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Abstract Background The distance matrix computed from multiple alignments of homologous sequences is widely used by distance-based phylogenetic methods to provide information on the evolution of protein families. This matrix can also be visualized in a low dimensional space by metric multidimensional scaling (MDS). Applied to protein families, MDS provides information complementary to the information derived from tree-based methods. Moreover, MDS gives a unique opportunity to compare orthologous sequence sets because it can add supplementary elements to a reference space. Results The R package bios2mds (from BIOlogical Sequences to MultiDimensional Scaling) has been designed to analyze multiple sequence alignments by MDS. Bios2mds starts with a sequence alignment, builds a matrix of distances between the aligned sequences, and represents this matrix by MDS to visualize a sequence space. This package also offers the possibility of performing K-means clustering in the MDS derived sequence space. Most importantly, bios2mds includes a function that projects supplementary elements (a.k.a. “out of sample” elements) onto the space defined by reference or “active” elements. Orthologous sequence sets can thus be compared in a straightforward way. The data analysis and visualization tools have been specifically designed for an easy monitoring of the evolutionary drift of protein sub-families. Conclusions The bios2mds package provides the tools for a complete integrated pipeline aimed at the MDS analysis of multiple sets of orthologous sequences in the R statistical environment. In addition, as the analysis can be carried out from user provided matrices, the projection function can be widely used on any kind of data.

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., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Ben Boubaker et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

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., (© 2023. Springer Science+Business Media, LLC, part of Springer Nature.)

Background: Identifying factors that are predictive for postpartum posttraumatic stress disorder (PTSD) is important to inform clinical and research practice. Yet prospective longitudinal studies investigating symptoms of postpartum PTSD and their prevalence, evolution, comorbidities, and predictors remain limited. The aim of this study was to estimate the prevalence of women's symptoms of PTSD at different times in the postpartum period and to identify comorbidities and predictive factors in a French-speaking sample., Methods: A total of 168 women participated in this longitudinal study, which included 3 assessment points: immediate postpartum, 2 months postpartum, and 6 months postpartum. Participants filled out questionnaires regarding sociodemographic characteristics, pregnancy, and birth outcomes; subjective perceptions of birth; and symptoms of depression, anxiety, and PTSD., Results: Among the participants, 11.7% reported having symptoms of PTSD 2 months after birth and 10.5% reported having symptoms 6 months after birth. Regarding comorbidities and associated factors, depressive symptoms, poor marital adjustment, and impaired maternal-infant bonding were significantly and positively correlated with symptoms of PTSD. Peritraumatic distress, negative perceived childbirth experience, and complications and perceived difficulties during birth were predictive factors for postpartum PTSD., Discussion: More than 1 in 10 women experienced symptoms of postpartum PTSD. Health care professionals need to be aware of symptoms of postpartum PTSD, predictive factors, and comorbidities to be able to better identify women presenting those symptoms and refer them for appropriate psychological support., (© 2022 by the American College of Nurse-Midwives.)

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., Competing Interests: The authors have declared that no competing interests exist.

Summary: 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., Availability and Implementation: The R package Bios2cor is available from the Comprehensive R Archive Network, at https://CRAN.R-project.org/package=Bios2cor., (© The Author(s) 2021. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Background: A woman's negative perception of her subjective childbirth experience can have consequences on the mother's psychological state and on early mother-baby relationships. To date, there is no validated tool in France allowing to evaluate childbirth experience in a multidimensional way. The aim of this study is to validate the Questionnaire Assessing the Childbirth Experience (QEVA) in a French sample of mothers. This tool was developed in a previous study where the authors combined 25 items into 6 dimensions: representations and expectations, sensory perceptions, feeling of control, perceived social support (medical staff and partner), emotions (positive and negative) and first moments with the baby., Methods: The sample included 256 women recruited in a maternity ward. Sociodemographic and obstetric characteristics of our sample were compared to those of the French national perinatal survey. The structure of the QEVA with 17 items was explored by an exploratory structural equation modeling (ESEM). An analysis of the internal consistency was conducted on the sub-scores of the identified factors, and the concurrent validity was assessed with the Peri-traumatic Distress Inventory (PDI) through a correlation and its associated t-test., Results: The characteristics of our sample and those of the national perinatal survey do not differ on age, marital status, parity, cannabis use, infertility treatment, epidural and baby weight, in favour of the good representativeness of our sample. The study of the QEVA structure revealed a 4-dimensional structure. Analysis of the psychometric qualities showed a good internal consistency, with an observed alpha value ranging from 0.69 to 0.86. The QEVA also shows a good concurrent validity with the peri-traumatic distress scores (r=0.51)., Conclusion: To date, the QEVA is the first standardized tool allowing a multidimensional evaluation of the subjective experience of childbirth. It has been validated on a French population using an exploratory structural equation modeling. This tool, which is simple to use and well accepted by mothers, enables health professionals not only to screen mothers experiencing difficult childbirth and in need of support, but also to adapt health care according to the dimensions of the birth experience and its associated difficulties (emotions during the birth, interactions with health professionals, first moments with the baby, or post-partum emotions)., (Copyright © 2020 L'Encéphale, Paris. Published by Elsevier Masson SAS. All rights reserved.)

Objectives: To describe the prevalence of negative childbirth experiences and to identify potential predictors, including demographic, prenatal, obstetric, and psychological factors, of these experiences among French women., Design: Descriptive, correlational, cross-sectional study., Setting: A maternity ward in a hospital located near Paris, France., Participants: A total of 256 women between the ages of 18 and 46 years were recruited while hospitalized in the maternity ward 1 to 6 days after childbirth., Methods: Participants completed a personal information form and five self-report scales: the Dyadic Adjustment Scale, the General Self-Efficacy Scale, the State-Trait Anxiety Inventory, the Relationship Scales Questionnaire, and the Questionnaire Assessing the Childbirth Experience. We performed linear regression analyses and used scores on the Questionnaire Assessing the Childbirth Experience as the dependent variable. We considered prenatal, psychological, and obstetric factors as independent variables and adjusted results for covariates., Results: The prevalence of a negative childbirth experience was 23.3% among our participants. We identified primiparity, high anxiety trait scores, and an anxious attachment style as the prenatal variables that contributed significantly to negative perceptions of childbirth based on the first step of the regression analysis (R 2  = .18; p < .001). We evaluated objective birth-related variables during the second step and found that mode of birth and use of epidural analgesia were significative predictors of the negative childbirth experience (R 2  = 0.36; p < .001). The last set of variables included subjective birth-related factors, such as absence of the partner during birth and low perceived sense of control; these variables increased the explained variance from 36% to 69% (p < .001) and showed that these aspects were powerful predictors of a negative childbirth experience., Conclusions: Given that some prenatal factors influence women's perceptions of their birth experiences, preventive measures can be implemented by health professionals to support women with risk factors. In the early postpartum period, health care professionals should focus on subjective variables of childbirth because they strongly predict the overall childbirth experience for women., Competing Interests: Conflict of Interest The authors report no conflicts of interest or relevant financial relationships., (Copyright © 2021 AWHONN, the Association of Women’s Health, Obstetric and Neonatal Nurses. Published by Elsevier Inc. All rights reserved.)

Objectives: The aims of this study were to identify the determinants and the vulnerability factors of women's mental health in the immediate postpartum period by investigating the first symptoms of anxiety, depression and peri-traumatic distress., Methods: 256 women participated in this cross-sectional and descriptive study. They responded during their stay in the maternity ward to a set of questionnaires between the 1st and the 6th day after delivery. This included an anamnestic questionnaire as well as different scales that evaluated generalized self-efficacy feeling (GSES), marital adjustment (DAS), perceived sense of control during labor and delivery (LAS), birth experience (QEVA), anxiety manifestations (STAI-Y), depressive symptoms (EPDS) and peri-traumatic distress (PDI)., Results: Symptoms of anxiety, depression and peri-traumatic distress in the immediate postpartum period, as indicators of women's mental health, are predicted by different determinants. An anxious personality and perceived complications during childbirth for the woman or baby have been shown to be significant predictors of postpartum anxiety. Symptoms of depression are related to a history of depression, a low overall sense of general efficacy and lower satisfaction in the marital relationship. Peri-traumatic distress is related to certain dimensions of the childbirth experience, such as perceived sense of control, perceived complications and emotions felt during birth., Conclusions: Symptoms of depression, anxiety and peritraumatic distress are linked to aspects of the prenatal period, but also to the experience of childbirth. More specific prevention, screening and care measures, depending on the woman's symptomatology, can be implemented during pregnancy or at the maternity. Further research seems essential to better understand the interactions between the prenatal period, childbirth and postpartum in explaining women's mental health in the immediate postpartum period., (Copyright © 2020 Elsevier Masson SAS. All rights reserved.)

Background : Up to 33% of women report a negative or traumatic childbirth experience. Given this high prevalence and its consistent association with adverse postpartum and child outcomes, it is essential to identify predictive factors and to improve the management of the childbirth experience. Objective : This systematic review explores and identifies risk and protective factors for women's subjective childbirth experience and birth satisfaction by reviewing original research. Methods : A systematic search was performed for childbirth experience literature on three online databases. Reviewed papers focused on women's subjective childbirth experience and its predictive factors. The articles were assessed with the Mixed Methods Appraisal Tool (MMAT). Results : Risk and protective factors are notably different depending on the study design, the country, or the method employed. The main risk factors are obstetric, such as emergency caesarean and highly perceived labour pain, and women's dissatisfaction with social support. The main protective factors are: obstetric, including highly perceived control during labour or satisfaction regarding partner's support. However, overall results are inconclusive for methodological or conceptual reasons. Conclusions : Several risk factors can be identified through pregnancy or childbirth. This underlines the importance of the quality of maternal interpersonal and professional relationships, especially with first-line perinatal health-care professionals, such as midwives.

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., (© 2021. Springer Science+Business Media, LLC, part of Springer Nature.)

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., Competing Interests: The authors have declared that no competing interests exist.

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 MEN2 A 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 variants-G691S (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.

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.

Aims: The angiotensin II type 1 receptor (AT1R) through the activation of immune cells plays a key role in arterial inward remodelling and reduced blood flow in cardiovascular disorders. On the other side, flow (shear stress)-mediated outward remodelling (FMR), involved in collateral arteries growth in ischaemic diseases, allows revascularization. We hypothesized that the type 2 receptor (AT2R), described as opposing the effects of AT1R, could be involved in FMR., Methods and Results: We studied FMR using a model of ligation of feed arteries supplying collateral pathways in the mouse mesenteric arterial bed in vivo. Seven days after ligation, diameter increased by 30% in high flow (HF) arteries compared with normal flow vessels. FMR was absent in mice lacking AT2R. At Day 2, T lymphocytes expressing AT2R were present preferentially around HF arteries. FMR did not occur in athymic (nude) mice lacking T cells and in mice treated with anti-CD3ε antibodies. AT2R activation induced interleukin-17 production by memory T cells. Treatment of nude mice or AT2R-deficient mice with interleukin-17 restored diameter enlargement in HF arteries. Interleukin-17 increased NO-dependent relaxation and matrix metalloproteinases activity, both important in FMR. Remodelling of feeding arteries in the skin flap model of ischaemia was also absent in AT2R-deficient mice and in anti-interleukin-17-treated mice. Finally, remodelling, absent in 12-month-old mice, was restored by a treatment with the AT2R non-peptidic agonist C21., Conclusion: AT2R-dependent interleukin-17 production by T lymphocyte is necessary for collateral artery growth and could represent a new therapeutic target in ischaemic disorders., (Published on behalf of the European Society of Cardiology. All rights reserved. © The Author 2016. For permissions please email: journals.permissions@oup.com.)

Background: 1-chloro-4-[2,2,2-trichloro-1-(4-chlorophenyl)ethyl]benzene (p,p'-DDT) is a persistent environmental endocrine disruptor (ED). Several studies have shown an association between p,p'-DDT exposure and reproductive abnormalities., Objectives: To investigate the putative effects of p,p'-DDT on the human follitropin receptor (FSHR) function., Methods and Results: We used Chinese hamster ovary (CHO) cells stably expressing human FSHR to investigate the impact of p,p'-DDT on FSHR activity and its interaction with the receptor. At a concentration of 5 μM p,p'-DDT increased the maximum response of the FSHR to follitropin by 32 ± 7.45%. However, 5 μM p,p'-DDT decreased the basal activity and did not influence the maximal response of the closely related LH/hCG receptor to human chorionic gonadotropin (hCG). The potentiating effect of p,p'-DDT was specific for the FSHR. Moreover, in cells that did not express FSHR, p,p'-DDT had no effect on cAMP response. Thus, the potentiating effect of p,p'-DDT was dependent on the FSHR. In addition, p,p'-DDT increased the sensitivity of FSHR to hCG and to a low molecular weight agonist of the FSHR, 3-((5methyl)-2-(4-benzyloxy-phenyl)-5-{[2-[3-ethoxy-4-methoxy-phenyl)-ethylcarbamoyl]-methyl}-4-oxo-thiazolidin-3-yl)-benzamide (16a). Basal activity in response to p,p'-DDT and potentiation of the FSHR response to FSH by p,p'-DDT varied among FSHR mutants with altered transmembrane domains (TMDs), consistent with an effect of p,p'-DDT via TMD binding. This finding was corroborated by the results of simultaneously docking p,p'-DDT and 16a into the FSHR transmembrane bundle., Conclusion: p,p'-DDT acted as a positive allosteric modulator of the FSHR in our experimental model. These findings suggest that G protein-coupled receptors are additional targets of endocrine disruptors., Citation: Munier M, Grouleff J, Gourdin L, Fauchard M, Chantreau V, Henrion D, Coutant R, Schiøtt B, Chabbert M, Rodien P. 2016. In vitro effects of the endocrine disruptor p,p'-DDT on human follitropin receptor. Environ Health Perspect 124:991-999; http://dx.doi.org/10.1289/ehp.1510006.

The thyrotropin receptor (TSHR) is a G protein-coupled receptor (GPCR) that is member of the leucine-rich repeat subfamily (LGR). In the absence of crystal structure, the success of rational design of ligands targeting the receptor internal cavity depends on the quality of the TSHR models built. In this subfamily, transmembrane helices (TM) 2 and 5 are characterized by the absence of proline compared to most receptors, raising the question of the structural conformation of these helices. To gain insight into the structural properties of these helices, we carried out bioinformatics and experimental studies. Evolutionary analysis of the LGR family revealed a deletion in TM5 but provided no information on TM2. Wild type residues at positions 2.58, 2.59 or 2.60 in TM2 and/or at position 5.50 in TM5 were substituted to proline. Depending on the position of the proline substitution, different effects were observed on membrane expression, glycosylation, constitutive cAMP activity and responses to thyrotropin. Only proline substitution at position 2.59 maintained complex glycosylation and high membrane expression, supporting occurrence of a bulged TM2. The TSHR transmembrane domain was modeled by homology with the orexin 2 receptor, using a protocol that forced the deletion of one residue in the TM5 bulge of the template. The stability of the model was assessed by molecular dynamics simulations. TM5 straightened during the equilibration phase and was stable for the remainder of the simulations. Our data support a structural model of the TSHR transmembrane domain with a bulged TM2 and a straight TM5 that is specific of glycoprotein hormone receptors.

Covariation between positions in a multiple sequence alignment may reflect structural, functional, and/or phylogenetic constraints and can be analyzed by a wide variety of methods. We explored several of these methods for their ability to identify covarying positions related to the divergence of a protein family at different hierarchical levels. Specifically, we compared seven methods on a model system composed of three nested sets of G-protein-coupled receptors (GPCRs) in which a divergence event occurred. The covariation methods analyzed were based on: χ2 test, mutual information, substitution matrices, and perturbation methods. We first analyzed the dependence of the covariation scores on residue conservation (measured by sequence entropy), and then we analyzed the networking structure of the top pairs. Two methods out of seven--OMES (Observed minus Expected Squared) and ELSC (Explicit Likelihood of Subset Covariation)--favored pairs with intermediate entropy and a networking structure with a central residue involved in several high-scoring pairs. This networking structure was observed for the three sequence sets. In each case, the central residue corresponded to a residue known to be crucial for the evolution of the GPCR family and the subfamily specificity. These central residues can be viewed as evolutionary hubs, in relation with an epistasis-based mechanism of functional divergence within a protein family., (© 2014 Wiley Periodicals, Inc.)

Class A G-protein-coupled receptors (GPCRs) provide a fascinating example of evolutionary success. In this review, we discuss how metric multidimensional scaling (MDS), a multivariate analysis method, complements traditional tree-based phylogenetic methods and helps decipher the mechanisms that drove the evolution of class A GPCRs. MDS provides low-dimensional representations of a distance matrix. Applied to a multiple sequence alignment, MDS represents the sequences in a Euclidean space as points whose interdistances are as close as possible to the distances in the alignment (the so-called sequence space). We detail how to perform the MDS analysis of a multiple sequence alignment and how to analyze and interpret the resulting sequence space. We also show that the projection of supplementary data (a property of the MDS method) can be used to straightforwardly monitor the evolutionary drift of specific subfamilies. The sequence space of class A GPCRs reveals the key role of mutations at the level of the TM2 and TM5 proline residues in the evolution of class A GPCRs., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Class A G-protein-coupled receptors (GPCRs) constitute the largest family of transmembrane receptors in the human genome. Understanding the mechanisms which drove the evolution of such a large family would help understand the specificity of each GPCR sub-family with applications to drug design. To gain evolutionary information on class A GPCRs, we explored their sequence space by metric multidimensional scaling analysis (MDS). Three-dimensional mapping of human sequences shows a non-uniform distribution of GPCRs, organized in clusters that lay along four privileged directions. To interpret these directions, we projected supplementary sequences from different species onto the human space used as a reference. With this technique, we can easily monitor the evolutionary drift of several GPCR sub-families from cnidarians to humans. Results support a model of radiative evolution of class A GPCRs from a central node formed by peptide receptors. The privileged directions obtained from the MDS analysis are interpretable in terms of three main evolutionary pathways related to specific sequence determinants. The first pathway was initiated by a deletion in transmembrane helix 2 (TM2) and led to three sub-families by divergent evolution. The second pathway corresponds to the differentiation of the amine receptors. The third pathway corresponds to parallel evolution of several sub-families in relation with a covarion process involving proline residues in TM2 and TM5. As exemplified with GPCRs, the MDS projection technique is an important tool to compare orthologous sequence sets and to help decipher the mutational events that drove the evolution of protein families.