OBJECTIVE:We aimed to characterize the phenotypic spectrum and functional consequences associated with variants in HCN2, encoding for the hyperpolarization-activated cyclic nucleotide (HCN) gated channel 2. METHODS:GeneMatcher facilitated the recruitment of 21 individuals with HCN2 variants from 15 unrelated families, carrying HCN2 variants. In vitro functional studies were performed by electrophysiology with Xenopus laevis oocytes and membrane trafficking was investigated in HEK cells by confocal imaging. Structural 3D-analysis of the HCN2 variants was performed. RESULTS:The phenotypic spectrum included developmental delay/intellectual disability (DD/ID, 17/21), epilepsy (10/21), language disorders (16/21), movement disorders (12/21), and axial hypotonia (10/21). Thirteen pathogenic variants (12 new and 1 already described) were identified: 11 missense (8 monoallelic and 3 biallelic), 1 recurrent inframe deletion (monoallelic), and 1 frameshift (biallelic). Functional analysis of p.(Arg324His) variant showed a strong increase of HCN2 conductance, whereas p.(Ala363Val) and p.(Met374Leu) exhibited dominant negative effects. The p.(Leu377His), p.(Pro493Leu), and p.(Gly587Asp) variants rendered HCN2 electrophysiologically silent and impaired membrane trafficking. Structural 3D-analysis revealed that, except for p.(Arg324His), all variants altered HCN2 stability. INTERPRETATION:Our findings broadened the HCN2 disease clinical spectrum to include DD/ID with or without epilepsy. Functional analysis in cellular models reveal that pathogenic HCN2 variants can cause either loss-of-function or gain-of-function, providing critical information for the development of targeted therapies for HCN2-related disorders. ANN NEUROL 2025;98:573-589.

Environmental factors, including mechanical stress and surrounding lipids, can influence the response of GPCRs, such as the mechanosensitive angiotensin II type 1 receptor (AT1). To investigate the impact of these factors on AT1 activation, we developed a steered molecular dynamics simulations protocol based on quaternion formalism. In this protocol, a pulling force was applied to the N-terminus of transmembrane helix 6 (TM6) to induce the TM6 opening characteristic of activation. Subsequently, the simulations were continued without constraints to allow the receptor to relax around the novel TM6 conformation under different conditions. We analyzed the responses of AT1 to membrane stretching, modeled by applying surface tension, in different bilayers. In phosphocholine bilayers without surface tension, we could observe a transient atypical structure of AT1, with an outward TM7 conformation, at the beginning of the activation process. This atypical structure then evolved toward a pre-active structure with outward TM6 and inward TM7. Strikingly, the presence of anionic phosphoglycerol lipids and application of surface tension synergistically favored the atypical structure, which led to an increase in the cross-section area of the receptor intracellular domain. Lipid internalization and H-bonds between lipid heads and the receptor C-terminus increased in phosphoglycerol vs phosphocholine bilayers, but did not depend on surface tension. The difference in the cross-section area of the atypical and pre-active conformations makes the conformational transition sensitive to lateral pressure, and favors the atypical conformation upon surface tension. Anionic lipids act as allosteric modulators of the conformational transition, by stabilizing the atypical conformation. These findings contribute to decipher the mechanisms underlying AT1 activation, highlighting the influence of environmental factors on GPCR responses. Moreover, our results reveal the existence of intermediary conformations that depend on receptor environment and could be targeted in drug design efforts.

Hyperpolarization activated Cyclic Nucleotide (HCN) gated channels are crucial for various neurophysiological functions, including learning and sensory functions, and their dysfunction are responsible for brain disorders, such as epilepsy. To date, HCN2 variants have only been associated with mild epilepsy and recently, one monoallelic missense variant has been linked to developmental and epileptic encephalopathy. Here, we expand the phenotypic spectrum of HCN2- related disorders by describing twenty-one additional individuals from fifteen unrelated families carrying HCN2 variants. Seventeen individuals had developmental delay/intellectual disability (DD/ID), two had borderline DD/ID, and one had borderline DD. Ten individuals had epilepsy with DD/ID, with median age of onset of 10 months, and one had epilepsy with normal development. Molecular diagnosis identified thirteen different pathogenic HCN2 variants, including eleven missense variants affecting highly conserved amino acids, one frameshift variant, and one in-frame deletion. Seven variants were monoallelic of which five occurred de novo, one was not maternally inherited, one was inherited from a father with mild learning disabilities, and one was of unknown inheritance. The remaining six variants were biallelic, with four homozygous and two compound heterozygous variants. Functional studies using two-electrode voltage-clamp recordings in Xenopus laevis oocytes were performed on three monoallelic variants, p.(Arg324His), p.(Ala363Val), and p.(Met374Leu), and three biallelic variants, p.(Leu377His), p.(Pro493Leu) and p.(Gly587Asp). The p.(Arg324His) variant induced a strong increase of HCN2 conductance, while p.(Ala363Val) and p.(Met374Leu) displayed dominant negative effects, leading to a partial loss of HCN2 channel function. By confocal imaging, we found that the p.(Leu377His), p.(Pro493Leu) and p.(Gly587Asp) pathogenic variants impaired membrane trafficking, resulting in a complete loss of HCN2 elicited currents in Xenopus oocytes. Structural 3D-analysis in depolarized and hyperpolarized states of HCN2 channels, revealed that the pathogenic variants p.(His205Gln), p.(Ser409Leu), p.(Arg324Cys), p.(Asn369Ser) and p.(Gly460Asp) modify molecular interactions altering HCN2 function. Taken together, our data broadens the clinical spectrum associated with HCN2 variants, and disclose that HCN2 is involved in developmental encephalopathy with or without epilepsy.

The analysis of the relationship between sequence and structure similarities during the evolution of a protein family has revealed a limit of sequence divergence for which structural conservation can be confidently assumed and homology modeling is reliable. Below this limit, the twilight zone corresponds to sequence divergence for which homology modeling becomes increasingly difficult and requires specific methods. Either with conventional threading methods or with recent deep learning methods, such as AlphaFold, the challenge relies on the identification of a template that shares not only a common ancestor (homology) but also a conserved structure with the query. As both homology and structural conservation are transitive properties, mining of sequence databases followed by multidimensional scaling (MDS) of the query sequence space can reveal intermediary sequences to infer homology and structural conservation between the query and the template. Here, as a case study, we studied the plethodontid receptivity factor isoform 1 (PRF1) from Plethodon jordani, a member of a pheromone protein family present only in lungless salamanders and weakly related to cytokines of the IL6 family. A variety of conventional threading methods led to the cytokine CNTF as a template. Sequence mining, followed by phylogenetic and MDS analysis, provided missing links between PRF1 and CNTF and allowed reliable homology modeling. In addition, we compared automated models obtained from web servers to a customized model to show how modeling can be improved by expert information.

In vertebrates, the octopeptide angiotensin II (AngII) is an important in vivo regulator of the cardiovascular system. It acts mainly through two G protein-coupled receptors, AT1 and AT2. To better understand distinctive features of these receptors, we carried out a phylogenetic analysis that revealed a mirror evolution of AT1 and AT2, each one split into two clades, separating fish from terrestrial receptors. It also revealed that hallmark mutations occurred at, or near, the sodium binding site in both AT1 and AT2. Electrostatics computations and molecular dynamics simulations support maintained sodium binding to human AT1 with slow ingress from the extracellular side and an electrostatic component of the binding free energy around -3kT, to be compared to around -2kT for human AT2 and the δ opioid receptor. Comparison of the sodium binding modes in wild type and mutated AT1 and AT2 from humans and eels indicates that the allosteric control by sodium in both AT1 and AT2 evolved during the transition from fish to amniota. The unusual S7.46N mutation in AT1 is mirrored by a L3.36M mutation in AT2. In the presence of sodium, the N7.46 pattern in amniota AT1 stabilizes the inward orientation of N3.35 in the apo receptor, which should contribute to efficient N3.35 driven biased signaling. The M3.36 pattern in amniota AT2 favours the outward orientation of N3.35 and the receptor promiscuity. Both mutations have physiological consequences for the regulation of the renin-angiotensin system.

With 700 members, G protein-coupled receptors (GPCRs) of the rhodopsin family (class A) form the largest membrane receptor family in humans and are the target of about 30% of presently available pharmaceutical drugs. The recent boom in GPCR structures led to the structural resolution of 57 unique receptors in different states (39 receptors in inactive state only, 2 receptors in active state only and 16 receptors in different activation states). In spite of these tremendous advances, most computational studies on GPCRs, including molecular dynamics simulations, virtual screening and drug design, rely on GPCR models obtained by homology modeling. In this protocol, we detail the different steps of homology modeling with the MODELLER software, from template selection to model evaluation. The present structure boom provides closely related templates for most receptors. If, in these templates, some of the loops are not resolved, in most cases, the numerous available structures enable to find loop templates with similar length for equivalent loops. However, simultaneously, the large number of putative templates leads to model ambiguities that may require additional information based on multiple sequence alignments or molecular dynamics simulations to be resolved. Using the modeling of the human bradykinin receptor B1 as a case study, we show how several templates are managed by MODELLER, and how the choice of template(s) and of template fragments can improve the quality of the models. We also give examples of how additional information and tools help the user to resolve ambiguities in GPCR modeling.

Both dynamic correlations in protein sidechain motions during molecular dynamics (MD) simulations and evolutionary correlations in multiple sequence alignments (MSAs) of homologous proteins may reveal functionally important residues. We developed the R package Bios2cor that provides a unique framework to investigate and, possibly, integrate both analyses. Bios2cor starts with an MSA or an MD trajectory and computes correlation/covariation scores between positions in the MSA or between sidechain dihedral angles or rotamers in the MD trajectory. In addition, Bios2cor provides a variety of tools for the analysis, the visualization and the interpretation of the data.

ABSTRACTIn vertebrates, the octopeptide angiotensin II (AngII) is an importantin vivoregulator of the cardiovascular system. It acts mainly through two G protein-coupled receptors, AT1 and AT2. To better understand the interplay between these receptors throughout the evolution of the renin-angiotensin system (RAS), we combined a phylogenetic study to electrostatics computations and molecular dynamics (MD) simulations of AT1 and AT2 receptors from different species. The phylogenetic analysis reveals a mirror evolution of AT1 and AT2 that are both split in two clades, separating fish from terrestrian receptors. It also indicates that the unusual allosteric sodium binding site of human AT1 is specific of amniota. Other AT1 and AT2 receptors display a canonical sodium binding site with a serine at position 7.46 (Ballesteros numbering). Electrostatics computations and MD simulations support maintained sodium binding to human AT1 with ingress from the extracellular side. Comparison of the sodium binding modes in AT1 and AT2 from humans and eels indicates that the allosteric control by sodium in both AT1 and AT2 evolved during the transition from an aqueous to a terrestrial environment. The unusual S7.46N mutation in amniota AT1 is mirrored by a L3.36M mutation in amniota AT2. The S7.46N mutation increases the specificity of AT1 for AngII relative to Ang derivatives, whereas the L3.36M mutation might have the opposite effect on AT2. Both mutations should contribute to the split of the renin-angiotensin system into the classical (AngII/AT1) and counter-regulatory (Ang1-7/AT2, Mas) arms in amniota.AUTHOR SUMMARYThe analysis of protein sequences from different species can reveal interesting trends in the structural and functional evolution of a protein family. Here, we analyze the evolution of two G protein-coupled receptors, AT1 and AT2, which bind the angiotensin II peptide and are important regulators of the cardiovascular system. We show that these receptors underwent a mirror evolution and that specific mutations of the sodium binding pocket in both AT1 and AT2 occurred in amniota. We underwent electrostatics computations and molecular dynamics simulations to decipher the details of the sodium binding mode in eel and human receptors, as prototype of fish and amniota receptors. Our results suggest that evolution favored an increased specificity of AT1 and a decreased specificity of AT2 for angiotensin II as compared to its derivatives. In turn, these data suggest that mutations in the sodium binding pocket of G protein-coupled receptors might be an efficient way to gain functional evolution.

The dynamic structure of proteins is essential for their functions and may include large conformational transitions which can be studied by molecular dynamics (MD) simulations. However, details of these transitions are difficult to automatically track. To facilitate their analysis, we developed two scores of correlation between sidechain dihedral angles. The CIRCULAR and OMES scores are computed from, respectively, dihedral angle values and rotamer distributions. As a case study, we applied our methods to an activation-like transition of the chemokine receptor CXCR4, observed during accelerated MD simulations. The principal component analysis of the correlation matrices was consistent with the networking structure of the top ranking pairs. Both scores identify a set of residues whose "collaborative" sidechain rotamerization immediately preceded or accompanied the conformational transition of CXCR4. Detailed analysis of the sequential order of these rotamerizations suggests that an allosteric mechanism, involving the outward motion of an asparagine residue in transmembrane helix 3, might be a prerequisite to the large scale conformational transition of CXCR4. This case study provides the proof-of-concept that the correlation methods developed here are valuable exploratory techniques to help decipher complex reactional pathways.

Chemokines and their receptors (members of the GPCR super-family) are involved in a wide variety of physiological processes and diseases; thus, understanding the specificity of the chemokine receptor family could help develop new receptor specific drugs. Here, we explore the evolutionary mechanisms that led to the emergence of the chemokine receptors. Based on GPCR hierarchical classification, we analyzed nested GPCR sets with an eigen decomposition approach of the sequence covariation matrix and determined three key residues whose mutation was crucial for the emergence of the chemokine receptors and their subsequent divergence into homeostatic and inflammatory receptors. These residues are part of the allosteric sodium binding site. Their structural and functional roles were investigated by molecular dynamics simulations of CXCR4 and CCR5 as prototypes of homeostatic and inflammatory chemokine receptors, respectively. This study indicates that the three mutations crucial for the evolution of the chemokine receptors dramatically altered the sodium binding mode. In CXCR4, the sodium ion is tightly bound by four protein atoms and one water molecule. In CCR5, the sodium ion is mobile within the binding pocket and moves between different sites involving from one to three protein atoms and two to five water molecules. Analysis of chemokine receptor evolution reveals that a highly constrained sodium binding site characterized most ancient receptors, and that the constraints were subsequently loosened during the divergence of this receptor family. We discuss the implications of these findings for the evolution of the chemokine receptor functions and mechanisms of action.

Searchable abstracts of presentations at key conferences in endocrinology ISSN 1470-3947 (print) | ISSN 1479-6848 (online)

La prévalence du diabète de type 2 (DT2) est en augmentation dans le monde. De nombreuses études suggèrent que l’exposition aux perturbateurs endocriniens (PE) puisse participer à cette évolution. Leur mode d’action est méconnu. Au-delà de leurs effets sur les récepteurs nucléaires, nous posons l’hypothèse que les PE peuvent agir en modulant la fonctionnalité des récepteurs couplés aux protéines G impliqués dans la physiopathologie du DT2, telle que le récepteur aux acides gras GPR40 (FFAR1). Celui-ci augmente la sécrétion d’insuline dépendante du glucose. Nous avons testé l’effet du bisphénol A (BPA), du phtalate MEHP et du pesticide p,p’DDT sur la modulation de la signalisation intracellulaire de GPR40 en présence de deux ligands : l’acide linoléique et le GW9508. Le récepteur est surexprimé après transfection transitoire dans des cellules HEK293 et son activité mesurée en évaluant la mobilisation calcique intracellulaire. Nous montrons que 10−11M de BPA inhibe de 30 % la mobilisation calcique stimulée par GW9508. Le MEHP a une action inhibitrice de 35 % avec le GW9508 pour des concentrations de 10−5 à 10−8M, et de 25 % avec l’acide linoléique pour une concentration de 10−6M. Le p,p’DDT réduit, en présence des deux ligands, de 30 à 35 % avec du GW9508 à des concentrations de 10−5 à 10−9M, de 20 à 30 % en présence d’acide linoléique pour des concentrations de 10−7 et 10−8M. Nous montrons donc un nouveau mécanisme d’action possible des PE dans la survenue du DT2 via GPR40.

The urotensinergic system was previously considered as being linked to numerous physiopathological states, including atherosclerosis, heart failure, hypertension, pre-eclampsia, diabetes, renal disease, as well as brain vascular lesions. Thus, it turns out that the actions of the urotensin II (UII)/G protein-coupled receptor UT system in animal models are currently not predictive enough in regard to their effects in human clinical trials and that UII analogs, established to target UT, were not as beneficial as expected in pathological situations. Thus, many questions remain regarding the overall signaling profiles of UT leading to complex involvement in cardiovascular and inflammatory responses as well as cancer. We address the potential UT chemotactic structural and functional definition under an evolutionary angle, by the existence of a common conserved structural feature among chemokine receptorsopioïdergic receptors and UT, i.e., a specific proline position in the transmembrane domain-2 TM2 (P2.58) likely responsible for a kink helical structure that would play a key role in chemokine functions. Even if the last decade was devoted to the elucidation of the cardiovascular control by the urotensinergic system, we also attempt here to discuss the role of UII on inflammation and migration, likely providing a peptide chemokine status for UII. Indeed, our recent work established that activation of UT by a gradient concentration of UII recruits Gαi/o and Gα13 couplings in a spatiotemporal way, controlling key signaling events leading to chemotaxis. We think that this new vision of the urotensinergic system should help considering UT as a chemotactic therapeutic target in pathological situations involving cell chemoattraction.

Diabetes Mellitus is associated with severe cardiovascular disorders involving the renin-angiotensin system, mainly through activation of the angiotensin II type 1 receptor (AT1R). Although the type 2 receptor (AT2R) opposes the effects of AT1R, with vasodilator and anti-trophic properties, its role in diabetes is debatable. Thus we investigated AT2R-mediated dilatation in a model of type 1 diabetes induced by streptozotocin in 5-month-old male mice lacking AT2R (AT2R −/y ). Glucose tolerance was reduced and markers of inflammation and oxidative stress (cyclooxygenase-2, gp91phox p22phox and p67phox) were increased in AT2R −/y mice compared to wild-type (WT) animals. Streptozotocin-induced hyperglycaemia was higher in AT2R −/y than in WT mice. Arterial gp91phox and MnSOD expression levels in addition to blood 8-isoprostane and creatinine were further increased in diabetic AT2R −/y mice compared to diabetic WT mice. AT2R-dependent dilatation in both isolated mesenteric resistance arteries and perfused kidneys was greater in diabetic mice than in non-diabetic animals. Thus, in type 1 diabetes, AT2R may reduce glycaemia and display anti-oxidant and/or anti-inflammatory properties in association with greater vasodilatation in mesenteric arteries and in the renal vasculature, a major target of diabetes. Therefore AT2R might represent a new therapeutic target in diabetes.

Background: The presence of single nucleotide polymorphisms (SNPs) in the REarranged during Transfection (RET) gene has been investigated with regard to their potential role in the development or progression of medullary thyroid cancer or pheochromocytomas (PHEO) in patients with the multiple endocrine neoplasia type 2 (MEN2) syndrome. The aim of this study was to evaluate the spectrum of RET variants in France between 2003 and 2013, and to evaluate the impact of SNPs on the MEN2A phenotype. Methods: In this retrospective cohort study, RET variants were screened in 5109 index cases, and RET pathogenic variants were screened in 2214 relatives. Exons 5, 8, 10, 11, 13, 14, 15, and 16 were characterized by Sanger sequencing. RET pathogenic variants, RET variants with unknown functional significance (VUS), and four RET SNP variantsG691S (rs1799939), L769L (rs1800861), S836S (rs1800862), and S904S (rs1800863)were characterized and are reported in index cases. In silico analysis and classification following the recommendation of the American College of Medical Genetics and Genomics was performed for RET VUS. Each patient's age at the time of diagnosis, sex, and the endocrine neoplasias present at molecular diagnosis were recorded. Results: Twenty-six single VUS in RET without any well-defined risk profiles were found in 33 patients. Nine of these were considered probably pathogenic, 11 of uncertain significance, and six as probably benign. Three double pathogenic variants found in three patients were classified as pathogenic. A study of the entire cohort showed that patients carrying pathogenic variants or VUS in RET together with PHEO were diagnosed earlier than the others. The presence of the G691S SNP, or a combination of SNPs, increased the risk of developing PHEO but did not modify the date of the diagnosis. No association was found between SNPs and medullary thyroid cancer or hyperparathyroidism. Conclusions: The findings propose a classification of 15 of the 26 VUS in RET without any well-defined risk profiles and suggest that the G691S SNP, or a combination of SNPs, may be associated with the development of PHEO.

In a multiple sequence alignment, sequence co-variations result from structural, functional, and/or phylogenetic constraints. Numerous methods have been developed to calculate co-variation scores, but few studies have compared these methods to identify which methods are best suited for the analysis of protein family divergence. Here, we give an overview of widely used methods and identify simple rules for selection of appropriate methods. Specifically, we found that methods such as OMES and ELSC-which favor pairs with intermediate entropy and covariation networks with hub structure-are well suited to reveal evolutionary information on family divergence. When applied to G protein-coupled receptors, these methods support an epistasis model of protein evolution in which, after a key mutation, co-evolution of several residues was necessary to restore and/or shift protein function.