Coactivator Peptide Research Articles

A Complex Interplay Between Melatonin and RORβ: RORβ is Unlikely a Putative Receptor for Melatonin as Revealed by Biophysical Assays.

A nuclear retinoic acid receptor (RAR)-related orphan receptor β (RORβ) is strictly expressed in the brain, particularly in the pineal gland where melatonin is primarily synthesized and concentrated. The controversial issues regarding the direct interaction of melatonin toward ROR receptors have prompted us to investigate the potential melatonin binding sites on different ROR isoforms. We adopted computational and biophysical approaches to investigate the potential of melatonin as the ligand for RORs, in particular RORβ. Herein, possible melatonin binding sites were predicted by molecular docking on human RORs. The results showed that melatonin might be able to bind within the ligand-binding domain (LBD) of all RORs, despite their difference in sequence homology. The predicted melatonin binding scores were comparable to binding energies with respect to those of melatonin interaction to the well-characterized membrane receptors, MT1 and MT2. Although the computational analyses suggested the binding potential of melatonin to the LBD of RORβ, biophysical validation failed to confirm the binding. Melatonin was unable to alter the stability of human RORβ as shown by the unaltered melting temperatures upon melatonin administration in differential scanning fluorometry (DSF). A thermodynamic isothermal titration calorimetry (ITC) profile showed that melatonin did not interact with human RORβ in solutions, even in the presence of SRC-1 co-activator peptide. Although the direct interaction between the LBD of RORβ could not be established, RORα and RORβ gene expressions were increased upon 24h treatment with μM-range melatonin. Our data, thus, support the studies that the nuclear effects of melatonin may not be directly mediated via its interaction with the RORβ. These findings warrant further investigation on how melatonin interacts with ROR signaling and urge the melatonin research community for a paradigm shift in the direct interaction of melatonin toward RORs. The quest to identify nuclear receptors for melatonin in neuronal cells remains valid for the community to achieve.

A structural basis for the activation of peroxisome proliferator-activated receptor gamma (PPARγ) by perfluorooctanoic acid (PFOA)

Perfluorooctanoic acid (PFOA) is a widespread environmental pollutant of the perfluoroalkyl substance (PFAS) class that is extremely resistant to environmental and metabolic degradation, leading to bioaccumulation. PFOA exposure has been linked to many health effects including endocrine disruption and metabolic dysregulation, but our understanding of the molecular mechanisms resulting in these outcomes remains incomplete. One target affected by PFOA is the ligand regulated nuclear receptor peroxisome proliferator-activated receptor gamma (PPARγ) which plays a critical role in controlling metabolic homeostasis through regulating processes such as adipogenesis, glucose homeostasis, inflammation and osteogenesis. It has been previously established that PFOA activates PPARγ through binding to the PPARγ ligand binding domain (PPARγ LBD) leading to increased expression of PPARγ controlled target genes. However, the mechanism by which PFOA achieves this has remained elusive. Here, we employed a combination of X-ray crystallography and fluorescence polarization assays to provide a structural basis for PFOA mediated activation of PPARγ via binding to the PPARγ LBD. Using X-ray crystallography, the cocrystal structure of the PPARγ LBD:PFOA complex was solved. This revealed that PFOA occupies three distinct sites, two within the PPARγ LBD and one within the activation function 2 (AF2) on the protein surface. Structural comparison of PFOA binding with previously reported PPARγ:ligand complexes supports that PFOA activates PPARγ by a partial agonist mechanism at micromolar concentrations. Fluorescence polarization assays also revealed that PFOA binding to the AF2 is unlikely to occur in a cellular context and confirmed that PFOA behaves as a partial agonist in vitro, weakly recruiting a coactivator peptide to the AF2 of the PPARγ LBD. This discovery provides an advancement in understanding PFOA mediated regulation of PPARγ, giving new insight regarding regulation of PPARγ by PFAS and PFAS substitutes in general and can be applied to the design and assessment of safer PFAS.

Different Coactivator Recruitment to Human PPARα/δ/γ Ligand-Binding Domains by Eight PPAR Agonists to Treat Nonalcoholic Fatty Liver Disease.

Three peroxisome proliferator-activated receptor subtypes, PPARα, PPAR(ß/)δ, and PPARγ, exert ligand-dependent transcriptional control in concert with retinoid X receptors (RXRs) on various gene sets harboring PPAR response elements (PPREs) in their promoter regions. Ligand-bound PPAR/RXR complexes do not directly regulate transcription; instead, they recruit multiprotein coactivator complexes to specific genomic regulatory loci to cooperatively activate gene transcription. Several coactivators are expressed in a single cell; however, a ligand-bound PPAR can be associated with only one coactivator through a consensus LXXLL motif. Therefore, altered gene transcription induced by PPAR subtypes/agonists may be attributed to the recruitment of various coactivator species. Using a time-resolved fluorescence resonance energy transfer assay, we analyzed the recruitment of four coactivator peptides (PGC1α, CBP, SRC1, and TRAP220) to human PPARα/δ/γ-ligand-binding domains (LBDs) using eight PPAR dual/pan agonists (bezafibrate, fenofibric acid, pemafibrate, pioglitazone, elafibranor, lanifibranor, saroglitazar, and seladelpar) that are/were anticipated to treat nonalcoholic fatty liver disease. These agonists all recruited four coactivators to PPARα/γ-LBD with varying potencies and efficacy. Only five agonists (bezafibrate, pemafibrate, elafibranor, lanifibranor, and seladelpar) recruited all four coactivators to PPARδ-LBD, and their concentration-dependent responses differed from those of PPARα/γ-LBD. These results indicate that altered gene expression through consensus PPREs by different PPAR subtypes/agonists may be caused, in part, by different coactivators, which may be responsible for the unique pharmacological properties of these PPAR agonists.

A structural mechanism of nuclear receptor biased agonism

Efforts to decrease the adverse effects of nuclear receptor (NR) drugs have yielded experimental agonists that produce better outcomes in mice. Some of these agonists have been shown to cause different, not just less intense, on-target transcriptomic effects; however, a structural explanation for such agonist-specific effects remains unknown. Here, we show that partial agonists of the NR peroxisome proliferator-associated receptor γ (PPARγ), which induce better outcomes in mice compared to clinically utilized type II diabetes PPARγ-binding drugs thiazolidinediones (TZDs), also favor a different group of coactivator peptides than the TZDs. We find that PPARγ full agonists can also be biased relative to each other in terms of coactivator peptide binding. We find differences in coactivator-PPARγ bonding between the coactivator subgroups which allow agonists to favor one group of coactivator peptides over another, including differential bonding to a C-terminal residue of helix 4. Analysis of all available NR-coactivator structures indicates that such differential helix 4 bonding persists across other NR-coactivator complexes, providing a general structural mechanism of biased agonism for many NRs. Further work will be necessary to determine if such bias translates into altered coactivator occupancy and physiology in cells.

Rational design of stapled helical peptides as antidiabetic PPARγ antagonists to target coactivator site by decreasing unfavorable entropy penalty instead of increasing favorable enthalpy contribution.

Peroxisome proliferator-activated receptor γ (PPARγ) is a ligand-activated transcription factor belonging to the nuclear hormone receptor and has been exploited as a well-established druggable target for the treatment of diabetes mellitus (DM). Traditionally, small-molecule compounds have been developed to attack at the ligand site and Ser273 phosphorylation site of PPARγ. In this study, we derived helical peptide segments from the LXXLL motif region of coactivator proteins as antidiabetic PPARγ antagonists, which were expected to competitively disrupt the native interaction between PPARγ and its cognate coactivators by rebinding at PPARγ coactivator site. Structural analysis, dynamics simulation and energetics dissection revealed that these peptides cannot be well folded into active helical structure when splitting from the protein context of their parent coactivators and exhibit a large flexibility and intrinsic disorder in the free state, which would, therefore, incur a considerable entropy penalty upon rebinding to PPARγ. Hydrocarbon stapling strategy was employed to constrain these free coactivator peptides into ordered helical conformation, thus largely minimizing unfavorable entropy penalty but having only a moderate effect on favorable enthalpy contribution. The computational findings were further substantiated by fluorescence-based assays; the binding affinity of three potent SRC1, NCoA6 and p300 coactivator peptides to PPARγ was observed to be improved by 7.2-fold, 4.2-fold and 5.7-fold upon the stapling, which were also measured to have an efficient competitive potency with their unstapled counterparts for PPARγ coactivator site, with CC50 = 0.096, 0.12 and 0.18μM, respectively.

3,4,5-Trimethoxycinnamate thymol ester inhibits melanogenesis in normal human melanocytes and 3D human epidermal equivalents via the PGC-1α-independent PPARγ partial agonism

Background3,4,5-Trimethoxycinnamate thymol ester (TCTE), an anti-melanogenic cosmetic agent prescribed currently, promotes adiponectin synthesis during adipogenesis in human bone marrow mesenchymal stem cells (hBM-MSCs). Adiponectin inhibits melanin biosynthesis and its biosynthesis is directly regulated by peroxisome proliferator-activated receptor (PPAR) γ. In this regard, TCTE may potentially affect PPARγ activity. However, contradicting effects of PPARγ agonists with different chemical structures on human melanogenesis have been reported. ObjectiveA molecular target of TCTE was investigated to elucidate the association of both adiponectin and PPARγ with anti-melanogenic activity. MethodsThe adiponectin secretion-promoting activity of TCTE was tested in an adipogenesis model of hBM-MSCs. A molecular target of TCTE for adiponectin secretion was evaluated via time-resolved fluorescence resonance energy transfer-based receptor binding and transactivation of PPARs. ResultsTCTE significantly promoted adiponectin secretion (EC50, 27.9 μM) during adipogenesis in hBM-MSCs and directly bound to PPARγ (Ki, 13.2 μM). The TCTE-bound PPARγ increased the recruitment of SRC-1, SRC-3, and TRAP220/DRIP-1 coactivator peptides without affecting PGC-1α coactivation. In the docking analysis, the optimal ligand binding mode of TCTE exhibited typical ligand-receptor interactions of PPARγ partial agonists. The PPARγ partial agonism of TCTE was established experimentally and the anti-melanogenic activity of TCTE was decreased by treatment with a PPARγ antagonist in cultured normal human melanocytes and a 3D model of human epidermis. ConclusionThe anti-melanogenic activity of TCTE was associated with a PGC-1α-independent PPARγ partial agonism.

The T850D Phosphomimetic Mutation in the Androgen Receptor Ligand Binding Domain Enhances Recruitment at Activation Function 2.

Several key functions of the androgen receptor (AR) such as hormone recognition and co-regulator recruitment converge in the ligand binding domain (LBD). Loss- or gain-of-function of the AR contributes to pathologies such as the androgen insensitivity syndrome and prostate cancer. Here, we describe a gain-of-function mutation of the surface-exposed threonine at position 850, located at the amino-terminus of Helix 10 (H10) in the AR LBD. Since T850 phosphorylation was reported to affect AR function, we created the phosphomimetic mutation T850D. The AR T850D variant has a 1.5- to 2-fold increased transcriptional activity with no effect on ligand affinity. In the androgen responsive LNCaP cell line grown in medium with low androgen levels, we observed a growth advantage for cells in which the endogenous AR was replaced by AR T850D. Despite the distance to the AF2 site, the AR T850D LBD displayed an increased affinity for coactivator peptides as well as the 23FQNLF27 motif of AR itself. Molecular Dynamics simulations confirm allosteric transmission of the T850D mutation towards the AF2 site via extended hydrogen bond formation between coactivator peptide and AF2 site. This mechanistic study thus confirms the gain-of-function character of T850D and T850 phosphorylation for AR activity and reveals details of the allosteric communications within the LBD.

Novel vitamin D3-hydroxyderivatives as candidates for the therapy against skin-aging and photo-aging: bioinformatical analysis

Vitamin D3 acts through its most active form, calcitriol, 1α,25-dihydroxyvitamin D3 [1,25(OH)2D3] as agonist of one of the receptors involved in this ligand action, vitamin D receptor (VDR), which is also a transcription factor. Numerous modifications of calcitriol at its side-chain, C-ring, A-ring, triene system, alone or in combination, as well as nonsteroidal mimics provided new VDR agonists and some antagonists with biological activity and possible therapeutical potential. Some of the D3 metabolites, including 20,23(OH)2D3 and 20(OH)D3 are able to inhibit RORE-mediated transactivation, as well as the interaction between the RORα/γ ligand-binding domain (LBD) with an LXXLL coactivator peptide. Our analysis of recently reported microarray data on vitamin D3 (D3) induced changes in cultured human keratinocytes indicated that D3 hydroxyderivatives stimulate the expression of genes involved in anti-aging activities. Furthermore, we noted upregulation of the kallikrein gene family by 1,25(OH)2D3 after 24-hour treatment, including stimulation of KLK6, KLK13, KLK3, KLK9, KLK5, KLK7, and KLK10. Also, after 6-hour incubation with 1,25(OH)2D3, the upregulation of KLK6, KLK13, and KLK3 was seen. Interestingly, ACEIs administered to hypertensive rats doubled the lifespan of these animals. In humans, ACEIs prevent hallmarks of aging, such as organ fibrosis and cardiac hypertrophy. We noted also that vitamin D3-hydroxyderivatives act against oxidative stress through upregulation of thioredoxin reductase (TXNRD1) and heme reductase-1 (HMOX-1) gene expression in keratinocytes treated for 24h. Another mechanism of anti-aging properties of inverse agonist RORa/γ is the resolution of inflammation caused by T helper (Th17) lymphocytes and switching the immune response into T regulatory (Treg) lymphocytes activation, with silencing of the inflammation state and reducing the inflammation process. The gene connected with inflammatory response, AKR1C3 (which encodes prostaglandin F synthase) is also strongly downregulated by 20,23(OH)2D3 in keratinocytes after incubation for 24 h. We suggest that vitamin D3 analogs, such as 20,23(OH)2D3, 1,25(OH)2D3, and 20(OH)D3 may have anti-aging properties through action on different pathways connected with DNA repair.

Bisphenol A derivatives act as novel coactivator-binding inhibitors for estrogen receptor β

Bisphenol A and its derivatives are recognized as endocrine disruptors based on their complex effects on estrogen receptor (ER) signaling. While the effects of bisphenol derivatives on ERα have been thoroughly evaluated, how these chemicals affect ERβ signaling is less well understood. Herein, we sought to identify novel ERβ ligands using a radioligand competitive binding assay to screen a chemical library of bisphenol derivatives. Many of the compounds identified showed intriguing dual activities as both ERα agonists and ERβ antagonists. Docking simulations of these compounds and ERβ suggested that they bound not only to the canonical binding site of ERβ but also to the coactivator binding site located on the surface of the receptor, suggesting that they act as coactivator-binding inhibitors (CBIs). Receptor–ligand binding experiments using WT and mutated ERβ support the presence of a second ligand-interaction position at the coactivator-binding site in ERβ, and direct binding experiments of ERβ and a coactivator peptide confirmed that these compounds act as CBIs. Our study is the first to propose that bisphenol derivatives act as CBIs, presenting critical insight for the future development of ER signaling–based drugs and their potential to function as endocrine disruptors.

Unraveling the Allosteric Cross-Talk between the Coactivator Peptide and the Ligand-Binding Site in the Glucocorticoid Receptor.

The glucocorticoid receptor (GR) is a nuclear receptor that controls critical biological processes by regulating the transcription of specific genes. There is a known allosteric cross-talk between the ligand and coregulator binding sites within the GR ligand-binding domain that is crucial for the control of the functional response. However, the molecular mechanisms underlying such an allosteric control remain elusive. Here, molecular dynamics (MD) simulations, bioinformatic analysis, and biophysical measurements are integrated to capture the structural and dynamic features of the allosteric cross-talk within the GR. We identified a network of evolutionarily conserved residues that enables the allosteric signal transduction, in agreement with experimental data. MD simulations clarify how such a network is dynamically interconnected and offer a mechanistic explanation of how different peptides affect the intensity of the allosteric signal. This study provides useful insights to elucidate the GR allosteric regulation, ultimately providing a foundation for designing novel drugs.

White Button Mushroom Disrupts AR Signaling in Prostate Cancer: The Scientific Basis for a Diet-Based Chemoprevention

Abstract White button mushroom (WBM) (Agaricus bisporus) is a potential prostate cancer (PCa) chemo-preventive and therapeutic agent. Our clinical phase trial of WBM powder in patients with biochemically recurrent PCa indicated that WBM intake reduced the circulating levels of prostate-specific antigen (PSA), with minimal side effects [1]. We hypothesized that WBM exerts its effects on PCa through the androgen receptor (AR) signaling axis. We thus conducted the reverse translational study. Androgen-sensitive PCa cell lines (LNCaP and VCaP) and patient-derived-xenografts (PDX), of a prostate tumor (TM00298) were used. In both LNCaP and VCaP cells, western blots and qRT-PCR assays indicated that WBM extract (6~30 mg/mL) suppressed DHT-induced PSA expression and cell proliferation in a dose-dependent manner. Immunofluorescence on AR revealed that the nuclear localization of AR was reduced upon WBM extract treatment, which agreed with the results of a PSA promotor-luciferase assay, suggesting that WBM extract inhibited DHT-induced luciferase activity. RNA-Seq on WBM-treated LNCaP cells confirmed that WBM treatment suppressed androgen response pathways and cell-cycle control pathways. Our prostate cancer PDX showed that oral intake of WBM extract (200 mg/kg/day) significantly suppressed tumor growth, as well as decreased PSA levels in both tumors and serum. Both in vitro and in vivo studies suggested that chemical(s) in WBM extract behave as AR antagonist(s). We previously identified a conjugated linoleic acid isomer (CLA-9Z11E) as an active component in WBM extract. In the present study, we extended these findings by performing LanthaScreen™ TR-FRET AR Coactivator Interaction Assays for a direct interaction of CLA-9Z11E with AR. We report here that CLA-9Z11E exerts a strong antagonist potency against the recruitment of an AR coactivator peptide towards AR. The inhibitory effect of CLA-9Z11E (IC50: 350 nM) was nearly two times stronger than the known AR antagonist, cyproterone acetate (IC50: 672 nM). The information gained from this study improves the overall understanding of how WBM may contribute to the prevention and treatment of PCa. It also serves as an important, scientific basis for developing diet-based chemoprevention and integrative therapeutic strategies for prostate cancer (supported by NIH R01 CA227230). Reference: [1] Twardowski P, et al. A phase I trial of mushroom powder in patients with biochemically recurrent prostate cancer: Roles of cytokines and myeloid-derived suppressor cells for Agaricus bisporus-induced prostate-specific antigen responses. Cancer. 2015.121(17):2942-50.

Binding affinity and synergy between agonists and peptides of the estrogen receptor‐beta (ERß) needed for arain‐selective SERB Activity

The estrogen receptor (ER) exist predominantly in two isoforms (ERα and ERß). Both are primarily activated by the naturally occurring hormone 17ß-Estradiol (E2) which regulates a variety of physiological processes. One of ER's major role is acting as key neuroprotective modulator for hippocampal plasticity in the brain. The ERß isoform is predominantly found in the brain, which makes it a promising drug target for memory consolidation. Selectivity for ERß over ERα is crucial to the development of drug leads, since ERα–induced cell proliferation results in risk of cancer. We have developed a selective and potent ERß agonist with in vivo efficacy for improving cognitive function within mice. A prior study determined that selectivity for ER is related to the ligand's ability to induce the conformational change necessary for the activation of transcription. Selective estrogen receptor modulators (SERMs) like raloxifene & tamoxifen are used to treat breast cancer by selectively targeting ERα in mammary tissue. This is done through the synergistic binding of coactivator proteins to the ER in the presence of an agonist. Similarly, it is predicted that selective estrogen beta agonists (SERBAs) have the ability to be tissue specific in the presence of specific coactivators. Through the use of computational modeling, a splice variant of SRC1 (steroid receptor coactivator-1), SRC1-4, was identified to be the most favorable brain-relevant peptide for ERß. This study determines the binding affinity of agonist for ERß and ERα in the presence and absence of SRC1-4. In addition we dive into the extent to which binding synergy between agonist and coactivator exist, which could lead to selective activity in the brain. The synergy associated with the conformational change is measured by ΔΔGsynergy based on binding affinity for both agonist and coactivator. The greater the synergy between the agonist and coactivator, the more promising the lead compound will be as a SERBA. Preliminary studies have been done for the binding of SRC1-4 using TR-FRET assays where the concentration of coactivator was varied. Fluorescence polarization (FP) studies were also utilized to determine a Kd. In these studies, FL-SRC1-4 remained constant while ERß-LBD concentrations varied in the presence of 1 μM (saturating) E2. The two methods are compared and contrasted. The same FP assay was completed again in the absence of E2 (no agonoism). This provides a binding affinity for SRC1-4 to the receptor when an agonist is not present. Computational modeling studies were performed to compare ER's affinity for SRC1-4 peptides over other coactivator peptides. 3 unlabeled peptides (SRC1-1, SRC1-3 and PGC1a) were used in a competitive FP binding assay with FL-SRC1-4. The data was consistent with prior data, indicating SRC1-4 has the highest affinity for ERß. The agonistic-synergy effect will be calculated through the measurement of E2 affinity for ERß in the presence and absence of SRC1-4. The comparison of ΔΔG(synergy) for ERß vs. ERα will ultimately provide a measurement of selectivity and synergy that is biologically relevant for SERB activity. Hanson, A. M. et al. A-C estrogens as potent and selective estrogen receptor-beta agonists (SERBAs) to enhance memory consolidation under low-estrogen conditions. J. Med. Chem.61, 4720–4738 (2018).

Discovery of a Novel Class of ERRα Agonists.

A novel class of estrogen-related receptor α (ERRα) agonists has been discovered. A structure-activity relationship study of high-throughput screening hits 1 and 2 led to the discovery of benzimidazole 3d (DS20362725) and acetophenone analogue 5c (DS45500853). The X-ray crystal structure of the ERRα ligand-binding domain in complex with 5c and PGC-1α coactivator peptide revealed conformational changes in the ligand-binding pocket to accommodate 5c and the key interaction between the protein and ligand. Since both analogues avoided PPARγ transcriptional activity, they can be useful tool compounds for investigating biological ERRα functions.

Stability of the Retinoid X Receptor-α Homodimer in the Presence and Absence of Rexinoid and Coactivator Peptide.

Differential scanning calorimetry and differential scanning fluorimetry were used to measure the thermal stability of human retinoid X receptor-α ligand binding domain (RXRα LBD) homodimer in the absence or presence of rexinoid and coactivator peptide, GRIP-1. The apo-RXRα LBD homodimer displayed a single thermal unfolding transition with a Tm of 58.7 °C and an unfolding enthalpy (ΔH) of 673 kJ/mol (12.5 J/g), much lower than average value (35 J/g) of small globular proteins. Using a heat capacity change (ΔCp) of 15 kJ/(mol K) determined by measurements at different pH values, the free energy of unfolding (ΔG) of the native state was 33 kJ/mol at 37 °C. Rexinoid binding to the apo-homodimer increased Tm by 5 to 9 °C and increased the ΔG of the native homodimer by 12 to 20 kJ/mol at 37 °C, consistent with the nanomolar dissociation constant (Kd) of the rexinoids. GRIP-1 binding to holo-homodimers containing rexinoid resulted in additional increases in ΔG of 14 kJ/mol, a value that was the same for all three rexinoids. Binding of rexinoid and GRIP-1 resulted in a combined 50% increase in unfolding enthalpy, consistent with reduced structural fluidity and more compact folding observed in other published structural studies. The complexes of UAB110 and UAB111 are each more stable than the UAB30 complex by 8 kJ/mol due to enhanced hydrophobic interactions in the binding pocket because of their larger end groups. This increase in thermodynamic stability positively correlates with their improved RXR activation potency. Thermodynamic measurements are thus valuable in predicting agonist potency.

Unravelling Allosteric Cross-Talk between Co-Activator Peptide and Ligand Binding Site in Glucocorticoid Receptor

Glucocorticoid receptor (GR) is a nuclear receptor that controls critical biological processes by regulating the transcription of specific genes. There is a known interconnection between the ligand and co-regulator binding sites of the GR ligand binding domain that is crucial for the functional differentiation of the downstream gene transcription. However, the molecular mechanisms underlying such an allosteric control remain elusive. The integration of molecular dynamics simulations, bioinformatic analysis and biophysical measurements led us to detect a network of dynamically interconnected evolutionary conserved residues that mediates the allosteric signal propagation between the co-regulator docking site and the ligand binding pocket. Co-regulator peptides alter the functionality of this network, explaining why the allosteric response depends on the peptide sequence. These findings provide useful insights for the comprehension of GR allosteric regulation that should help the design of novel drugs.

Single-Molecule Insights into Allosteric Regulation of the Nuclear Receptor Protein RXRα

Nuclear receptors are crucial transcription factors that respond to a broad range of external signaling ligands by binding cognate DNA response elements and switching on transcription. As such, these proteins form tightly regulated, multi-component allosteric systems. RXRα is one of the key players in this protein family for its role as a promiscuous heterodimerization partner. We employ single-molecule fluorescence techniques to investigate the conformational dynamics of RXRα's ligand binding domain (LBD)—the essential regulatory domain. It has been proposed that ligand binding changes the conformation of helix 12 of the LBD and positions it in a conformation favoring coactivator peptide binding. Our single-molecule FRET studies, however, demonstrate that helix 12 undergoes a substantial conformational change only after the coactivator peptide binds. This suggests an unexpected allosteric mechanism of nuclear receptor activation, which we investigate. In particular, we address the questions: on what time scales do the conformational changes of helix 12 take place? and how are its dynamics affected by modulations in other parts of RXRα and in its binding partners? Our studies will pave the way to better understanding of the complex regulatory machinery embodied in nuclear receptor function.

The acyl chains of phosphoinositide PIP3 alter the structure and function of nuclear receptor steroidogenic factor-1

AbstractNuclear receptors are transcription factors that bind lipids, an event that induces a structural conformation of the receptor that favors interaction with transcriptional coactivators. The nuclear receptor steroidogenic factor-1 (SF-1, NR5A1) binds the signaling phosphoinositides PI(4,5)P2 (PIP2) and PI(3,4,5)P3 (PIP3), and our previous crystal structures showed how the phosphoinositide headgroups regulate SF-1 function. However, what role the acyl chains play in regulating SF-1 structure remains unaddressed. Here, we used X-ray crystallography with in vitro binding and functional assays to examine how the acyl chains of PIP3 regulate human SF-1 ligand-binding domain structure and function. Altering acyl chain length and unsaturation regulates apparent binding of all tested phosphoinositides to SF-1. Mass spectrometry–based lipidomics data suggest C16 and C18 phospholipids preferentially associate with SF-1 expressed ectopically in bacteria. We then solved the 2.5 Å crystal structure of SF-1 bound to dioleoyl PIP3(18:1/18:1) to compare it with a matched structure of SF-1 bound to dipalmitoyl PIP3(16:0/16:0). The dioleoyl-bound structure was severely disordered in a specific SF-1 region associated with pathogenic human polymorphisms and within the coactivator-binding region critical for SF-1 function while inducing increased sensitivity to protease digestion in solution. Validating these structural observations, in vitro functional studies showed dioleoyl PIP3 induced 6-fold poorer affinity of a peroxisome proliferator-activated receptor gamma coactivator 1-alpha coactivator peptide for SF-1 compared with dipalmitoyl PIP3. Together, these data suggest the chemical nature of the phosphoinositide acyl chains controls the ordered state of specific, clinically important structural regions in SF-1, regulating SF-1 function in vitro.

Rational design and chemical modification of TEAD coactivator peptides to target hippo signaling pathway against gastrointestinal cancers

Human Hippo signaling pathway has been recognized as a new and promising therapeutic target of gastrointestinal cancers, which is regulated by the intermolecular recognition between the TEA domain (TEAD) transcription factor and its prime coactivators. The coactivator proteins adopt two hotspot sites, namely α-helix and Ω-loop, to interact with TEAD. Here, we demonstrate that both the α-helix and Ω-loop peptides cannot maintain in structured state when splitting from the full-length coactivator proteins; they exhibit a large intrinsic disorder in free state that prevents the coactivator peptide recognition by TEAD. Rational design is used to optimize the interfacial residues of coactivator α-helix peptides, which can effectively improve the favorable direct readout effect upon the peptide binding to TEAD. Chemical modification is employed to constrain the free α-helix peptide into native ordered conformation. The method introduces an all-hydrocarbon bridge across i and i + 4 residues to stabilize the helical structure of a free coactivator peptide, which can considerably reduce the unfavorable indirect readout effect upon the peptide binding to TEAD. The all-hydrocarbon bridge is designed to point out of the TEAD–peptide complex interface, which would not disrupt the direct intermolecular interaction between the TEAD and peptide. Therefore, the stapling only improves peptide affinity, but does not alter peptide specificity, to TEAD. Affinity assay confirms that the binding potency of coactivator α-helix peptides is improved substantially by >5-fold upon the rational design and chemical modification. Structural analysis reveals that the optimized/stapled peptides can form diverse nonbonded interactions such as hydrogen bonds and hydrophobic contacts with TEAD, thus conferring stability and specificity to the TEAD–peptide complex systems.

Elucidation of Molecular Mechanism of a Selective PPARα Modulator, Pemafibrate, through Combinational Approaches of X-ray Crystallography, Thermodynamic Analysis, and First-Principle Calculations.

The selective PPARα modulator (SPPARMα) is expected to medicate dyslipidemia with minimizing adverse effects. Recently, pemafibrate was screened from the ligand library as an SPPARMα bearing strong potency. Several clinical pieces of evidence have proved the usefulness of pemafibrate as a medication; however, how pemafibrate works as a SPPARMα at the molecular level is not fully known. In this study, we investigate the molecular mechanism behind its novel SPPARMα character through a combination of approaches of X-ray crystallography, isothermal titration calorimetry (ITC), and fragment molecular orbital (FMO) analysis. ITC measurements have indicated that pemafibrate binds more strongly to PPARα than to PPARγ. The crystal structure of PPARα-ligand binding domain (LBD)/pemafibrate/steroid receptor coactivator-1 peptide (SRC1) determined at 3.2 Å resolution indicates that pemafibrate binds to the ligand binding pocket (LBP) of PPARα in a Y-shaped form. The structure also reveals that the conformation of the phenoxyalkyl group in pemafibrate is flexible in the absence of SRC1 coactivator peptide bound to PPARα; this gives a freedom for the phenoxyalkyl group to adopt structural changes induced by the binding of coactivators. FMO calculations have indicated that the accumulation of hydrophobic interactions provided by the residues at the LBP improve the interaction between pemafibrate and PPARα compared with the interaction between fenofibrate and PPARα.

Abstract C53: Liver X receptor ligand targets glutamine metabolism in pancreatic cancer

Abstract Pancreatic ductal adenocarcinoma (PDAC) is the most prevalent form of pancreatic cancer. It is predicted to become the third leading cause of cancer-related deaths by 2030. PDAC has a poor survival rate of 5 years with a 7% success rate. Current treatments include nontargeted highly toxic chemotherapeutic agents. More than 95% of PDAC patients have activating mutations of Kristen RAt Sarcoma virus (KRAS) gene, and it is known to drive metabolic reprogramming that increases cancer cell survival and resistance against the chemotherapeutic drugs. KRAS promotes noncanonical consumption of glutamine by upregulating the expression of glutaminase 1 (GLS1) enzyme. This, in turn, supports PDAC growth and survival by maintaining redox homeostasis and also by replenishing citric acid cycle intermediates. Liver X receptor beta (LXRβ), a nuclear receptor, is a ligand-dependent transcription factor that is ubiquitously expressed in all tissues. It plays an important role in maintaining cholesterol, lipid, and glucose homeostasis under normal physiologic conditions. Agonist-bound LXRβ activates the transcription of genes involved in lipogenesis, reverse cholesterol transport, and glucose metabolism. Our group has previously shown that LXRβ is expressed in PDAC tissues, and treatment with GW3965, an LXRβ agonist, reduced PDAC proliferation. However, LXRβ agonists have adverse side effects of increasing liver and circulating triglycerides. This study identified a novel ligand of LXRβ 1E5, which significantly reduced PDAC cell proliferation. 1E5 decreased the basal expression of LXRβ target genes such as sterol regulatory element-binding protein 1 (SREBP1c) and ATP binding cassette subfamily A member 1 (ABCA1). It also increased the binding of co-repressor peptides such as nuclear receptor co-repressor (NCoR) and silencing mediator of retinoic acid and thyroid hormone receptor (SMRT) to the LXRβ ligand binding domain, whereas it decreased the binding of the co-activator peptide as demonstrated by the time-resolved fluorescence energy transfer (TR-FRET) assay, thereby inhibiting the transcriptional activity of LXRβ. Therefore, 1E5 acts as an LXRβ inverse agonist. Furthermore, the metabolomics study revealed that 1E5 disrupts glutamine metabolism in PDAC cells by downregulating the glutamine, aspartate, and alpha-ketoglutarate levels in cells. Gene expression analysis showed that it significantly downregulated GLS1 expression. These results demonstrate that the novel inverse agonist is a candidate agent in the treatment of PDAC by targeting glutamine metabolism. Citation Format: Shivangi Srivastava, Scott Widmann, Chin-Yo Lin. Liver X receptor ligand targets glutamine metabolism in pancreatic cancer [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Advances in Science and Clinical Care; 2019 Sept 6-9; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2019;79(24 Suppl):Abstract nr C53.