Phosphorylation of PPAR? Affects the Collective Motions of the PPAR?-RXR?-DNA Complex.
ABSTRACT: Peroxisome-proliferator activated receptor-? (PPAR?) is a nuclear hormone receptor that forms a heterodimeric complex with retinoid X receptor-? (RXR?) to regulate transcription of genes involved in fatty acid storage and glucose metabolism. PPAR? is a target for pharmaceutical intervention in type 2 diabetes, and insight into interactions between PPAR?, RXR?, and DNA is of interest in understanding the function and regulation of this complex. Phosphorylation of PPAR? by cyclin-dependent kinase 5 (Cdk5) has been shown to dysregulate the expression of metabolic regulation genes, an effect that is counteracted by PPAR? ligands. We applied molecular dynamics (MD) simulations to study the relationship between the ligand-binding domains of PPAR? and RXR? with their respective DNA-binding domains. Our results reveal that phosphorylation alters collective motions within the PPAR?-RXR? complex that affect the LBD-LBD dimerization interface and the AF-2 coactivator binding region of PPAR?.
Project description:Nuclear receptors are multi-domain transcription factors that bind to DNA elements from which they regulate gene expression. The peroxisome proliferator-activated receptors (PPARs) form heterodimers with the retinoid X receptor (RXR), and PPAR-gamma has been intensively studied as a drug target because of its link to insulin sensitization. Previous structural studies have focused on isolated DNA or ligand-binding segments, with no demonstration of how multiple domains cooperate to modulate receptor properties. Here we present structures of intact PPAR-gamma and RXR-alpha as a heterodimer bound to DNA, ligands and coactivator peptides. PPAR-gamma and RXR-alpha form a non-symmetric complex, allowing the ligand-binding domain (LBD) of PPAR-gamma to contact multiple domains in both proteins. Three interfaces link PPAR-gamma and RXR-alpha, including some that are DNA dependent. The PPAR-gamma LBD cooperates with both DNA-binding domains (DBDs) to enhance response-element binding. The A/B segments are highly dynamic, lacking folded substructures despite their gene-activation properties.
Project description:Nuclear receptors retinoic X receptor ? (RXR?) and peroxisome proliferator activated receptor ? (PPAR?) function potently in metabolic diseases, and are both important targets for anti-diabetic drugs. Coactivation of RXR? and PPAR? is believed to synergize their effects on glucose and lipid metabolism. Here we identify the natural product magnolol as a dual agonist targeting both RXR? and PPAR?. Magnolol was previously reported to enhance adipocyte differentiation and glucose uptake, ameliorate blood glucose level and prevent development of diabetic nephropathy. Although magnolol can bind and activate both of these two nuclear receptors, the transactivation assays indicate that magnolol exhibits biased agonism on the transcription of PPAR-response element (PPRE) mediated by RXR?:PPAR? heterodimer, instead of RXR-response element (RXRE) mediated by RXR?:RXR? homodimer. To further elucidate the molecular basis for magnolol agonism, we determine both the co-crystal structures of RXR? and PPAR? ligand-binding domains (LBDs) with magnolol. Structural analyses reveal that magnolol adopts its two 5-allyl-2-hydroxyphenyl moieties occupying the acidic and hydrophobic cavities of RXR? L-shaped ligand-binding pocket, respectively. While, two magnolol molecules cooperatively accommodate into PPAR? Y-shaped ligand-binding pocket. Based on these two complex structures, the key interactions for magnolol activating RXR? and PPAR? are determined. As the first report on the dual agonist targeting RXR? and PPAR? with receptor-ligand complex structures, our results are thus expected to help inspect the potential pharmacological mechanism for magnolol functions, and supply useful hits for nuclear receptor multi-target ligand design.
Project description:The nuclear receptor retinoid X receptor-alpha (RXR-alpha)-peroxisome proliferator-activated receptor-gamma (PPAR-gamma) heterodimer was recently reported to have a crucial function in mediating the deleterious effects of organotin compounds, which are ubiquitous environmental contaminants. However, because organotins are unrelated to known RXR-alpha and PPAR-gamma ligands, the mechanism by which these compounds bind to and activate the RXR-alpha-PPAR-gamma heterodimer at nanomolar concentrations has remained elusive. Here, we show that tributyltin (TBT) activates all three RXR-PPAR-alpha, -gamma, -delta heterodimers, primarily through its interaction with RXR. In addition, the 1.9 A resolution structure of the RXR-alpha ligand-binding domain in complex with TBT shows a covalent bond between the tin atom and residue Cys 432 of helix H11. This interaction largely accounts for the high binding affinity of TBT, which only partly occupies the RXR-alpha ligand-binding pocket. Our data allow an understanding of the binding and activation properties of the various organotins and suggest a mechanism by which these tin compounds could affect other nuclear receptor signalling pathways.
Project description:PPAR? is a key regulator of glucose homeostasis and insulin sensitization. PPAR? must heterodimerize with its dimeric partner, the retinoid X receptor (RXR), to bind DNA and associated coactivators such as p160 family members or PGC-1? to regulate gene networks. To understand how coactivators are recognized by the functional heterodimer PPAR?/RXR? and to determine the topological organization of the complexes, we performed a structural study using small angle X-ray scattering of PPAR?/RXR? in complex with DNA from regulated gene and the TIF2 receptor interacting domain (RID). The solution structures reveal an asymmetry of the overall structure due to the crucial role of the DNA in positioning the heterodimer and indicate asymmetrical binding of TIF2 to the heterodimer.
Project description:The nuclear receptor peroxisome proliferator-activated receptor ? (PPAR?) is the master transcriptional regulator in adipogenesis. PPAR? forms a heterodimer with another nuclear receptor, retinoid X receptor (RXR), to form an active transcriptional complex, and their transcriptional activity is tightly regulated by the association with either coactivators or corepressors. In this study, we identified T-cell death-associated gene 51 (TDAG51) as a novel corepressor of PPAR?-mediated transcriptional regulation. We showed that TDAG51 expression is abundantly maintained in the early stage of adipogenic differentiation. Forced expression of TDAG51 inhibited adipocyte differentiation in 3T3-L1 cells. We found that TDAG51 physically interacts with PPAR? in a ligand-independent manner. In deletion mutant analyses, large portions of the TDAG51 domains, including the pleckstrin homology-like, glutamine repeat and proline-glutamine repeat domains but not the proline-histidine repeat domain, are involved in the interaction with the region between residues 140 and 506, including the DNA binding domain, hinge, ligand binding domain and activation function-2 domain, in PPAR?. The heterodimer formation of PPAR?-RXR? was competitively inhibited in a ligand-independent manner by TDAG51 binding to PPAR?. Thus, our data suggest that TDAG51, which could determine adipogenic cell fate, acts as a novel negative regulator of PPAR? by blocking RXR? recruitment to the PPAR?-RXR? heterodimer complex in adipogenesis.
Project description:Genome-wide profiling of PPAR?:RXR and RNA polymerase II reveals temporal activation of distinct metabolic pathways in RXR dimer composition during adipogenesis. Chromatin immunoprecipitation combined with deep sequencing was performed to generate genome-wide maps of peroxisome prolifelator-activated receptor gamma (PPARg) and retinoid X receptor (RXR) binding sites, and RNA polymerase II (RNAPII) occupancy at high resolution throughout adipocyte differentiation of 3T3-L1 cells. The data provides the first positional and temporal map PPAR? and RXR occupancy during adipocyte differentiation at a global scale. The number of PPAR?:RXR shared binding sites is steadily increasing from D0 to D6. At Day6 there are over 5000 high confidence shared PPARy:RXR binding sites. We show that at the early days of differentiation several of these sites bind not only PPAR?:RXR but also other RXR dimers. The data also provides a comprehensive temporal map of RNAPII occupancy at genes throughout 3T3-L1 adipogenesis thereby uncovering groups of similarly regulated genes belonging to glucose and lipid metabolic pathways. The majority of the upregulated but very few downregulated genes have assigned PPAR?:RXR target sites, thereby underscoring the importance of PPAR?:RXR in gene activation during adipogenesis and indicating that a hitherto unrecognized high number of adipocyte genes are directly activated by PPAR?:RXR Examination of PPARg and RXR bindingsites during adipocyte differentiation (day 0 to 6) and association with transcription via RNAPII occupancy.
Project description:The ability of a retinoid X receptor (RXR) to heterodimerize with many nuclear receptors, including LXR, PPAR, NGF1B and RAR, underscores its pivotal role within the nuclear receptor superfamily. Among these heterodimers, PPAR:RXR is considered an important signalling mediator of both PPAR ligands, such as fatty acids, and 9-cis retinoic acid (9-cis RA), an RXR ligand. In contrast, the existence of an RXR/9-cis RA signalling pathway independent of PPAR or any other dimerization partner remains disputed. Using in vivo chromatin immunoprecipitation, we now show that RXR homodimers can selectively bind to functional PPREs and induce transactivation. At the molecular level, this pathway requires stabilization of the homodimer-DNA complexes through ligand-dependent interaction with the coactivator SRC1 or TIF2. This pathway operates both in the absence and in the presence of PPAR, as assessed in cells carrying inactivating mutations in PPAR genes and in wild-type cells. In addition, this signalling pathway via PPREs is fully functional and can rescue the severe hypothermia phenotype observed in fasted PPARalpha-/- mice. These observations have important pharmacological implications for the development of new rexinoid-based treatments.
Project description:Liver X receptor (LXR) and peroxisome proliferator-activated receptor (PPAR) are two members of nuclear receptors involved in the nutrient metabolisms of dietary fatty acid and cholesterol. They are found to be of cross-talk function in that LXR regulates fatty acid synthesis and PPAR controls fatty acid degradation. LXRs (LXRalpha and LXRbeta) function by forming obligate heterodimers with the retinoid X receptor (RXR), and subsequently binding to specific DNA response elements within the regulatory regions of their target genes. In this work, the kinetic features concerning LXR/RXR and LXR/PPAR interactions have been fully investigated using surface plasmon resonance (SPR) technology. It is found that LXRs could bind to all the three PPAR subtypes, PPARalpha, PPARgamma and PPARdelta with different binding affinities, and such receptor/receptor interactions could be regulated by ligand binding. Moreover, molecular dynamics (MD) simulations were performed on six typical complex models. The results revealed that ligands may increase the interaction energies between the receptor interfaces of the simulated receptor/receptor complexes. The MD results are in agreement with the SPR data. Further analyses on the MD results indicated that the ligand binding might increase the hydrogen bonds between the interfaces of the receptor/receptor complex.
Project description:Peroxisome proliferator-activated receptor gamma (PPAR?) has recently been revealed to regulate tumor microenvironments. In particular, genetic alterations of PPAR? found in various cancers have been reported to play important roles in tumorigenesis by affecting PPAR? transactivation. In this study, we found that helix H3 of the PPAR? ligand-binding domain (LBD) has a number of sites that are mutated in cancers. To uncover underlying molecular mechanisms between helix H3 mutations and tumorigenesis, we performed structure?function studies on the PPAR? LBDs containing helix H3 mutations found in cancers. Interestingly, PPAR? Q286E found in bladder cancer induces a constitutively active conformation of PPAR? LBD and thus abnormal activation of PPAR?/RXR? pathway, which suggests tumorigenic roles of PPAR? in bladder cancer. In contrast, other helix H3 mutations found in various cancers impair ligand binding essential for transcriptional activity of PPAR?. These data indicate that cancer-associated mutations clustered in helix H3 of PPAR? LBD exhibit differential effects in PPAR?-mediated tumorigenesis and provide a basis for the development of new biomarkers targeting tumor microenvironments.
Project description:The peroxisome proliferator-activated receptors (PPARs) regulate genes involved in lipid and carbohydrate metabolism, and are targets of drugs approved for human use. Whereas the crystallographic structure of the complex of full length PPAR? and RXR? is known, structural alterations induced by heterodimer formation and DNA contacts are not well understood. Herein, we report a small-angle X-ray scattering analysis of the oligomeric state of hPPAR? alone and in the presence of retinoid X receptor (RXR). The results reveal that, in contrast with other studied nuclear receptors, which predominantly form dimers in solution, hPPAR? remains in the monomeric form by itself but forms heterodimers with hRXR?. The low-resolution models of hPPAR?/RXR? complexes predict significant changes in opening angle between heterodimerization partners (LBD) and extended and asymmetric shape of the dimer (LBD-DBD) as compared with X-ray structure of the full-length receptor bound to DNA. These differences between our SAXS models and the high-resolution crystallographic structure might suggest that there are different conformations of functional heterodimer complex in solution. Accordingly, hydrogen/deuterium exchange experiments reveal that the heterodimer binding to DNA promotes more compact and less solvent-accessible conformation of the receptor complex.