Comparison of activity, structure, and dynamics of SF-1 and LRH-1 complexed with small molecule modulators.

Steroidogenic factor-1 (SF-1) is a phospholipid-sensing nuclear receptor expressed in the adrenal glands, gonads, and hypothalamus which controls steroidogenesis and metabolism. There is significant therapeutic interest in SF-1 because of its oncogenic properties in adrenocortical cancer. Synthetic modulators are attractive for targeting SF-1 for clinical and laboratory purposes due to the poor pharmaceutical properties of its native phospholipid ligands. While small molecule agonists targeting SF-1 have been synthesized, no crystal structures have been reported of SF-1 in complexes with synthetic compounds. This has prevented the establishment of structure-activity relationships that would enable better characterization of ligand-mediated activation and improvement in current chemical scaffolds. Here, we compare the effects of small molecules in SF-1 and its close homolog, liver receptor homolog-1 (LRH-1), and identify several molecules that specifically activate LRH-1. We also report the first crystal structure of SF-1 in complex with a synthetic agonist that displays low nanomolar affinity and potency for SF-1. We use this structure to explore the mechanistic basis for small molecule agonism of SF-1, especially compared to LRH-1, and uncover unique signaling pathways that drive LRH-1 specificity. Molecular dynamics simulations reveal differences in protein dynamics at the pocket mouth as well as ligand-mediated allosteric communication from this region to the coactivator binding interface. Our studies, therefore, shed important insight into the allostery driving SF-1 activity and show potential for modulation of LRH-1 over SF-1.

A peroxisomal ubiquitin ligase complex forms a retrotranslocation channel.

Peroxisomes are ubiquitous organelles that house various metabolic reactions and are essential for human health1-4. Luminal peroxisomal proteins are imported from the cytosol by mobile receptors, which then recycle back to the cytosol by a poorly understood process1-4. Recycling requires receptor modification by a membrane-embedded ubiquitin ligase complex comprising three RING finger domain-containing proteins (Pex2, Pex10 and Pex12)5,6. Here we report a cryo-electron microscopy structure of the ligase complex, which together with biochemical and in vivo experiments reveals its function as a retrotranslocation channel for peroxisomal import receptors. Each subunit of the complex contributes five transmembrane segments that co-assemble into an open channel. The three ring finger domains form a cytosolic tower, with ring finger 2 (RF2) positioned above the channel pore. We propose that the N terminus of a recycling receptor is inserted from the peroxisomal lumen into the pore and monoubiquitylated by RF2 to enable extraction into the cytosol. If recycling is compromised, receptors are polyubiquitylated by the concerted action of RF10 and RF12 and degraded. This polyubiquitylation pathway also maintains the homeostasis of other peroxisomal import factors. Our results clarify a crucial step during peroxisomal protein import and reveal why mutations in the ligase complex cause human disease.

Structural basis of Tom20 and Tom22 cytosolic domains as the human TOM complex receptors.

Mitochondrial preproteins synthesized in cytosol are imported into mitochondria by a multisubunit translocase of the outer membrane (TOM) complex. Functioned as the receptor, the TOM complex components, Tom 20, Tom22, and Tom70, recognize the presequence and further guide the protein translocation. Their deficiency has been linked with neurodegenerative diseases and cardiac pathology. Although several structures of the TOM complex have been reported by cryoelectron microscopy (cryo-EM), how Tom22 and Tom20 function as TOM receptors remains elusive. Here we determined the structure of TOM core complex at 2.53 Å and captured the structure of the TOM complex containing Tom22 and Tom20 cytosolic domains at 3.74 Å. Structural analysis indicates that Tom20 and Tom22 share a similar three-helix bundle structural feature in the cytosolic domain. Further structure-guided biochemical analysis reveals that the Tom22 cytosolic domain is responsible for binding to the presequence, and the helix H1 is critical for this binding. Altogether, our results provide insights into the functional mechanism of the TOM complex recognizing and transferring preproteins across the mitochondrial membrane.

Binding Affinity Measurement of Nuclear Export Signal Peptides to Their Exporter CRM1.

CRM1 recognizes hundreds to thousands of protein cargoes by binding to the eight to fifteen residue-long nuclear export signals (NESs) within their polypeptide chains. Various assays to measure the binding affinity of NESs for CRM1 have been developed. CRM1 binds to NESs with a wide range of binding affinities, with dissociation constants that span from low nanomolar to tens of micromolar. An optimized binding affinity assay with improved throughput was recently developed to measure binding affinities of NES peptides for CRM1 in the presence of excess RanGTP. The assay can measure affinities, with multiple replicates, for up to seven different NES peptides per screening plate. Here, we present a protocol for the purification of the necessary proteins and for measuring CRM1-NES binding affinities.

Crystallization of Nuclear Export Signals or Small-Molecule Inhibitors Bound to Nuclear Exporter CRM1.

The Karyopherin protein CRM1 or XPO1 is the major nuclear export receptor that regulates nuclear exit of thousands of macromolecules in the cell. CRM1 recognizes protein cargoes by binding to their 8-15 residue-long nuclear export signals (NESs). A ternary CRM1-Ran-RanBP1 complex engineered to be suitable for crystallization has enabled structure determination by X-ray crystallography of CRM1 bound to many NES peptides and small-molecule inhibitors. Here, we present a protocol for the purification of the individual proteins, formation of the ternary CRM1-Ran-RanBP1 complex and crystallization of this complex for X-ray crystallography.

Proglumide Reverses Nonalcoholic Steatohepatitis by Interaction with the Farnesoid X Receptor and Altering the Microbiome.

Nonalcoholic steatohepatitis (NASH) is associated with obesity, metabolic syndrome, and dysbiosis of the gut microbiome. Cholecystokinin (CCK) is released by saturated fats and plays an important role in bile acid secretion. CCK receptors are expressed on cholangiocytes, and CCK-B receptor expression increases in the livers of mice with NASH. The farnesoid X receptor (FXR) is involved in bile acid transport and is a target for novel therapeutics for NASH. The aim of this study was to examine the role of proglumide, a CCK receptor inhibitor, in a murine model of NASH and its interaction at FXR. Mice were fed a choline deficient ethionine (CDE) diet to induce NASH. Some CDE-fed mice received proglumide-treated drinking water. Blood was collected and liver tissues were examined histologically. Proglumide's interaction at FXR was evaluated by computer modeling, a luciferase reporter assay, and tissue FXR expression. Stool microbiome was analyzed by RNA-Sequencing. CDE-fed mice developed NASH and the effect was prevented by proglumide. Computer modeling demonstrated specific binding of proglumide to FXR. Proglumide binding in the reporter assay was consistent with a partial agonist at the FXR with a mean binding affinity of 215 nM. FXR expression was significantly decreased in livers of CDE-fed mice compared to control livers, and proglumide restored FXR expression to normal levels. Proglumide therapy altered the microbiome signature by increasing beneficial and decreasing harmful bacteria. These data highlight the potential novel mechanisms by which proglumide therapy may improve NASH through interaction with the FXR and consequent alteration of the gut microbiome.

Structural insight into the molecular mechanism of cilofexor binding to the farnesoid X receptor.

Farnesoid X receptor (FXR) is a bile acid-related nuclear receptor and is considered a promising target to treat several liver disorders. Cilofexor is a selective FXR agonist and has already entered phase III trials in primary sclerosing cholangitis (PSC) patients. Pruritis caused by cilofexor treatment is dose dependent. The binding characteristics of cilofexor with FXR and its pruritogenic mechanism remain unclear. In our research, the affinity of cilofexor bound to FXR was detected using an isothermal titration calorimetry (ITC) assay. The binding mechanism between cilofexor and FXR-LBD is explained by the cocrystal structure of the FXR/cilofexor complex. Structural models indicate the possibility that cilofexor activates Mas-related G protein-coupled receptor X4 (MRGPRX4) or G protein-coupled bile acid receptor 1 (GPBAR1), leading to pruritus. In summary, our analyses provide a molecular mechanism of cilofexor binding to FXR and provide a possible explanation for the dose-dependent pruritis of cilofexor.

Co-Translational Membrane Targeting and Holo-Translocon Docking of Ribosomes Translating the SRP Receptor.

Many integral membrane proteins are produced by translocon-associated ribosomes. The assembly of ribosomes translating membrane proteins on the translocons is mediated by a conserved system, composed of the signal recognition particle and its receptor (FtsY in Escherichia coli). FtsY is a peripheral membrane protein, and its role late during membrane protein targeting involves interactions with the translocon. However, earlier stages in the pathway have remained obscure, namely, how FtsY targets the membrane in vivo and where it initially docks. Our previous studies have demonstrated co-translational membrane-targeting of FtsY translation intermediates and identified a nascent FtsY targeting-peptide. Here, in a set of in vivo experiments, we utilized tightly stalled FtsY translation intermediates, pull-down assays and site-directed cross-linking, which revealed FtsY-nascent chain-associated proteins in the cytosol and on the membrane. Our results demonstrate interactions between the FtsY-translating ribosomes and cytosolic chaperones, which are followed by directly docking on the translocon. In support of this conclusion, we show that translocon over-expression increases dramatically the amount of membrane associated FtsY-translating ribosomes. The co-translational contacts of the FtsY nascent chains with the translocon differ from its post-translational contacts, suggesting a major structural maturation process. The identified interactions led us to propose a model for how FtsY may target the membrane co-translationally. On top of our past observations, the current results may add another tier to the hypothesis that FtsY acts stoichiometrically in targeting ribosomes to the membrane in a constitutive manner.

A study of a hierarchical structure of proteins and ligand binding sites of receptors using the triangular spatial relationship-based structure comparison method and development of a size-filtering feature designed for comparing different sizes of protein structures.

The presence of receptors and the specific binding of the ligands determine nearly all cellular responses. Binding of a ligand to its receptor causes conformational changes of the receptor that triggers the subsequent signaling cascade. Therefore, systematically studying structures of receptors will provide insight into their functions. We have developed the triangular spatial relationship (TSR)-based method where all possible triangles are constructed with Cα atoms of a protein as vertices. Every triangle is represented by an integer denoted as a "key" computed through the TSR algorithm. A structure is thereby represented by a vector of integers. In this study, we have first defined substructures using different types of keys. Second, using different types of keys represents a new way to interpret structure hierarchical relations and differences between structures and sequences. Third, we demonstrate the effects of sequence similarity as well as sample size on the structure-based classifications. Fourth, we show identification of structure motifs, and the motifs containing multiple triangles connected by either an edge or a vertex are mapped to the ligand binding sites of the receptors. The structure motifs are valuable resources for the researchers in the field of signal transduction. Next, we propose amino-acid scoring matrices that capture "evolutionary closeness" information based on BLOSUM62 matrix, and present the development of a new visualization method where keys are organized according to evolutionary closeness and shown in a 2D image. This new visualization opens a window for developing tools with the aim of identification of specific and common substructures by scanning pixels and neighboring pixels. Finally, we report a new algorithm called as size filtering that is designed to improve structure comparison of large proteins with small proteins. Collectively, we provide an in-depth interpretation of structure relations through the detailed analyses of different types of keys and their associated key occurrence frequencies, geometries, and labels. In summary, we consider this study as a new computational platform where keys are served as a bridge to connect sequence and structure as well as structure and function for a deep understanding of sequence, structure, and function relationships of the protein family.

CRM1 Promotes Capsid Disassembly and Nuclear Envelope Translocation of Adenovirus Independently of Its Export Function.

After receptor-mediated endocytosis and endosomal escape, adenoviral capsids can travel via microtubule organizing centers to the nuclear envelope. Upon capsid disassembly, viral genome import into nuclei of interphase cells then occurs through nuclear pore complexes, involving the nucleoporins Nup214 and Nup358. Import also requires the activity of the classic nuclear export receptor CRM1, as it is blocked by the selective inhibitor leptomycin B. We have now used artificially enucleated as well as mitotic cells to analyze the role of an intact nucleus in different steps of the viral life cycle. In enucleated U2OS cells, viral capsids traveled to the microtubule organizing center, whereas their removal from this complex was blocked, suggesting that this step required nuclear factors. In mitotic cells, on the other hand, CRM1 promoted capsid disassembly and genome release, suggesting a role of this protein that does not require intact nuclear envelopes or nuclear pore complexes and is distinct from its function as a nuclear export receptor. Similar to enucleation, inhibition of CRM1 by leptomycin B also leads to an arrest of adenoviral capsids at the microtubule organizing center. In a small-scale screen using leptomycin B-resistant versions of CRM1, we identified a mutant, CRM1 W142A P143A, that is compromised with respect to adenoviral capsid disassembly in both interphase and mitotic cells. Strikingly, this mutant is capable of exporting cargo proteins out of the nucleus of living cells or digitonin-permeabilized cells, pointing to a role of the mutated region that is not directly linked to nuclear export. IMPORTANCE A role of nucleoporins and of soluble transport factors in adenoviral genome import into the nucleus of infected cells in interphase has previously been established. The nuclear export receptor CRM1 promotes genome import, but its precise function is not known. Using enucleated and mitotic cells, we showed that CRM1 does not simply function by exporting a crucial factor out of the nucleus that would then trigger capsid disassembly and genome import. Instead, CRM1 has an export-independent role, a notion that is also supported by a mutant, CRM1 W142A P143A, which is export competent but deficient in viral capsid disassembly, in both interphase and mitotic cells.

Recent Advances in the Medicinal Chemistry of Farnesoid X Receptor.

Farnesoid X receptor (FXR) is an important regulator of bile acid, lipid, amino acid, and glucose homeostasis, hepatic inflammation, regeneration, and fibrosis. FXR has been recognized as a promising drug target for various metabolic diseases such as lipid disorders, nonalcoholic fatty liver disease (NAFLD), nonalcoholic steatohepatitis (NASH), and chronic kidney disease. A large number of FXR ligands have been developed by pharmaceutical companies and academic institutions, and several candidates have progressed into clinical trials in the past decade. However, it is continually a challenge to discover drugs targeting FXR due to side effects associated with long-term administration. In this perspective, we summarize the research progress on medicinal chemistry of FXR modulators from 2018 to the present by discussing the diverse structures of synthetic FXR modulators including steroidal and non-steroidal ligands, their structure-activity relationships (SARs), and their therapeutic applications.

Atg39 selectively captures inner nuclear membrane into lumenal vesicles for delivery to the autophagosome.

Mechanisms that turn over components of the nucleus and inner nuclear membrane (INM) remain to be fully defined. We explore how components of the INM are selected by a cytosolic autophagy apparatus through a transmembrane nuclear envelope-localized cargo adaptor, Atg39. A split-GFP reporter showed that Atg39 localizes to the outer nuclear membrane (ONM) and thus targets the INM across the nuclear envelope lumen. Consistent with this, sequence elements that confer both nuclear envelope localization and a membrane remodeling activity are mapped to the Atg39 lumenal domain; these lumenal motifs are required for the autophagy-mediated degradation of integral INM proteins. Interestingly, correlative light and electron microscopy shows that the overexpression of Atg39 leads to the expansion of the ONM and the enclosure of a network of INM-derived vesicles in the nuclear envelope lumen. Thus, we propose an outside-in model of nucleophagy where INM is delivered into vesicles in the nuclear envelope lumen, which can be targeted by the autophagosome.

Prediction of Drug-Target Interactions by Combining Dual-Tree Complex Wavelet Transform with Ensemble Learning Method.

Identification of drug-target interactions (DTIs) is vital for drug discovery. However, traditional biological approaches have some unavoidable shortcomings, such as being time consuming and expensive. Therefore, there is an urgent need to develop novel and effective computational methods to predict DTIs in order to shorten the development cycles of new drugs. In this study, we present a novel computational approach to identify DTIs, which uses protein sequence information and the dual-tree complex wavelet transform (DTCWT). More specifically, a position-specific scoring matrix (PSSM) was performed on the target protein sequence to obtain its evolutionary information. Then, DTCWT was used to extract representative features from the PSSM, which were then combined with the drug fingerprint features to form the feature descriptors. Finally, these descriptors were sent to the Rotation Forest (RoF) model for classification. A 5-fold cross validation (CV) was adopted on four datasets (Enzyme, Ion Channel, GPCRs (G-protein-coupled receptors), and NRs (Nuclear Receptors)) to validate the proposed model; our method yielded high average accuracies of 89.21%, 85.49%, 81.02%, and 74.44%, respectively. To further verify the performance of our model, we compared the RoF classifier with two state-of-the-art algorithms: the support vector machine (SVM) and the k-nearest neighbor (KNN) classifier. We also compared it with some other published methods. Moreover, the prediction results for the independent dataset further indicated that our method is effective for predicting potential DTIs. Thus, we believe that our method is suitable for facilitating drug discovery and development.

Identification of intracellular glycosaminoglycan-interacting proteins by affinity purification mass spectrometry.

Glycosaminoglycans (GAGs) are essential functional components of the extracellular matrix (ECM). Artificial GAGs like sulfated hyaluronan (sHA) exhibit pro-osteogenic properties and boost healing processes. Hence, they are of high interest for supporting bone regeneration and wound healing. Although sulfated GAGs (sGAGs) appear intracellularly, the knowledge about intracellular effects and putative interaction partners is scarce. Here we used an affinity-purification mass spectrometry-based (AP-MS) approach to identify novel and particularly intracellular sGAG-interacting proteins in human bone marrow stromal cells (hBMSC). Overall, 477 proteins were found interacting with at least one of four distinct sGAGs. Enrichment analysis for protein localization showed that mainly intracellular and cell-associated interacting proteins were identified. The interaction of sGAG with α2-macroglobulin receptor-associated protein (LRPAP1), exportin-1 (XPO1), and serine protease HTRA1 (HTRA1) was confirmed in reverse assays. Consecutive pathway and cluster analysis led to the identification of biological processes, namely processes involving binding and processing of nucleic acids, LRP1-dependent endocytosis, and exosome formation. Respecting the preferentially intracellular localization of sGAG in vesicle-like structures, also the interaction data indicate sGAG-specific modulation of vesicle-based transport processes. By identifying many sGAG-specific interacting proteins, our data provide a resource for upcoming studies aimed at molecular mechanisms and understanding of sGAG cellular effects.

Molecular mechanism of cargo recognition and handover by the mammalian signal recognition particle.

Co-translational protein targeting to membranes by the signal recognition particle (SRP) is a universally conserved pathway from bacteria to humans. In mammals, SRP and its receptor (SR) have many additional RNA features and protein components compared to the bacterial system, which were recently shown to play regulatory roles. Due to its complexity, the mammalian SRP targeting process is mechanistically not well understood. In particular, it is not clear how SRP recognizes translating ribosomes with exposed signal sequences and how the GTPase activity of SRP and SR is regulated. Here, we present electron cryo-microscopy structures of SRP and SRP·SR in complex with the translating ribosome. The structures reveal the specific molecular interactions between SRP and the emerging signal sequence and the elements that regulate GTPase activity of SRP·SR. Our results suggest the molecular mechanism of how eukaryote-specific elements regulate the early and late stages of SRP-dependent protein targeting.

Function and therapeutic potential of N-acyl amino acids.

N-acyl amino acids (NAAs) are amphiphilic molecules, with different potential fatty acid and head group moieties. NAAs are the largest family of anandamide congener lipids discovered to date. In recent years, several NAAs have been identified as potential ligands, engaging novel binding sites and mechanisms for modulation of membrane proteins such as G-protein coupled receptors (GPRs), nuclear receptors, ion channels, and transporters. NAAs play a key role in a variety of physiological functions as lipid signaling molecules. Understanding the structure, function roles, and pharmacological potential of these NAAs is still in its infancy, and the biochemical roles are also mostly unknown. This review will provide a summary of the literature on NAAs and emphasize their therapeutic potential.

An optimized protocol with a stepwise approach to identify specific nuclear receptor ligands from cultured mammalian cells.

Here, we describe an optimized protocol to identify specific nuclear receptor ligands. First, to rule out any compound interference with luciferase activity per se, we describe an in vitro assay assessing potential inhibition or activation of luciferase enzymatic activity. Second, to comply with EMA and FDA guidelines to mitigate drug-drug interactions, we detail assays assessing constitutive androstane receptor (CAR) and pregnane X receptor (PXR) activation ability. Finally, to minimize off-target detection effects, we describe the use of mammalian one- (or two-) hybrid systems. For complete details on the use and execution of this protocol, please refer to Hering et al. (2018).

Molecular Dynamics of Cobalt Protoporphyrin Antagonism of the Cancer Suppressor REV-ERBß.

Nuclear receptor REV-ERBß is an overexpressed oncoprotein that has been used as a target for cancer treatment. The metal-complex nature of its ligand, iron protoporphyrin IX (Heme), enables the REV-ERBß to be used for multiple therapeutic modalities as a photonuclease, a photosensitizer, or a fluorescence imaging agent. The replacement of iron with cobalt as the metal center of protoporphyrin IX changes the ligand from an agonist to an antagonist of REV-ERBß. The mechanism behind that phenomenon is still unclear, despite the availability of crystal structures of REV-ERBß in complex with Heme and cobalt protoporphyrin IX (CoPP). This study used molecular dynamic simulations to compare the effects of REV-ERBß binding to Heme and CoPP, respectively. The initial poses of Heme and CoPP in complex with agonist and antagonist forms of REV-ERBß were predicted using molecular docking. The binding energies of each ligand were calculated using the MM/PBSA method. The computed binding affinity of Heme to REV-ERBß was stronger than that of CoPP, in agreement with experimental results. CoPP altered the conformation of the ligand-binding site of REV-ERBß, disrupting the binding site for nuclear receptor corepressor, which is required for REV-ERBß to regulate the transcription of downstream target genes. Those results suggest that a subtle change in the metal center of porphyrin can change the behavior of porphyrin in cancer cell signaling. Therefore, modification of porphyrin-based agents for cancer therapy should be conducted carefully to avoid triggering unfavorable effects.

Dolomiaea souliei ethyl acetate extract protected against α-naphthylisothiocyanate-induced acute intrahepatic cholestasis through regulation of farnesoid x receptor-mediated bile acid metabolism.

BACKGROUND: Cholestasis is characterized by accumulation of bile components in liver and systemic circulation. Restoration of bile acid homeostasis via activating farnesoid x receptor (FXR) is a promising strategy for the treatment of cholestasis. FXR-SHP (small heterodimer partner) axis plays an important role in maintaining bile acid homeostasis. PURPOSE: To investigate the anti-cholestasis effect of Dolomiaea souliei (Franch.) C.Shih (D. souliei) and clarify its underlying mechanism against α-naphthylisothiocyanate (ANIT) induced acute intrahepatic cholestasis. METHODS: ANIT-induced Sprague-Dawley rats were employed to investigate the anti-cholestasis effect of D. souliei ethyl acetate extract (DSE). Ursodeoxycholic acid (UDCA) was used as positive control. Bile flow and blood biochemical parameters were measured. Liver histopathological examination was conducted via hematoxylin-eosin staining. Western blot analysis was carried out to evaluate the protein levels related to bile acids metabolism and inflammation. The interactions between FXR and costunolide or dehydrocostus lactone, were conducted by molecular docking experiments. The effect of costunolide and dehydrocostus lactone on aspartate aminotransferase (AST), alanine aminotransferase (ALT) levels and FXR expression were also evaluated using guggulsterone-induced L02 cells. RESULTS: DSE could promote bile excretions and protect against ANIT-induced liver damage in cholestasis rats. Protein levels of FXR, SHP, Na+/taurocholate cotransporter (NTCP), bile salt export pump (BSEP), multidrug resistance-associated protein 2 (MRP2) were increased and the expressions of cholesterol 7α-hydroxylase (CYP7A1) and sterol 27-hydroxylase (CYP27A1) were decreased by DSE. Meanwhile, the anti-inflammatory factors, tumor necrosis factor-α (TNF-α), interleukin-1ß (IL-1ß), interleukin-6 (IL-6) were also significantly increased, and the pro-inflammatory factor, interleukin-10 (IL-10), was significantly decreased in rats of DSE groups. Molecular docking revealed that costunolide and dehydrocostus lactone could be well docked into the FXR protein molecule, and hydrophobic interactions played the main function. Costunolide could reverse the increased AST and ALT levels and increase the FXR expression in guggulsterone-induced L02 cells. CONCLUSION: DSE had an anti-cholestasis effect by activating FXR-SHP axis, inhibiting synthesis of bile acid, and increasing bile secretion, together with inflammatory response and improving liver injury. Costunolide may be the main active component. This study provided a potential therapeutic mechanism for D. souliei as an anti-cholestasis medicine in the treatment of cholestasis liver diseases.

Endogenous vitamin E metabolites mediate allosteric PPARγ activation with unprecedented co-regulatory interactions.

Vitamin E exhibits pharmacological effects beyond established antioxidant activity suggesting involvement of unidentified mechanisms. Here, we characterize endogenously formed tocopherol carboxylates and the vitamin E mimetic garcinoic acid (GA) as activators of the peroxisome proliferator-activated receptor gamma (PPARγ). Co-stimulation of PPARγ with GA and the orthosteric agonist pioglitazone resulted in additive transcriptional activity. In line with this, the PPARγ-GA complex adopted a fully active conformation and interestingly contained two bound GA molecules with one at an allosteric site. A co-regulator interaction scan demonstrated an unanticipated co-factor recruitment profile for GA-bound PPARγ compared with canonical PPARγ agonists and gene expression analysis revealed different effects of GA and pioglitazone on PPAR signaling in hepatocytes. These observations reveal allosteric mechanisms of PPARγ modulation as an alternative avenue to PPARγ targeting and suggest contributions of PPARγ activation by α-13-tocopherolcarboxylate to the pharmacological effects of vitamin E.