Search

International audience; Understanding the transcriptional function of thyroid hormone receptors implies a precise analysis of their interactions with chromatin and other protein components of the cells. We present here two protocols that are routinely used in our laboratory. The first co-immunoprecipitation procedure allows addressing the capacity of proteins to form stable multiprotein complexes with TRs in cells. The chromatin affinity purification enables us to define the sites occupied by TRs on chromatin. In this case the lack of high quality antibodies is circumvented by introducing an N-terminal tag in TR, with unspecific affinity for immunoglobulins.

International audience; Bisphenol-A (BPA) is one of the highest-volume chemicals produced worldwide and the widespread exposure of individuals to BPA is suspected to affect a variety of physiological functions, including reproduction, development, and metabolism. Its estrogenic activity has been well documented in the last 15 years. In addition to estrogen receptors, BPA has been also shown to bind to and activate the estrogen-related receptor γ and pregnane X receptor and inhibit the androgen receptor. Halogenated BPAs were also shown to activate the peroxisome proliferator-activated receptor γ and inhibit thyroid hormone receptors. In this chapter, we review recent studies shedding light on the structural and molecular mechanisms by which BPA and its halogenated derivatives interfere with nuclear hormone receptor signaling. These data provide guidelines for the development of safer substitutes devoid of hormonal activity and may help environmental risk assessment.

International audience; Bisphenol-A (BPA) is one of the highest-volume chemicals produced worldwide and the widespread exposure of individuals to BPA is suspected to affect a variety of physiological functions, including reproduction, development, and metabolism. Its estrogenic activity has been well documented in the last 15 years. In addition to estrogen receptors, BPA has been also shown to bind to and activate the estrogen-related receptor γ and pregnane X receptor and inhibit the androgen receptor. Halogenated BPAs were also shown to activate the peroxisome proliferator-activated receptor γ and inhibit thyroid hormone receptors. In this chapter, we review recent studies shedding light on the structural and molecular mechanisms by which BPA and its halogenated derivatives interfere with nuclear hormone receptor signaling. These data provide guidelines for the development of safer substitutes devoid of hormonal activity and may help environmental risk assessment.

International audience; The capability of the flame retardants tetrabromobisphenol A (TBBPA) and tetrachlorobisphenol A (TCBPA) to activate peroxysome proliferator-activated receptors (PPARs) alpha, beta, and gamma and estrogen receptors (ERs) alpha and beta has been recently investigated, but the activity of their biotransformation products and of their lower molecular weight analogues formed in the environment remains unexplored. The aim of this study was to investigate the relationship between the degree of halogenation of BPA analogues and their affinity and activity towards human PPAR gamma and ERs and to characterize active metabolites of major marketed halogenated bisphenols. The biological activity of all compounds was studied using reporter cell lines expressing these nuclear receptors (NRs). We used NR-based affinity columns to rapidly evaluate the binding affinity of halogenated bisphenols for PPAR gamma and ERs and to trap active metabolites of TBBPA and TCBPA formed in HepG2 cells. The agonistic potential of BPA analogs highly depends on their halogenation degree: the bulkier halogenated BPA analogs, the greater their capability to activate PPAR gamma. In addition, PPAR gamma-based affinity column, HGELN-PPAR gamma reporter cell line and crystallographic analysis clearly demonstrate that the sulfation pathway, usually considered as a detoxification process, leads for TBBPA and TCBPA, to the formation of sulfate conjugates which possess a residual PPAR gamma-binding activity. Our results highlight the effectiveness NR-based affinity columns to trap and characterize biologically active compounds from complex matrices. Polyhalogenated bisphenols, but also some of their metabolites, are potential disrupters of PPAR gamma activity.

Human KIN17 (hKIN17) is highly conserved, more abundant in proliferating cellsthan in quiescent cells and overexpressed after UV irradiation and ionizing radiation. All thesesfeatures raised our interest for its structural and functional analysis at the molecular level in orderto describe its functions at the cellular level. Several properties have been described for thehKIN17 protein in different cellular mechanisms such as DNA replication, DNA repair and RNAmetabolism. During my project, I showed by combining data from CD, NMR and SAXS thathKIN17 has a structured domain probably exhibiting a zinc finger fold and a winged helixdomain in its N-terminus region. Only the first domain binds to nucleic acids. The second domainbinds to several RNA helicases involved in transcription and translation. Using X-raycrystallography, I solved the structure of the C-terminal domain which is found only in highereukaryotes and folds into a double SH3-like domain. I showed using different biochemicalmethods (molecular filtration, non denaturing IEF) that this domain anchors the hKIN17 proteinin a nuclear and acidic complex of high molecular weight, whose components are currentlyidentified by mass spectrometry. Moreover, this domain binds to RNA and modified histones.Taken together, these results confirm the involvement of hKIN17 in RNA metabolism and clarifythe role of each domain within the protein.; Très conservée, plus abondante dans les cellules en prolifération que dans les cellulesnormales, stimulée par les rayonnements UV et ionisants, autant de caractéristiques qui ontsuscité notre intérêt pour l'analyse structurale et fonctionnelle de la protéine KIN17 humaine(hKIN17) dans le but de caractériser sa ou ses fonction(s) dans la cellule. Plusieurs propriétés ontété décrites pour la protéine hKIN17 dans différents mécanismes cellulaires tels que la réplicationde l'ADN, la réparation de l'ADN et le métabolisme de l'ARN. Pendant ma thèse, j'ai montré encombinant des données de DC, RMN et SAXS que la protéine hKIN17 possède un domainestructuré adoptant probablement le repliement d'un doigt de zinc et un domaine winged helixdans sa région N-terminale. Seul le premier domaine lie les acides nucléiques, le deuxièmedomaine ayant pour partenaires plusieurs hélicases à ARN impliquées dans la transcription et latraduction. J'ai résolu par cristallographie aux rayons X la structure du domaine C-terminal quiest retrouvé uniquement chez les eucaryotes supérieurs et se replie en un double domaine de typeSH3. J'ai montré par différentes méthodes biochimiques (filtration sur gel, gel IEF natif) que cedomaine ancre la protéine hKIN17 à un complexe nucléaire acide de haut poids moléculaire dontles composants sont en cours d'identification par spectrométrie de masse. De plus, il lie l'ARN etles histones modifiées. L'ensemble de ces résultats confirment l'implication de la protéinehKIN17 dans le métabolisme de l'ARN et précisent le rôle de chacun des domaines au sein de laprotéine.

Human KIN17 (hKIN17) is highly conserved, more abundant in proliferating cellsthan in quiescent cells and overexpressed after UV irradiation and ionizing radiation. All thesesfeatures raised our interest for its structural and functional analysis at the molecular level in orderto describe its functions at the cellular level. Several properties have been described for thehKIN17 protein in different cellular mechanisms such as DNA replication, DNA repair and RNAmetabolism. During my project, I showed by combining data from CD, NMR and SAXS thathKIN17 has a structured domain probably exhibiting a zinc finger fold and a winged helixdomain in its N-terminus region. Only the first domain binds to nucleic acids. The second domainbinds to several RNA helicases involved in transcription and translation. Using X-raycrystallography, I solved the structure of the C-terminal domain which is found only in highereukaryotes and folds into a double SH3-like domain. I showed using different biochemicalmethods (molecular filtration, non denaturing IEF) that this domain anchors the hKIN17 proteinin a nuclear and acidic complex of high molecular weight, whose components are currentlyidentified by mass spectrometry. Moreover, this domain binds to RNA and modified histones.Taken together, these results confirm the involvement of hKIN17 in RNA metabolism and clarifythe role of each domain within the protein., Très conservée, plus abondante dans les cellules en prolifération que dans les cellulesnormales, stimulée par les rayonnements UV et ionisants, autant de caractéristiques qui ontsuscité notre intérêt pour l'analyse structurale et fonctionnelle de la protéine KIN17 humaine(hKIN17) dans le but de caractériser sa ou ses fonction(s) dans la cellule. Plusieurs propriétés ontété décrites pour la protéine hKIN17 dans différents mécanismes cellulaires tels que la réplicationde l'ADN, la réparation de l'ADN et le métabolisme de l'ARN. Pendant ma thèse, j'ai montré encombinant des données de DC, RMN et SAXS que la protéine hKIN17 possède un domainestructuré adoptant probablement le repliement d'un doigt de zinc et un domaine winged helixdans sa région N-terminale. Seul le premier domaine lie les acides nucléiques, le deuxièmedomaine ayant pour partenaires plusieurs hélicases à ARN impliquées dans la transcription et latraduction. J'ai résolu par cristallographie aux rayons X la structure du domaine C-terminal quiest retrouvé uniquement chez les eucaryotes supérieurs et se replie en un double domaine de typeSH3. J'ai montré par différentes méthodes biochimiques (filtration sur gel, gel IEF natif) que cedomaine ancre la protéine hKIN17 à un complexe nucléaire acide de haut poids moléculaire dontles composants sont en cours d'identification par spectrométrie de masse. De plus, il lie l'ARN etles histones modifiées. L'ensemble de ces résultats confirment l'implication de la protéinehKIN17 dans le métabolisme de l'ARN et précisent le rôle de chacun des domaines au sein de laprotéine.

Journal of molecular biology 433(9), 166899 (2021). doi:10.1016/j.jmb.2021.166899, Retinoic acid receptors (RARs) and retinoid X receptors (RXRs) form heterodimers that activate target gene transcription by recruiting co-activator complexes in response to ligand binding. The nuclear receptor (NR) co-activator TIF2 mediates this recruitment by interacting with the ligand-binding domain (LBD) of NRs trough the nuclear receptor interaction domain (TIF2NRID) containing three highly conserved ��-helical LxxLL motifs (NR-boxes). The precise binding mode of this domain to RXR/RAR is not clear due to the disordered nature of TIF2. Here we present the structural characterization of TIF2NRID by integrating several experimental (NMR, SAXS, Far-UV CD, SEC-MALS) and computational data. Collectively, the data are in agreement with a largely disordered protein with partially structured regions, including the NR-boxes and their flanking regions, which are evolutionary conserved. NMR and X-ray crystallographic data on TIF2NRID in complex with RXR/RAR reveal a multisite binding of the three NR-boxes as well as an active role of their flanking regions in the interaction., Published by Elsevier, Amsterdam [u.a.]

Orientadores: Ana Carolina Migliorini Figueira, Albane le Maire Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia Resumo: O Receptor de Ácido Retinóico (RAR) é uma proteína pertencente à família dos receptores nucleares que atua na transcrição de genes relacionados ao crescimento e proliferação celular, homeostase e no metabolismo. Essa regulação da transcrição é mediada pela interação com seu ligante natural, o ácido retinóico. Na presença desse ligante, o RAR é capaz de recrutar moléculas coativadoras que interagem com outras proteínas da maquinaria de transcrição. Porém, na ausência do ligante, o RAR consegue reprimir a transcrição ao interagir com moléculas correpressoras, que são proteínas multidomínios capazes de recrutar enzimas que impedem a maquinaria de transcrição de atuar em determinado gene. Anomalias neste tipo de regulação podem relacionar o RAR e outros RNs a doenças, como é o caso da Leucemia Promielocítica Aguda (APL), uma condição patológica que resulta em uma alta proliferação de células promielóides anormais. Nesta condição, o RAR é expresso fusionado à Proteína da Leucemia Mielóide (PML) e se mantem inativo, sendo que a hipótese mais aceita é que esta fusão PML-RAR dificulta o desligamento de moléculas correpressoras, e por consequência, impede que haja a ativação de genes que diferenciem essas células promielóides. Esta tese buscou compreender detalhes moleculares do mecanismo de repressão nos quais essas proteínas estão envolvidas. Para isso foram realizados diversos ensaios que consistem primeiramente na (a) expressão das proteínas normais (selvagem) e de condição patológica (mutante PLM-RAR), (b) estudos de interação entre RAR/PML-RAR e correpressores, bem como sua interação nos complexos com RXR e DNA, (c) montagem dos complexos com correpressores, e (d) estudos estruturais dessas proteínas e complexos.Os resultados obtidos nesta pesquisa apontam para uma nova abordagem no entendimento de como a presença da proteína PML-RAR atua na APL. Ensaios de DLS e de estabilidade térmica apontam para a capacidade do PML-RAR de gerar grandes complexos organizados, com características beta-amiloides, como identificado em ensaios de ThT. Através destes resultados levantamos a hipótese de que o PML-RAR, ao formar estes grandes complexos, impede a ação do RAR nas vias de regulação gênica (interação com DNA e correguladores) e do PML nas suas funções nucleares, através do sequestro de proteínas normais/selvagens (RAR e PML) Abstract: The Retinoic Acid Receptor (RAR) is part of the nuclear receptors superfamily , and acts as transcription regulators of genes related to cell growth and proliferation, homeostasis and metabolism. This process is mediated by the interaction of RAR with its natural ligand, the retinoic acid. In the presence of its ligand, RAR is able to recruit coactivators that are capable of interacting with other proteins from the transcription machinery. However, in the absence of ligand, RAR can repress transcription by interacting with corepressors, which are multidomain proteins capable of recruiting enzymes that prevent transcription machinery to function in a particular gene. In this way many diseases are related to RAR and to nuclear receptors in general. This is the case of acute promyelocytic leukemia (APL), a pathological condition which presents a high promieloids abnormal cell proliferation . This occurs because the RAR is expressed fused to Protein Myelogenous Leukemia (PML), and behaves repressed. Until now, the most accepted explanation for this repression is that RAR-PML impairs the corepressors' dissociation and, consequently, prevents the activation of genes that are involved in these promieloid cells differentiation. In this sense, this study aimed to understand the molecular mechanism of repression in which RAR is involved. For this purpose, several tests which consist primarily on (a) expression of normal proteins (wild type) and in pathological conditions (PLM-RAR mutant) were performed together with (b) the study of the nuclear receptors and corepressores interactions , as well as, the interaction of these complexes with DNA, (c) the assembling of the NR-corepressores complexes , and (d) the structural studies of these complexes. The results obtained in this research point to a new approach in understanding how the presence of the PML-RAR protein acts in the APL. DLS and thermal stability assays point to the ability of PML-RAR to generate large organized complexes, with beta-amyloid characteristics, as identified in ThT assay. Through these results we raise the hypothesis that PML-RAR, when forming these large complexes, prevents the action of RAR in the pathways of gene regulation (interaction with DNA and co-regulators) and PML in its nuclear functions, through the sequestration of normal/wild proteins (RAR and PML) Doutorado Fármacos, Medicamentos e Insumos para Saúde Doutora em Ciências FAPESP 2016/02348-2 CAPES 001

International audience; Background: The occurrence of halogenated analogs of the xenoestrogen bisphenol A (BPA) has been recently demonstrated both in environmental and human samples. These analogs include brominated [e.g., tetrabromobisphenol A (TBBPA)] and chlorinated [e.g., tetrachlorobisphenol A (TCBPA)] bisphenols, which are both flame retardants. Because of their structural homology with BPA, such chemicals are candidate endocrine disruptors. However, their possible target(s) within the nuclear hormone receptor superfamily has remained unknown.Objectives: We investigated whether BPA and its halogenated analogs could be ligands of estrogen receptors (ERs) and peroxisome proliferator–activated receptors (PPARs) and act as endocrine-disrupting chemicals.Methods: We studied the activity of compounds using reporter cell lines expressing ERs and PPARs. We measured the binding affinities to PPARγ by competitive binding assays with [3H]‑rosiglitazone and investigated the impact of TBBPA and TCBPA on adipocyte differentiation using NIH3T3-L1 cells. Finally, we determined the binding mode of halogenated BPAs to PPARγ by X‑ray crystallography.Results: We observed that TBBPA and TCBPA are human, zebrafish, and Xenopus PPARγ ligands and determined the mechanism by which these chemicals bind to and activate PPARγ. We also found evidence that activation of ERα, ERβ, and PPARγ depends on the degree of halogenation in BPA analogs. We observed that the bulkier brominated BPA analogs, the greater their capability to activate PPARγ and the weaker their estrogenic potential.Conclusions: Our results strongly suggest that polyhalogenated bisphenols could function as obesogens by acting as agonists to disrupt physiological functions regulated by human or animal PPARγ.