Valerie Cavett

A coactivator trap identifies NONO (p54nrb) as a component of the cAMP-signaling pathway

Signal transduction pathways often use a transcriptional component to mediate adaptive cellular responses. Coactivator proteins function prominently in these pathways as the conduit to the basic transcriptional machinery. Here we present a high-throughput cell-based screening strategy, termed the “coactivator trap,” to study the functional interactions of coactivators with transcription factors. We applied this strategy to the cAMP signaling pathway, which utilizes two families of coactivators, the cAMP response element binding protein (CREB) binding protein (CBP)/p300 family and the recently identified transducers of regulated CREB activity family (TORCs1–3). In addition to identifying numerous known interactions of these coactivators, this analysis identified NONO (p54nrb) as a TORC-interacting protein. RNA interference experiments demonstrate that NONO is necessary for cAMP-dependent activation of CREB target genes in vivo. Furthermore, TORC2 and NONO complex on cAMP-responsive promoters, and NONO acts as a bridge between the CREB/TORC complex and RNA polymerase II. These data demonstrate the utility of the coactivator trap by identification of a component of cAMP-mediated transcription.

Mapping the phosphorylation sites of Ulk1.

Ulk1 is a serine/threonine kinase that controls macroautophagy, an essential homeostatic recycling pathway that degrades bulk cytoplasmic material and directs the turnover of organelles such as peroxisomes and mitochondria. Further, macroautophagy is potently induced by signals that trigger metabolic stress, such as hypoxia and amino acid starvation, where its recycling functions provide macromolecules necessary to maintain catabolic metabolism and cell survival. Substrates for Ulk1 have not been identified, and little is known regarding post-translational control of Ulk1 kinase activity and function. To gain insights into the regulatory mechanisms of Ulk1, we developed a robust purification protocol for Ulk1 and demonstrated that Ulk1 is highly phosphorylated and requires autophosphorylation for stability. Importantly, high-resolution, tandem mass spectrometry identified multiple sites of phosphorylation on Ulk1, including several within domains known to regulate macroautophagy. Differential phosphorylation analyses also identified sites of phosphorylation in the C-terminal domain that depend upon or require Ulk1 autophosphorylation.

Resveratrol modulates the inflammatory response via an estrogen receptor-signal integration network

Resveratrol has beneficial effects on aging, inflammation and metabolism, which are thought to result from activation of the lysine deacetylase, sirtuin 1 (SIRT1), the cAMP pathway, or AMP-activated protein kinase. In this study, we report that resveratrol acts as a pathway-selective estrogen receptor-α (ERα) ligand to modulate the inflammatory response but not cell proliferation. A crystal structure of the ERα ligand-binding domain (LBD) as a complex with resveratrol revealed a unique perturbation of the coactivator-binding surface, consistent with an altered coregulator recruitment profile. Gene expression analyses revealed significant overlap of TNFα genes modulated by resveratrol and estradiol. Furthermore, the ability of resveratrol to suppress interleukin-6 transcription was shown to require ERα and several ERα coregulators, suggesting that ERα functions as a primary conduit for resveratrol activity. DOI: http://dx.doi.org/10.7554/eLife.02057.001

Ligand-binding dynamics rewire cellular signaling via estrogen receptor-α

Ligand-binding dynamics control allosteric signaling through the estrogen receptor-α (ERα), but the biological consequences of such dynamic binding orientations are unknown. Here, we compare a set of ER ligands having dynamic binding orientation (dynamic ligands) with a control set of isomers that are constrained to bind in a single orientation (constrained ligands). Proliferation of breast cancer cells directed by constrained ligands is associated with DNA binding, coactivator recruitment and activation of the estrogen-induced gene GREB1, reflecting a highly interconnected signaling network. In contrast, proliferation driven by dynamic ligands is associated with induction of ERα-mediated transcription in a DNA-binding domain (DBD)-dependent manner. Further, dynamic ligands displayed enhanced anti-inflammatory activity. The DBD-dependent profile was predictive of these signaling patterns in a larger diverse set of natural and synthetic ligands. Thus, ligand dynamics directs unique signaling pathways, and reveals a novel role of the DBD in allosteric control of ERα-mediated signaling.

Strategies to recover proteins from ocular tissues for proteomics

We present here the results of protein extraction from different ocular regions using different detergents. Extraction strategies used to determine optimal protein extraction included: pressure cycling and aqueous–organic phase extraction in combination with electrophoretic fractionation for anterior, posterior, and peripapillary sclera. Detergent extraction of proteins from freshly enucleated porcine eyes (n = 8) showed significant differences for different eye regions. Protein yield ranged from 2.3 to 50.7 μg protein/mg for different ocular tissues, with the lens yielding the most protein. ASB‐14 and Triton X‐100 provided the best protein yields (n = 10) for anterior and posterior sclera. The spectrophotometric measurements for ASB‐14 were not consistent with SDS‐PAGE densitometry. A combination of 0.5% Triton X‐100, 0.5% Tween‐20, and 0.1% Genapol C‐100 was found optimal for extraction from sclera. Proteins from different regions of the eye are best extracted with different detergents. The pressure cycling technology provided superior extraction compared to the other methods. Additional aqueous–organic phase partitioning enables superior fractionation when compared to SDS‐PAGE alone. Organic phase fractionation is compatible with MS and allowed identification of 34, 71, and 77 proteins respectively from anterior, posterior, and peripapillary sclera. The extraction strategy may affect the final outcome in protein profiling by MS or by other methods.

Structural mechanism for signal transduction in RXR nuclear receptor heterodimers

A subset of nuclear receptors (NRs) function as obligate heterodimers with retinoid X receptor (RXR), allowing integration of ligand-dependent signals across the dimer interface via an unknown structural mechanism. Using nuclear magnetic resonance (NMR) spectroscopy, x-ray crystallography and hydrogen/deuterium exchange (HDX) mass spectrometry, here we show an allosteric mechanism through which RXR co-operates with a permissive dimer partner, peroxisome proliferator-activated receptor (PPAR)-γ, while rendered generally unresponsive by a non-permissive dimer partner, thyroid hormone (TR) receptor. Amino acid residues that mediate this allosteric mechanism comprise an evolutionarily conserved network discovered by statistical coupling analysis (SCA). This SCA network acts as a signalling rheostat to integrate signals between dimer partners, ligands and coregulator-binding sites, thereby affecting signal transmission in RXR heterodimers. These findings define rules guiding how NRs integrate two ligand-dependent signalling pathways into RXR heterodimer-specific responses.

DNA-Encoded Solid-Phase Synthesis: Encoding Language Design and Complex Oligomer Library Synthesis

The promise of exploiting combinatorial synthesis for small molecule discovery remains unfulfilled due primarily to the “structure elucidation problem”: the back-end mass spectrometric analysis that significantly restricts one-bead-one-compound (OBOC) library complexity. The very molecular features that confer binding potency and specificity, such as stereochemistry, regiochemistry, and scaffold rigidity, are conspicuously absent from most libraries because isomerism introduces mass redundancy and diverse scaffolds yield uninterpretable MS fragmentation. Here we present DNA-encoded solid-phase synthesis (DESPS), comprising parallel compound synthesis in organic solvent and aqueous enzymatic ligation of unprotected encoding dsDNA oligonucleotides. Computational encoding language design yielded 148 thermodynamically optimized sequences with Hamming string distance ≥ 3 and total read length <100 bases for facile sequencing. Ligation is efficient (70% yield), specific, and directional over 6 encoding positions. A series of isomers served as a testbed for DESPS’s utility in split-and-pool diversification. Single-bead quantitative PCR detected 9 × 104 molecules/bead and sequencing allowed for elucidation of each compound’s synthetic history. We applied DESPS to the combinatorial synthesis of a 75 645-member OBOC library containing scaffold, stereochemical and regiochemical diversity using mixed-scale resin (160-μm quality control beads and 10-μm screening beads). Tandem DNA sequencing/MALDI-TOF MS analysis of 19 quality control beads showed excellent agreement (<1 ppt) between DNA sequence-predicted mass and the observed mass. DESPS synergistically unites the advantages of solid-phase synthesis and DNA encoding, enabling single-bead structural elucidation of complex compounds and synthesis using reactions normally considered incompatible with unprotected DNA. The widespread availability of inexpensive oligonucleotide synthesis, enzymes, DNA sequencing, and PCR make implementation of DESPS straightforward, and may prompt the chemistry community to revisit the synthesis of more complex and diverse libraries.

Development of selective estrogen receptor modulator (SERM)-like activity through an indirect mechanism of estrogen receptor antagonism: defining the binding mode of 7-oxabicyclo[2.2.1]hept-5-ene scaffold core ligands.

Previously, we discovered estrogen receptor (ER) ligands with a novel three‐dimensional oxabicyclo[2.2.1]heptene core scaffold and good ER binding affinity act as partial agonists via small alkyl ester substitutions on the bicyclic core that indirectly modulate the critical switch helix in the ER ligand binding domain, helix 12, by interactions with helix 11. This contrasts with the mechanism of action of tamoxifen, which directly pushes helix 12 out of the conformation required for gene activation. We now report that a much larger substitution can be tolerated at this position of the bicyclic core scaffold, namely a phenyl sulfonate group, which defines a novel binding epitope for the estrogen receptor. We prepared an array of 14 oxabicycloheptene sulfonates, varying the phenyl sulfonate group. As with the parent compound, 5,6‐bis‐(4‐hydroxyphenyl)‐7‐oxabicyclo[2.2.1]hept‐5‐ene‐2‐sulfonic acid phenyl ester (OBHS), these compounds showed preferential affinity for ERα, and the disposition and size of the phenyl substituents were important determinants of the binding affinity and selectivity of these compounds, with those having ortho substituents giving the highest, and para substituents the lowest affinities for ERα. A few analogues exhibit ERα binding affinities that are comparable to or, in the case of the ortho‐chloro analogue, higher than that of OBHS itself. In cell‐based studies, we found several compounds with activity profiles comparable to tamoxifen, but acting entirely as indirect antagonists, allosterically interfering with recruitment of coactivator proteins to the receptor. Thus, the OBHS binding epitope represents a novel approach to the development of estrogen receptor antagonists via an indirect mechanism of antagonism.

Identification and Structure-Activity Relationships of a Novel Series of Estrogen Receptor Ligands Based on 7-Thiabicyclo[2.2.1]hept-2-ene-7-oxide

To develop estrogen receptor (ER) ligands having novel structures and activities, we have explored compounds in which the central hydrophobic core has a more three-dimensional topology than typically found in estrogen ligands and thus exploits the unfilled space in the ligand-binding pocket. Here, we build upon our previous investigations of 7-oxabicyclo[2.2.1]heptene core ligands, by replacing the oxygen bridge with a sulfoxide. These new 7-thiabicyclo[2.2.1]hept-2-ene-7-oxides were conveniently prepared by a Diels-Alder reaction of 3,4-diarylthiophenes with dienophiles in the presence of an oxidant and give cycloadducts with endo stereochemistry. Several new compounds demonstrated high binding affinities with excellent ERα selectivity, but unlike oxabicyclic compounds, which are transcriptional antagonists, most thiabicyclic compounds are potent, ERα-selective agonists. Modeling suggests that the gain in activity of the thiabicyclic compounds arises from their endo stereochemistry that stabilizes an active ER conformation. Further, the disposition of methyl substituents in the phenyl groups attached to the bicyclic core unit contributes to their binding affinity and subtype selectivity.

Full antagonism of the estrogen receptor without a prototypical ligand side chain.

Resistance to endocrine therapies remains a significant clinical problem for estrogen receptor-α (ERα)-positive breast cancer. On-target side effects limit therapeutic compliance and use for chemoprevention, highlighting an unmet need for new therapies. Here we present a full-antagonist ligand series lacking the prototypical ligand side chain that has been universally used to engender antagonism of ERα through poorly understood structural mechanisms. A series of crystal structures and phenotypic assays reveal a structure-based design strategy with separate design elements for antagonism and degradation of the receptor and access to a structurally distinct space for further improvements in ligand design. Understanding structural rules that guide ligands to produce diverse ERα-mediated phenotypes has broad implications for the treatment of breast cancer and other estrogen-sensitive aspects of human health including bone homeostasis, energy metabolism, and autoimmunity.