The TOPOVIBL meiotic DSB formation protein: new insights from its biochemical and structural characterization.

The TOPOVIL complex catalyzes the formation of DNA double strand breaks (DSB) that initiate meiotic homologous recombination, an essential step for chromosome segregation and genetic diversity during gamete production. TOPOVIL is composed of two subunits (SPO11 and TOPOVIBL) and is evolutionarily related to the archaeal TopoVI topoisomerase complex. SPO11 is the TopoVIA subunit orthologue and carries the DSB formation catalytic activity. TOPOVIBL shares homology with the TopoVIB ATPase subunit. TOPOVIBL is essential for meiotic DSB formation, but its molecular function remains elusive, partly due to the lack of biochemical studies. Here, we purified TOPOVIBLΔC25 and characterized its structure and mode of action in vitro. Our structural analysis revealed that TOPOVIBLΔC25 adopts a dynamic conformation in solution and our biochemical study showed that the protein remains monomeric upon incubation with ATP, which correlates with the absence of ATP binding. Moreover, TOPOVIBLΔC25 interacted with DNA, with a preference for some geometries, suggesting that TOPOVIBL senses specific DNA architectures. Altogether, our study identified specific TOPOVIBL features that might help to explain how TOPOVIL function evolved toward a DSB formation activity in meiosis.

Design and in vitro characterization of RXR variants as tools to investigate the biological role of endogenous rexinoids.

Retinoid X receptors (RXRα, ß, and γ) are essential members of the nuclear receptor (NR) superfamily of ligand-dependent transcriptional regulators that bind DNA response elements and control the expression of large gene networks. As obligate heterodimerization partners of many NRs, RXRs are involved in a variety of pathophysiological processes. However, despite this central role in NR signaling, there is still no consensus regarding the precise biological functions of RXRs and the putative role of the endogenous ligands (rexinoids) previously proposed for these receptors. Based on available crystal structures, we introduced a series of amino acid substitutions into the ligand-binding pocket of all three RXR subtypes in order to alter their binding properties. Subsequent characterization using a battery of cell-based and in vitro assays led to the identification of a double mutation abolishing the binding of any ligand while keeping the other receptor functions intact and a triple mutation that selectively impairs interaction with natural rexinoids but not with some synthetic ligands. We also report crystal structures that help understand the specific ligand-binding capabilities of both variants. These RXR variants, either fully disabled for ligand binding or retaining the property of being activated by synthetic compounds, represent unique tools that could be used in future studies to probe the presence of active endogenous rexinoids in tissues/organs and to investigate their role in vivo. Last, we provide data suggesting a possible involvement of fatty acids in the weak interaction of RXRs with corepressors.

Structural Insights into the Interaction of the Intrinsically Disordered Co-activator TIF2 with Retinoic Acid Receptor Heterodimer (RXR/RAR).

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 α-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.

PPARγ S273 Phosphorylation Modifies the Dynamics of Coregulator Proteins Recruitment.

The nuclear receptor PPARγ is essential to maintain whole-body glucose homeostasis and insulin sensitivity, acting as a master regulator of adipogenesis, lipid, and glucose metabolism. Its activation through natural or synthetic ligands induces the recruitment of coactivators, leading to transcription of target genes such as cytokines and hormones. More recently, post translational modifications, such as PPARγ phosphorylation at Ser273 by CDK5 in adipose tissue, have been linked to insulin resistance trough the dysregulation of expression of a specific subset of genes. Here, we investigate how this phosphorylation may disturb the interaction between PPARγ and some coregulator proteins as a new mechanism that may leads to insulin resistance. Through cellular and in vitro assays, we show that PPARγ phosphorylation inhibition increased the activation of the receptor, therefore the increased recruitment of PGC1-α and TIF2 coactivators, whilst decreases the interaction with SMRT and NCoR corepressors. Moreover, our results show a shift in the coregulators interaction domains preferences, suggesting additional interaction interfaces formed between the phosphorylated PPARγ and some coregulator proteins. Also, we observed that the CDK5 presence disturb the PPARγ-coregulator's synergy, decreasing interaction with PGC1-α, TIF2, and NCoR, but increasing coupling of SMRT. Finally, we conclude that the insulin resistance provoked by PPARγ phosphorylation is linked to a differential coregulators recruitment, which may promote dysregulation in gene expression.

IDPs and their complexes in GPCR and nuclear receptor signaling.

G protein-coupled receptors (GPCRs) and Nuclear Receptors (NRs) are two signaling machineries that are involved in major physiological processes and, as a consequence, in a substantial number of diseases. Therefore, they actually represent two major targets for drugs with potential applications in almost all public health issues. Full exploitation of these targets for therapeutic purposes nevertheless requires opening original avenues in drug design, and this in turn implies a better understanding of the molecular mechanisms underlying their functioning. However, full comprehension of how these complex systems function and how they are deregulated in a physiopathological context is obscured by the fact that these proteins include a substantial number of disordered regions that are central to their mechanism of action but whose structural and functional properties are still largely unexplored. In this chapter, we describe how these intrinsically disordered regions (IDR) or proteins (IDP) intervene, control and finely modulate the thermodynamics of complexes involved in GPCR and NR regulation, which in turn triggers a multitude of cascade of events that are exquisitely orchestrated to ultimately control the biological output.

Protein-protein interactions in the regulation of RAR-RXR heterodimers transcriptional activity.

The three retinoic acid receptor subtypes (RARα, RARß and RARγ) act as ligand-inducible transcription factors binding to DNA regulatory elements in the promoter regions of target genes by forming heterodimers with the retinoid X receptors (RXRα, RXRß and RXRγ). They act as ligand-dependent transcription factors that regulate a large variety of genes involved in cell growth, differentiation, survival and death. The (patho)physiological functions of RAR-RXR heterodimers rely on a dynamic sequence of protein-protein interactions, many of which being modulated by natural (retinoic acid) or synthetic ligands. Direct protein-protein interactions include heterodimerization between RARs and RXRs, recruitment (and release) of transcriptional coactivators and corepressors, cross-talk with other transcription factors, including nuclear receptors, or transient association with many enzymes involved in post-translational modifications to cite the most prominent ones. This chapter describes structural, biochemical, biophysical and cell-based assays to monitor protein-protein interactions relevant to the retinoic acid signaling pathways with a focus on those for which a structural description has been provided.

Two Novel Cases of Resistance to Thyroid Hormone Due to THRA Mutation.

Resistance to thyroid hormone alpha (RTHα) is a rare and under-recognized genetic disease caused by mutations of THRA, the gene encoding thyroid hormone receptor α1 (TRα1). We report here two novel THRA missense mutations (M259T, T273A) in patients with RTHα. We combined biochemical and cellular assays with in silico modeling to assess the capacity of mutant TRα1 to bind triiodothyronine (T3), to heterodimerize with RXR, to interact with transcriptional coregulators, and to transduce a T3 transcriptional response. M259T, and to a lower extent T273A, reduces the affinity of TRα1 for T3. Their negative influence is only reverted by large excess of T3. The severity of the two novel RTHα cases originates from a reduction in the binding affinity of TRα1 mutants to T3 and thus correlates with the incapacity of corepressors to dissociate from TRα1 mutants in the presence of T3.

Regulation of RXR-RAR Heterodimers by RXR- and RAR-Specific Ligands and Their Combinations.

The three subtypes (α, ß, and γ) of the retinoic acid receptor (RAR) are ligand-dependent transcription factors that mediate retinoic acid signaling by forming heterodimers with the retinoid X receptor (RXR). Heterodimers are functional units that bind ligands (retinoids), transcriptional co-regulators and DNA, to regulate gene networks controlling cell growth, differentiation, and death. Using biochemical, crystallographic, and cellular approaches, we have set out to explore the spectrum of possibilities to regulate RXR-RAR heterodimer-dependent transcription through various pharmacological classes of RAR- and RXR- specific ligands, alone or in combination. We reveal the molecular details by which these compounds direct specificity and functionality of RXR-RAR heterodimers. Among these ligands, we have reevaluated and improved the molecular and structural definition of compounds CD2665, Ro41-5253, LE135, or LG100754, highlighting novel functional features of these molecules. Our analysis reveals a model of RXR-RAR heterodimer action in which each subunit retains its intrinsic properties in terms of ligand and co-regulator binding. However, their interplay upon the combined action of RAR- and RXR-ligands allows for the fine tuning of heterodimer activity. It also stresses the importance of accurate ligand characterization to use synthetic selective retinoids appropriately and avoid data misinterpretations.

Interplay of Protein Disorder in Retinoic Acid Receptor Heterodimer and Its Corepressor Regulates Gene Expression.

In its unliganded form, the retinoic acid receptor (RAR) in heterodimer with the retinoid X receptor (RXR) exerts a strong repressive activity facilitated by the recruitment of transcriptional corepressors in the promoter region of target genes. By integrating complementary structural, biophysical, and computational information, we demonstrate that intrinsic disorder is a required feature for the precise regulation of RAR activity. We show that structural dynamics of RAR and RXR H12 regions is an essential mechanism for RAR regulation. Unexpectedly we found that, while mainly disordered, the corepressor N-CoR presents evolutionary conserved structured regions involved in transient intramolecular contacts. In the presence of RXR/RAR, N-CoR exploits its multivalency to form a cooperative multisite complex that displays equilibrium between different conformational states that can be tuned by cognate ligands and receptor mutations. This equilibrium is key to preserving the repressive basal state while allowing the conversion to a transcriptionally active form.

Pathological Interactions Between Mutant Thyroid Hormone Receptors and Corepressors and Their Modulation by a Thyroid Hormone Analogue with Therapeutic Potential.

BACKGROUND: Thyroid hormone receptors (TRs) are tightly regulated by the corepressors nuclear receptor corepressor (NCoR) and silencing mediator of retinoic acid and thyroid hormone receptors. Three conserved corepressor/NR signature box motifs (CoRNR1-3) forming the nuclear receptor interaction domain have been identified in these corepressors. Whereas TRs regulate multiple normal physiological and developmental pathways, mutations in TRs can result in endocrine diseases and be associated with cancers due to impairment of corepressor release. Three mutants that are located in helix H11 of TRs are of special interest: TRα-M388I, a mutant associated with the development of renal clear cell carcinomas (RCCCs), and TRß-Δ430 and TRß-Δ432, two deletion mutants causing resistance to thyroid hormone syndrome. METHODS: Several cell-based and biophysical methods were used to measure the affinity between wild-type and mutant TRα and TRß and all the CoRNR motifs from corepressors to quantify the effects of different thyroid hormone analogues on these interactions. This study was coupled with the measurement of interactions between wild-type and mutant TRs in the context of a heterodimer with RXR to a NCoR fragment in the presence of the same ligands. Structural insights into the binding mode of corepressors to TRs were assessed in parallel by nuclear magnetic resonance spectroscopy. RESULTS: The study shows that TRs interact more avidly with the silencing mediator of retinoic acid and thyroid hormone receptors than with NCoR peptides, and that TRα binds most avidly to S-CoRNR3, whereas TRß binds preferentially to S-CoRNR2. In the studied TR mutants, a transfer of the CoRNR-specificity toward CoRNR1 was observed, coupled with a significant increase in the binding strength. In contrast to 3,5,3'-triiodothyronine (T3), the agonist TRIAC and the antagonist NH-3 were very efficient at dissociating the abnormally strong interactions between mutant TRßs and corepressors. A strong impairment of T3-binding for TRß mutants was shown compared to TRIAC and NH-3 and could explain the different efficiencies of the different ligands in releasing corepressors from the studied TRß mutants. Consequently, TRIAC was found to be more effective than T3 in facilitating coactivator recruitment and decreasing the dominant activity of TRß-Δ430. CONCLUSION: This study helps to clarify the specific interaction surfaces involved in the pathologic phenotype of TR mutants and demonstrates that TRIAC is a potential therapeutic agent for patients suffering from resistance to thyroid hormone syndromes.

Screening for PPAR Non-Agonist Ligands Followed by Characterization of a Hit, AM-879, with Additional No-Adipogenic and cdk5-Mediated Phosphorylation Inhibition Properties.

Peroxisome proliferator-activated receptor gamma (PPARγ) is a member of a nuclear receptor superfamily and acts as a ligand-dependent transcription factor, playing key roles in maintenance of adipose tissue and in regulation of glucose and lipid homeostasis. This receptor is the target of thiazolidinediones, a class of antidiabetic drugs, which improve insulin sensitization and regulate glycemia in type 2 diabetes. Despite the beneficial effects of drugs, such as rosiglitazone and pioglitazone, their use is associated with several side effects, including weight gain, heart failure, and liver disease, since these drugs induce full activation of the receptor. By contrast, a promising activation-independent mechanism that involves the inhibition of cyclin-dependent kinase 5 (CDK5)-mediated PPARγ phosphorylation has been related to the insulin-sensitizing effects induced by these drugs. Thus, we aimed to identify novel PPARγ ligands that do not possess agonist properties by conducting a mini-trial with 80 compounds using the sequential steps of thermal shift assay, 8-anilino-1-naphthalenesulfonic acid fluorescence quenching, and a cell-based transactivation assay. We identified two non-agonist PPARγ ligands, AM-879 and P11, and one partial-agonist, R32. Using fluorescence anisotropy, we show that AM-879 does not dissociate the NCOR corepressor in vitro, and it has only a small effect on TRAP coactivator recruitment. In cells, AM-879 could not induce adipocyte differentiation or positively regulate the expression of genes associated with adipogenesis. In addition, AM-879 inhibited CDK5-mediated phosphorylation of PPARγ in vitro. Taken together, these findings supported an interaction between AM-879 and PPARγ; this interaction was identified by the analysis of the crystal structure of the PPARγ:AM-879 complex and evidenced by AM-879's mechanism of action as a putative PPARγ non-agonist with antidiabetic properties. Moreover, we present an optimized assay pipeline capable of detecting ligands that physically bind to PPARγ but do not cause its activation as a new strategy to identify ligands for this nuclear receptor.

The Human Mixed Lineage Leukemia 5 (MLL5), a Sequentially and Structurally Divergent SET Domain-Containing Protein with No Intrinsic Catalytic Activity.

Mixed Lineage Leukemia 5 (MLL5) plays a key role in hematopoiesis, spermatogenesis and cell cycle progression. Chromatin binding is ensured by its plant homeodomain (PHD) through a direct interaction with the N-terminus of histone H3 (H3). In addition, MLL5 contains a Su(var)3-9, Enhancer of zeste, Trithorax (SET) domain, a protein module that usually displays histone lysine methyltransferase activity. We report here the crystal structure of the unliganded SET domain of human MLL5 at 2.1 Å resolution. Although it shows most of the canonical features of other SET domains, both the lack of key residues and the presence in the SET-I subdomain of an unusually large loop preclude the interaction of MLL5 SET with its cofactor and substrate. Accordingly, we show that MLL5 is devoid of any in vitro methyltransferase activity on full-length histones and histone H3 peptides. Hence, the three dimensional structure of MLL5 SET domain unveils the structural basis for its lack of methyltransferase activity and suggests a new regulatory mechanism.

Molecular mechanisms of transcriptional control by Rev-erbα: An energetic foundation for reconciling structure and binding with biological function.

Rev-erbα and ß are nuclear receptors that function as transcriptional repressors of genes involved in regulating circadian rhythms, glucose, and cholesterol metabolism and the inflammatory response. Given these key functions, Rev-erbs are important drug targets for treatment of a number of human pathologies, including cancer, heart disease, and type II diabetes. Transcriptional repression by the Rev-erbs involves direct competition with transcriptional activators for target sites, but also recruitment by the Rev-erbs of the NCoR corepressor protein. Interestingly, Rev-erbs do not appear to interact functionally with a very similar corepressor, Smrt. Transcriptional repression by Rev-erbs is thought to occur in response to the binding of heme, although structural, and ligand binding studies in vitro show that heme and corepressor binding are antagonistic. We carried out systematic studies of the ligand and corepressor interactions to address the molecular basis for corepressor specificity and the energetic consequences of ligand binding using a variety of biophysical approaches. Highly quantitative fluorescence anisotropy assays in competition mode revealed that the Rev-erb specificity for the NCoR corepressor lies in the first two residues of the ß-strand in Interaction Domain 1 of NCoR. Our studies confirmed and quantitated the strong antagonism of heme and corepressor binding and significant stabilization of the corepressor complex by a synthetic ligand in vitro. We propose a model which reconciles the contradictory observations concerning the effects of heme binding in vitro and in live cells.

Retinoic acid receptors: structural basis for coregulator interaction and exchange.

In the form of heterodimers with retinoid X receptors (RXRs), retinoic acid receptors (RARs) are master regulators of gene expression in humans and important drug targets. They act as ligand-dependent transcription factors that regulate a large variety of gene networks controlling cell growth, differentiation, survival and death. The biological functions of RARs rely on a dynamic series of coregulator exchanges controlled by ligand binding. Unliganded RARs exert a repressor activity by interacting with transcriptional corepressors which themselves serve as docking platforms for the recruitment of histone deacetylases that impose a higher order structure on chromatin which is not permissive to gene transcription. Upon ligand binding, the receptor undergoes conformational changes inducing corepressor release and the recruitment of coactivators with histone acetylase activities allowing chromatin decompaction and gene transcription. In the following, we review the structural determinants of the interaction between RAR and either type of coregulators both at the level of the individual receptor and in the context of the RAR-RXR heterodimers. We also discuss the molecular details of the fine tuning of these associations by the various pharmacological classes of ligands.

Nuclear receptor profiling of bisphenol-A and its halogenated analogues.

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.

Structural and mechanistic insights into bisphenols action provide guidelines for risk assessment and discovery of bisphenol A substitutes.

Bisphenol A (BPA) is an industrial compound and a well known endocrine-disrupting chemical with estrogenic activity. The widespread exposure of individuals to BPA is suspected to affect a variety of physiological functions, including reproduction, development, and metabolism. Here we report that the mechanisms by which BPA and two congeners, bisphenol AF and bisphenol C (BPC), bind to and activate estrogen receptors (ER) α and ß differ from that used by 17ß-estradiol. We show that bisphenols act as partial agonists of ERs by activating the N-terminal activation function 1 regardless of their effect on the C-terminal activation function 2, which ranges from weak agonism (with BPA) to antagonism (with BPC). Crystallographic analysis of the interaction between bisphenols and ERs reveals two discrete binding modes, reflecting the different activities of compounds on ERs. BPA and 17ß-estradiol bind to ERs in a similar fashion, whereas, with a phenol ring pointing toward the activation helix H12, the orientation of BPC accounts for the marked antagonist character of this compound. Based on structural data, we developed a protocol for in silico evaluation of the interaction between bisphenols and ERs or other members of the nuclear hormone receptor family, such as estrogen-related receptor γ and androgen receptor, which are two known main targets of bisphenols. Overall, this study provides a wealth of tools and information that could be used for the development of BPA substitutes devoid of nuclear hormone receptor-mediated activity and more generally for environmental risk assessment.

Retinoid receptors and therapeutic applications of RAR/RXR modulators.

Retinoic acid receptors (RARs) are ligand-controlled transcription factors that function as heterodimers with retinoid X receptors (RXRs) to regulate cell growth, differentiation, survival and death. Due to their regulatory potential, these nuclear receptors (NRs) are major drug targets for a variety of pathologies, including cancer and metabolic diseases. A large amount of RAR- and RXR-selective ligands, ranging from (partial) agonists to antagonists and inverse agonists, have been designed and the corresponding structural and functional analyses have provided deep insight into the molecular basis of ligand action. Ligands regulate, via allosteric conformational changes, the ability of these NRs to interact with different sets of coregulators, which in turn recruit enzymatically active complexes/machineries. Here, we describe strategies in the design of selective RXR and RAR modulators and review the structural mechanisms by which the diverse pharmacological classes of compounds modulate receptor functions. Finally, we discuss the perspectives for retinoid- and rexinoid-based therapies.

In-plate protein crystallization, in situ ligand soaking and X-ray diffraction.

X-ray crystallography is now a recognized technique for ligand screening, especially for fragment-based drug design. However, protein crystal handling is still tedious and limits further automation. An alternative method for the solution of crystal structures of proteins in complex with small ligands is proposed. Crystallization drops are directly exposed to an X-ray beam after cocrystallization or soaking with the desired ligands. The use of dedicated plates in connection with an optimal parametrization of the G-rob robot allows efficient data collection. Three proteins currently under study in our laboratory for ligand screening by X-ray crystallography were used as validation test cases. The protein crystals belonged to different space groups, including a challenging monoclinic case. The resulting diffraction data can lead to clear ligand recognition, including indication of alternating conformations. These results demonstrate a possible method for automation of ligand screening by X-ray crystallography.

Characterization of novel ligands of ERα, Erß, and PPARγ: the case of halogenated bisphenol A and their conjugated metabolites.

The capability of the flame retardants tetrabromobisphenol A (TBBPA) and tetrachlorobisphenol A (TCBPA) to activate peroxysome proliferator-activated receptors (PPARs) α, ß, and γ and estrogen receptors (ERs) α and ß 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 PPARγ 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 PPARγ 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 PPARγ. In addition, PPARγ-based affinity column, HGELN-PPARγ 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 PPARγ-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 PPARγ activity.

Peroxisome proliferator-activated receptor γ is a target for halogenated analogs of bisphenol A.

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, ERß, 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.