SSFE are useful for investigating general and detailed sequence-structure-function relationships of GPCRs, performing structure-based drug design and for better understanding the molecular mechanisms underlying disease-associated mutations in GPCRs. The effectiveness of our multiple template and fragment approach is demonstrated by the accuracy of our predicted homology models compared to recently published crystal structures.

From molecular details of the interplay between transmembrane helices of the thyrotropin receptor to general aspects of signal transduction in family a G-protein-coupled receptors (GPCRs).

Transmembrane helices (TMHs) 5 and 6 are known to be important for signal transduction by G-protein-coupled receptors (GPCRs). Our aim was to characterize the interface between TMH5 and TMH6 of the thyrotropin receptor (TSHR) to gain molecular insights into aspects of signal transduction and regulation. A proline at TMH5 position 5.50 is highly conserved in family A GPCRs and causes a twist in the helix structure. Mutation of the TSHR-specific alanine (Ala-5935·5°) at this position to proline resulted in a 20-fold reduction of cell surface expression. This indicates that TMH5 in the TSHR might have a conformation different from most other family A GPCRs by forming a regular α-helix. Furthermore, linking our own and previous data from directed mutagenesis with structural information led to suggestions of distinct pairs of interacting residues between TMH5 and TMH6 that are responsible for stabilizing either the basal or the active state. Our insights suggest that the inactive state conformation is constrained by a core set of polar interactions among TMHs 2, 3, 6, and 7 and in contrast that the active state conformation is stabilized mainly by non-polar interactions between TMHs 5 and 6. Our findings might be relevant for all family A GPCRs as supported by a statistical analysis of residue properties between the TMHs of a vast number of GPCR sequences.

SDM--a server for predicting effects of mutations on protein stability and malfunction.

The sheer volume of non-synonymous single nucleotide polymorphisms that have been generated in recent years from projects such as the Human Genome Project, the HapMap Project and Genome-Wide Association Studies means that it is not possible to characterize all mutations experimentally on the gene products, i.e. elucidate the effects of mutations on protein structure and function. However, automatic methods that can predict the effects of mutations will allow a reduced set of mutations to be studied. Site Directed Mutator (SDM) is a statistical potential energy function that uses environment-specific amino-acid substitution frequencies within homologous protein families to calculate a stability score, which is analogous to the free energy difference between the wild-type and mutant protein. Here, we present a web server for SDM (http://www-cryst.bioc.cam.ac.uk/~sdm/sdm.php), which has obtained more than 10,000 submissions since being online in April 2008. To run SDM, users must upload a wild-type structure and the position and amino acid type of the mutation. The results returned include information about the local structural environment of the wild-type and mutant residues, a stability score prediction and prediction of disease association. Additionally, the wild-type and mutant structures are displayed in a Jmol applet with the relevant residues highlighted.

Research resource: Update and extension of a glycoprotein hormone receptors web application.

The SSFA-GPHR (Sequence-Structure-Function-Analysis of Glycoprotein Hormone Receptors) database provides a comprehensive set of mutation data for the glycoprotein hormone receptors (covering the lutropin, the FSH, and the TSH receptors). Moreover, it provides a platform for comparison and investigation of these homologous receptors and helps in understanding protein malfunctions associated with several diseases. Besides extending the data set (> 1100 mutations), the database has been completely redesigned and several novel features and analysis tools have been added to the web site. These tools allow the focused extraction of semiquantitative mutant data from the GPHR subtypes and different experimental approaches. Functional and structural data of the GPHRs are now linked interactively at the web interface, and new tools for data visualization (on three-dimensional protein structures) are provided. The interpretation of functional findings is supported by receptor morphings simulating intramolecular changes during the activation process, which thus help to trace the potential function of each amino acid and provide clues to the local structural environment, including potentially relocated spatial counterpart residues. Furthermore, double and triple mutations are newly included to allow the analysis of their functional effects related to their spatial interrelationship in structures or homology models. A new important feature is the search option and data visualization by interactive and user-defined snake-plots. These new tools allow fast and easy searches for specific functional data and thereby give deeper insights in the mechanisms of hormone binding, signal transduction, and signaling regulation. The web application "Sequence-Structure-Function-Analysis of GPHRs" is accessible on the internet at http://www.ssfa-gphr.de/.

Meet me halfway: when genomics meets structural bioinformatics.

The DNA sequencing technology developed by Frederick Sanger in the 1970s established genomics as the basis of comparative genetics. The recent invention of next-generation sequencing (NGS) platform has added a new dimension to genome research by generating ultra-fast and high-throughput sequencing data in an unprecedented manner. The advent of NGS technology also provides the opportunity to study genetic diseases where sequence variants or mutations are sought to establish a causal relationship with disease phenotypes. However, it is not a trivial task to seek genetic variants responsible for genetic diseases and even harder for complex diseases such as diabetes and cancers. In such polygenic diseases, multiple genes and alleles, which can exist in healthy individuals, come together to contribute to common disease phenotypes in a complex manner. Hence, it is desirable to have an approach that integrates omics data with both knowledge of protein structure and function and an understanding of networks/pathways, i.e. functional genomics and systems biology; in this way, genotype-phenotype relationships can be better understood. In this review, we bring this 'bottom-up' approach alongside the current NGS-driven genetic study of genetic variations and disease aetiology. We describe experimental and computational techniques for assessing genetic variants and their deleterious effects on protein structure and function.