Rescue of Function of Mutant Luteinising Hormone Receptors with Deficiencies in Cell Surface Expression, Hormone Binding, and Hormone Signalling.

INTRODUCTION: G protein-coupled receptor (GPCR) mutations are implicated in many diseases. Most inactivating mutations cause receptor misfolding and prevent trafficking to the plasma membrane. Pharmacological chaperones can "rescue" cell surface expression of such mutants, presumably by stabilising correct folding of the nascent protein. OBJECTIVE: Here we examine the scope of intracellularly retained luteinising hormone receptor (LHR) mutants that can be "rescued" by the pharmacological chaperone LHR-Chap, and whether this allosteric agonist can also restore the function of mutant LHRs with deficiencies in hormone binding or hormone-induced signalling. METHODS: Mutant LHRs were expressed in HEK 293-T cells. Cell surface expression/localisation, hormone binding, and hCG/LHR-Chap signalling were determined by ELISA, radioligand binding, and inositol phosphate accumulation assays, respectively. Molecular modelling predicted LHR-Chap interactions. RESULTS: LHR-Chap increased cell surface expression of a subset of retained mutants located in transmembrane helices predicted to be stabilised by LHR-Chap binding. For 3 (T4613.47I, L5024.61P, and S6167.46Y) hCG-responsiveness was increased following treatment. LHRs with mutations in the hormone-binding site (C131ECDR and I152ECDT) or in the hinge region (E354HingeK) had good cell surface expression but poor response to hormone stimulation, yet were responsive to allosteric activation by LHR-Chap. CONCLUSIONS: LHR-Chap, in addition to rescuing cell surface expression of intracellularly retained LHR mutants, can rescue function in mutant receptors with binding and signalling deficiencies that have normal cell surface expression. This demonstration of rescue of multiple elements of LHR dysfunction arising from inactivating mutations offers exceptional potential for treating patients with diseases arising from GPCR mutations in general.

AhR sensing of bacterial pigments regulates antibacterial defence.

The aryl hydrocarbon receptor (AhR) is a highly conserved ligand-dependent transcription factor that senses environmental toxins and endogenous ligands, thereby inducing detoxifying enzymes and modulating immune cell differentiation and responses. We hypothesized that AhR evolved to sense not only environmental pollutants but also microbial insults. We characterized bacterial pigmented virulence factors, namely the phenazines from Pseudomonas aeruginosa and the naphthoquinone phthiocol from Mycobacterium tuberculosis, as ligands of AhR. Upon ligand binding, AhR activation leads to virulence factor degradation and regulated cytokine and chemokine production. The relevance of AhR to host defence is underlined by heightened susceptibility of AhR-deficient mice to both P. aeruginosa and M. tuberculosis. Thus, we demonstrate that AhR senses distinct bacterial virulence factors and controls antibacterial responses, supporting a previously unidentified role for AhR as an intracellular pattern recognition receptor, and identify bacterial pigments as a new class of pathogen-associated molecular patterns.

Identification of Key Receptor Residues Discriminating Human Chorionic Gonadotropin (hCG)- and Luteinizing Hormone (LH)-Specific Signaling.

(1) The human luteinizing hormone (LH)/chorionic gonadotropin (hCG) receptor (LHCGR) discriminates its two hormone ligands and differs from the murine receptor (Lhr) in amino acid residues potentially involved in qualitative discerning of LH and hCG. The latter gonadotropin is absent in rodents. The aim of the study is to identify LHCGR residues involved in hCG/LH discrimination. (2) Eight LHCGR cDNAs were developed, carrying "murinizing" mutations on aminoacidic residues assumed to interact specifically with LH, hCG, or both. HEK293 cells expressing a mutant or the wild type receptor were treated with LH or hCG and the kinetics of cyclic adenosine monophosphate (cAMP) and phosphorylated extracellular signal-regulated kinases 1/2 (pERK1/2) activation was analyzed by bioluminescence resonance energy transfer (BRET). (3) Mutations falling within the receptor leucine reach repeat 9 and 10 (LRR9 and LRR10; K225S +T226I and R247T), of the large extracellular binding domain, are linked to loss of hormone-specific induced cAMP increase, as well as hCG-specific pERK1/2 activation, leading to a Lhr-like modulation of the LHCGR-mediated intracellular signaling pattern. These results support the hypothesis that LHCGR LRR domain is the interaction site of the hormone ß-L2 loop, which differs between LH and hCG, and might be fundamental for inducing gonadotropin-specific signals. (4) Taken together, these data identify LHCGR key residues likely evolved in the human to discriminate LH/hCG specific binding.

Thyrotropin Receptor: Allosteric Modulators Illuminate Intramolecular Signaling Mechanisms at the Interface of Ecto- and Transmembrane Domain.

The large TSH-bound ectodomain of the thyrotropin receptor (TSHR) activates the transmembrane domain (TMD) indirectly via an internal agonist (IA). The ectodomain/TMD interface consists of a converging helix, a Cys-Cys-bridge-linked IA, and extracellular loops (ECL). To investigate the intramolecular course of molecular activation, especially details of the indirect activation, we narrowed down allosteric inhibition sites of negative allosteric modulator (NAM) by mutagenesis, homology modeling, and competition studies with positive allosteric modulator (PAM). From the inhibitory effects of NAM S37a on: 1) chimeras with swapped ectodomain, 2) stepwise N-terminal truncations, 3) distinct constitutively active mutations distributed across the hinge region and ECL, but not across the TMD, we conclude that S37a binds at the ectodomain/TMD interface, between the converging helix, ECL1, and the IA. This is also supported by the noncompetitive inhibition of PAM-C2-activation by S37a in the TSHR-TMD construct lacking the ectodomain. Mutagenesis studies on the IA and ECL were guided by our refined model of the ectodomain/TMD interface and indicate an interaction with the TSHR-specific residues E404 (preceding IA) and H478 (ECL1). At this new allosteric interaction site, NAM S37a blocks both TSH- and PAM-induced activation of the TSHR. Our refined models, mutations, and new allosteric binding pocket helped us to gain more detailed insights into the intramolecular course of TSHR activation at the ectodomain/TMD interface, including the delocalization of the converging helix and rearrangement of the conformation of IA. These changes are embedded between the ECL and cooperatively trigger active conformations of TMD. SIGNIFICANCE STATEMENT: The intramolecular activation mechanisms of the TSHR appear to be distinct from those of other G protein-coupled receptors, as the TSHR has a uniquely large N-terminal ectodomain that includes the hormone binding site and an internal agonist sequence. We present new molecular and structural insights into the interface between ectodomain and transmembrane domain in the TSHR, as well as the transfer of activation to the transmembrane domain. This knowledge is critical for understanding activation or inhibition of the receptor by allosteric ligands. We have identified a new allosteric antagonist binding pocket that is located exactly at this interface and possesses specific features that may allow the generation of potent highly TSHR-selective drugs, of potential value for the treatment of Graves' orbitopathy.

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.

AKAP18:PKA-RIIα structure reveals crucial anchor points for recognition of regulatory subunits of PKA.

A-kinase anchoring proteins (AKAPs) interact with the dimerization/docking (D/D) domains of regulatory subunits of the ubiquitous protein kinase A (PKA). AKAPs tether PKA to defined cellular compartments establishing distinct pools to increase the specificity of PKA signalling. Here, we elucidated the structure of an extended PKA-binding domain of AKAP18ß bound to the D/D domain of the regulatory RIIα subunits of PKA. We identified three hydrophilic anchor points in AKAP18ß outside the core PKA-binding domain, which mediate contacts with the D/D domain. Such anchor points are conserved within AKAPs that bind regulatory RII subunits of PKA. We derived a different set of anchor points in AKAPs binding regulatory RI subunits of PKA. In vitro and cell-based experiments confirm the relevance of these sites for the interaction of RII subunits with AKAP18 and of RI subunits with the RI-specific smAKAP. Thus we report a novel mechanism governing interactions of AKAPs with PKA. The sequence specificity of each AKAP around the anchor points and the requirement of these points for the tight binding of PKA allow the development of selective inhibitors to unequivocally ascribe cellular functions to the AKAP18-PKA and other AKAP-PKA interactions.

The Pseudo signal peptide of the corticotropin-releasing factor receptor type 2A prevents receptor oligomerization.

N-terminal signal peptides mediate the interaction of native proteins with the translocon complex of the endoplasmic reticulum membrane and are cleaved off during early protein biogenesis. The corticotropin-releasing factor receptor type 2a (CRF(2(a))R) possesses an N-terminal pseudo signal peptide, which represents a so far unique domain within the large protein family of G protein-coupled receptors (GPCRs). In contrast to a conventional signal peptide, the pseudo signal peptide remains uncleaved and consequently forms a hydrophobic extension at the N terminus of the receptor. The functional consequence of the presence of the pseudo signal peptide is not understood. Here, we have analyzed the significance of this domain for receptor dimerization/oligomerization in detail. To this end, we took the CRF(2(a))R and the homologous corticotropin-releasing factor receptor type 1 (CRF(1)R) possessing a conventional cleaved signal peptide and conducted signal peptide exchange experiments. Using single cell and single molecule imaging methods (fluorescence resonance energy transfer and fluorescence cross-correlation spectroscopy, respectively) as well as biochemical experiments, we obtained two novel findings; we could show that (i) the CRF(2(a))R is expressed exclusively as a monomer, and (ii) the presence of the pseudo signal peptide prevents its oligomerization. Thus, we have identified a novel functional domain within the GPCR protein family, which plays a role in receptor oligomerization and which may be useful to study the functional significance of this process in general.

Molecular sampling of the allosteric binding pocket of the TSH receptor provides discriminative pharmacophores for antagonist and agonists.

The TSHR (thyrotropin receptor) is activated endogenously by the large hormone thyrotropin and activated pathologically by auto-antibodies. Both activate and bind at the extracellular domain. Recently, SMLs (small-molecule ligands) have been identified, which bind in an allosteric binding pocket within the transmembrane domain. Modelling driven site-directed mutagenesis of amino acids lining this pocket led to the delineation of activation and inactivation sensitive residues. Modified residues showing CAMs (constitutively activating mutations) indicate signalling-sensitive positions and mark potential trigger points for agonists. Silencing mutations lead to an impairment of basal activity and mark contact points for antagonists. Mapping these residues on to a structural model of TSHR indicates locations where an SML may switch the receptor to an inactive or active conformation. In the present article, we report the effects of SMLs on these signalling-sensitive amino acids at the TSHR. Surprisingly, the antagonistic effect of SML compound 52 was reversed to an agonistic effect, when tested at the CAM Y667A. Switching agonism to antagonism and the reverse by changing either SMLs or residues covering the binding pocket provides detailed knowledge about discriminative pharmacophores. It prepares the basis for rational optimization of new high-affinity antagonists to interfere with the pathogenic activation of the TSHR.

Using ortholog sequence data to predict the functional relevance of mutations in G-protein-coupled receptors.

Evaluating the functional relevance of naturally occurring gene variants usually requires experimental testing or is even impossible because of the lack of appropriate functional assays. Here we have analyzed whether comparative sequence data from orthologs are suitable to predict the functional relevance of mutations in a model protein, a G-protein-coupled receptor for ADP (P2Y(12)). The functional effect of every possible substitution at each amino acid position within a portion of P2Y(12) (1254 mutants) was individually determined. Sequence analysis of >70 P2Y(12) vertebrate orthologs revealed that this amino acid variability ensuring proper receptor function in vivo highly correlates (>90%) with the in vitro experimental data. Therefore, ortholog sequence data are helpful to predict the functional relevance of individual positions and mutations for P2Y(12). It is likely that similar conclusions may be extended for other GPCRs and conserved proteins as well.

Perspectives of data science in preclinical safety assessment.

The data landscape in preclinical safety assessment is fundamentally changing because of not only emerging new data types, such as human systems biology, or real-world data (RWD) from clinical trials, but also technological advancements in data-processing software and analytical tools based on deep learning approaches. The recent developments of data science are illustrated with use cases for the three factors: predictive safety (new in silico tools), insight generation (new data for outstanding questions); and reverse translation (extrapolating from clinical experience to resolve preclinical questions). Further advances in this field can be expected if companies focus on overcoming identified challenges related to a lack of platforms and data silos and assuring appropriate training of data scientists within the preclinical safety teams.

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.

PTGL: a database for secondary structure-based protein topologies.

With growing amount of experimental data, the number of known protein structures also increases continuously. Classification of protein structures helps to understand relationships between protein structure and function. The main classification methods based on secondary structures are SCOP, CATH and TOPS, which all classify under different aspects, and therefore can lead to different results. We developed a mathematically unique representation of protein structure topologies at a higher abstraction level providing new aspects of classification and enabling for a fast search through the data. Protein Topology Graph Library (PTGL; http://ptgl.zib.de) aims at providing a database on protein secondary structure topologies, including search facilities, the visualization as intuitive topology diagrams as well as in the 3D structure, and additional information. Secondary structure-based protein topologies are represented uniquely as undirected labeled graphs in four different ways allowing for exploration under different aspects. The linear notations, and the 2D and 3D diagrams of each notation facilitate a deeper understanding of protein topologies. Several search functions for topologies and sub-topologies, BLAST search possibility, and links to SCOP, CATH and PDBsum support individual and large-scale investigation of protein structures. Currently, PTGL comprises topologies of 54,859 protein structures. Main structural patterns for common structural motifs like TIM-barrel or Jelly Roll are pre-implemented, and can easily be searched.

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.

Small-molecule inhibitors of the PDZ domain of Dishevelled proteins interrupt Wnt signalling.

Dishevelled (Dvl) proteins are important regulators of the Wnt signalling pathway, interacting through their PDZ domains with the Wnt receptor Frizzled. Blocking the Dvl PDZ-Frizzled interaction represents a potential approach for cancer treatment, which stimulated the identification of small-molecule inhibitors, among them the anti-inflammatory drug Sulindac and Ky-02327. Aiming to develop tighter binding compounds without side effects, we investigated structure-activity relationships of sulfonamides. X-ray crystallography showed high complementarity of anthranilic acid derivatives in the GLGF loop cavity and space for ligand growth towards the PDZ surface. Our best binding compound inhibits Wnt signalling in a dose-dependent manner as demonstrated by TOP-GFP assays (IC50∼50 µM) and Western blotting of ß-catenin levels. Real-time PCR showed reduction in the expression of Wnt-specific genes. Our compound interacted with Dvl-1 PDZ (KD=2.4 µM) stronger than Ky-02327 and may be developed into a lead compound interfering with the Wnt pathway.

An interactive web-tool for molecular analyses links naturally occurring mutation data with three-dimensional structures of the rhodopsin-like glycoprotein hormone receptors.

The collection, description and molecular analysis of naturally occurring (pathogenic) mutations are important for understanding the functional mechanisms and malfunctions of biological units such as proteins. Numerous databases collate a huge amount of functional data or descriptions of mutations, but tools to analyse the molecular effects of genetic variations are as yet poorly provided. The goal of this work was therefore to develop a translational web-application that facilitates the interactive linkage of functional and structural data and which helps improve our understanding of the molecular basis of naturally occurring gain- or loss- of function mutations. Here we focus on the human glycoprotein hormone receptors (GPHRs), for which a huge number of mutations are known to cause diseases. We describe new options for interactive data analyses within three-dimensional structures, which enable the assignment of molecular relationships between structure and function. Strikingly, as the functional data are converted into relational percentage values, the system allows the comparison and classification of data from different GPHR subtypes and different experimental approaches. Our new application has been incorporated into a freely available database and website for the GPHRs (http://www.ssfa-gphr.de), but the principle development would also be applicable to other macromolecules.

Introducing the concept of virtual control groups into preclinical toxicology testing.

Sharing legacy data from in vivo toxicity studies offers the opportunity to analyze the variability of control groups stratified for strain, age, duration of study, vehicle and other experimental conditions. Historical animal control group data may lead to a repository, which could be used to construct virtual control groups (VCGs) for toxicity studies. VCGs are an established concept in clinical trials, but the idea of replacing living beings with virtual data sets has so far not been introduced into the design of regulatory animal studies. The use of VCGs has the potential of a 25% reduction in animal use by replacing the control group animals with existing randomized data sets. Prerequisites for such an approach are the availability of large and well-structured control data sets as well as thorough statistical evaluations. the foundation of data sharing has been laid within the Innovative Medicines Initiatives projects eTOX and eTRANSAFE. For a proof of principle participating companies have started to collect control group data for subacute (4-week) GLP studies with Wistar rats (the strain preferentially used in Europe) and are characterizing these data for its variability. In a second step, the control group data will be shared among the companies and cross-company variability will be investigated. In a third step, a set of studies will be analyzed to assess whether the use of VCG data would have influenced the outcome of the study compared to the real control group.

Aryl Hydrocarbon Receptor Modulation by Tuberculosis Drugs Impairs Host Defense and Treatment Outcomes.

Antimicrobial resistance in tuberculosis (TB) is a public health threat of global dimension, worsened by increasing drug resistance. Host-directed therapy (HDT) is an emerging concept currently explored as an adjunct therapeutic strategy for TB. One potential host target is the ligand-activated transcription factor aryl hydrocarbon receptor (AhR), which binds TB virulence factors and controls antibacterial responses. Here, we demonstrate that in the context of therapy, the AhR binds several TB drugs, including front line drugs rifampicin (RIF) and rifabutin (RFB), resulting in altered host defense and drug metabolism. AhR sensing of TB drugs modulates host defense mechanisms, notably impairs phagocytosis, and increases TB drug metabolism. Targeting AhR in vivo with a small-molecule inhibitor increases RFB-treatment efficacy. Thus, the AhR markedly impacts TB outcome by affecting both host defense and drug metabolism. As a corollary, we propose the AhR as a potential target for HDT in TB in adjunct to canonical chemotherapy.

Author Correction: The Henna pigment Lawsone activates the Aryl Hydrocarbon Receptor and impacts skin homeostasis.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Host monitoring of quorum sensing during Pseudomonas aeruginosa infection.

Pseudomonas aeruginosa rapidly adapts to altered conditions by quorum sensing (QS), a communication system that it uses to collectively modify its behavior through the production, release, and detection of signaling molecules. QS molecules can also be sensed by hosts, although the respective receptors and signaling pathways are poorly understood. We describe a pattern of regulation in the host by the aryl hydrocarbon receptor (AhR) that is critically dependent on qualitative and quantitative sensing of P. aeruginosa quorum. QS molecules bind to AhR and distinctly modulate its activity. This is mirrored upon infection with P. aeruginosa collected from diverse growth stages and with QS mutants. We propose that by spying on bacterial quorum, AhR acts as a major sensor of infection dynamics, capable of orchestrating host defense according to the status quo of infection.

The Henna pigment Lawsone activates the Aryl Hydrocarbon Receptor and impacts skin homeostasis.

As a first host barrier, the skin is constantly exposed to environmental insults that perturb its integrity. Tight regulation of skin homeostasis is largely controlled by the aryl hydrocarbon receptor (AhR). Here, we demonstrate that Henna and its major pigment, the naphthoquinone Lawsone activate AhR, both in vitro and in vivo. In human keratinocytes and epidermis equivalents, Lawsone exposure enhances the production of late epidermal proteins, impacts keratinocyte differentiation and proliferation, and regulates skin inflammation. To determine the potential use of Lawsone for therapeutic application, we harnessed human, murine and zebrafish models. In skin regeneration models, Lawsone interferes with physiological tissue regeneration and inhibits wound healing. Conversely, in a human acute dermatitis model, topical application of a Lawsone-containing cream ameliorates skin irritation. Altogether, our study reveals how a widely used natural plant pigment is sensed by the host receptor AhR, and how the physiopathological context determines beneficial and detrimental outcomes.