Characterization of activating mutations of NOTCH3 in T-cell acute lymphoblastic leukemia and anti-leukemic activity of NOTCH3 inhibitory antibodies.

Notch receptors have been implicated as oncogenic drivers in several cancers, the most notable example being NOTCH1 in T-cell acute lymphoblastic leukemia (T-ALL). To characterize the role of activated NOTCH3 in cancer, we generated an antibody that detects the neo-epitope created upon gamma-secretase cleavage of NOTCH3 to release its intracellular domain (ICD3), and sequenced the negative regulatory region (NRR) and PEST (proline, glutamate, serine, threonine) domain coding regions of NOTCH3 in a panel of cell lines. We also characterize NOTCH3 tumor-associated mutations that result in activation of signaling and report new inhibitory antibodies.

Notch2 is required for inflammatory cytokine-driven goblet cell metaplasia in the lung.

The balance and distribution of epithelial cell types is required to maintain tissue homeostasis. A hallmark of airway diseases is epithelial remodeling, leading to increased goblet cell numbers and an overproduction of mucus. In the conducting airway, basal cells act as progenitors for both secretory and ciliated cells.

Interaction with both ZNRF3 and LGR4 is required for the signalling activity of R-spondin.

R-spondin proteins sensitize cells to Wnt signalling and act as potent stem cell growth factors. Various membrane proteins have been proposed as potential receptors of R-spondin, including LGR4/5, membrane E3 ubiquitin ligases ZNRF3/RNF43 and several others proteins. Here, we show that R-spondin interacts with ZNRF3/RNF43 and LGR4 through distinct motifs.

TEM8/ANTXR1 blockade inhibits pathological angiogenesis and potentiates tumoricidal responses against multiple cancer types.

Current antiangiogenic agents used to treat cancer only partially inhibit neovascularization and cause normal tissue toxicities, fueling the need to identify therapeutic agents that are more selective for pathological angiogenesis. Tumor endothelial marker 8 (TEM8), also known as anthrax toxin receptor 1 (ANTXR1), is a highly conserved cell-surface protein overexpressed on tumor-infiltrating vasculature. Here we show that genetic disruption of Tem8 results in impaired growth of human tumor xenografts of diverse origin including melanoma, breast, colon, and lung cancer.

Purification of the large ribosomal subunit via its association with the small subunit.

We have developed an affinity purification of the large ribosomal subunit from Deinococcus radiodurans that exploits its association with FLAG-tagged 30S subunits. Thus, capture is indirect so that no modification of the 50S is required and elution is achieved under mild conditions (low magnesium) that disrupt the association, avoiding the addition of competitor ligands or coelution of common contaminants. Efficient purification of highly pure 50S is achieved, and the chromatography simultaneously sorts the 50S into three classes according to their association status (unassociated, loosely associated, or tightly associated), improving homogeneity.

Structural basis for the catalytic mechanism of human phosphodiesterase 9.

The phosphodiesterases (PDEs) are metal ion-dependent enzymes that regulate cellular signaling by metabolic inactivation of the ubiquitous second messengers cAMP and cGMP. In this role, the PDEs are involved in many biological and metabolic processes and are proven targets of successful drugs for the treatments of a wide range of diseases. However, because of the rapidity of the hydrolysis reaction, an experimental knowledge of the enzymatic mechanisms of the PDEs at the atomic level is still lacking.

The 2.0 A crystal structure of the ERalpha ligand-binding domain complexed with lasofoxifene.

Lasofoxifene is a new and potent selective estrogen receptor modulator (SERM). The structural basis of its interaction with the estrogen receptor has been investigated by crystallographic analysis of its complex with the ligand-binding domain of estrogen receptor alpha at a resolution of 2.0 A.

Theoretical and experimental design of atypical kinase inhibitors: application to p38 MAP kinase.

Mimics of the benzimidazolone nucleus found in inhibitors of p38 kinase are proposed, and their theoretical potential as bioisosteres is described. A set of calculated descriptors relevant to the anticipated binding interaction for the fragments 1-methyl-1H-benzotriazole 5, 3-methyl-benzo[d]isoxazole 3, and 3-methyl-[1,2,4]triazolo[4,3-a]pyridine 4, pyridine 1, and 1,3-dimethyl-1,3-dihydro-benzoimidazol-2-one 2 are reported. The design considerations and synthesis of p38 inhibitors based on these H-bond acceptor fragments is detailed.

Phosphorylation of serine residues in histidine-tag sequences attached to recombinant protein kinases: a cause of heterogeneity in mass and complications in function.

High-level recombinant expression of protein kinases in eukaryotic cells or Escherichia coli commonly gives products that are phosphorylated by autocatalysis or by the action of endogenous kinases. Here, we report that phosphorylation occurred on serine residues adjacent to hexahistidine affinity tags (His-tags) derived from several commercial expression vectors and fused to overexpressed kinases. The result was observed with a variety of recombinant kinases expressed in either insect cells or E.

Discovery of a novel series of 6-azauracil-based thyroid hormone receptor ligands: potent, TR beta subtype-selective thyromimetics.

In this communication, we wish to describe the discovery of a novel series of 6-azauracil-based thyromimetics that possess up to 100-fold selectivities for binding and functional activation of the beta(1)-isoform of the thyroid receptor family. Structure-activity relationship studies on the 3,5- and 3'-positions provided compounds with enhanced TR beta affinity and selectivity. Key binding interactions between the 6-azauracil moiety and the receptor have been determined through of X-ray crystallographic analysis.

Kinetic analysis of estrogen receptor/ligand interactions.

Surface plasmon resonance biosensor technology was used to directly measure the binding interactions of small molecules to the ligand-binding domain of human estrogen receptor. In a screening mode, specific ligands of the receptor were easily discerned from nonligands. In a high-resolution mode, the association and dissociation phase binding responses were shown to be reproducible and could be fit globally to a simple interaction model to extract reaction rate constants.

Activation of human liver glycogen phosphorylase by alteration of the secondary structure and packing of the catalytic core.

Glycogen phosphorylases catalyze the breakdown of glycogen to glucose-1-phosphate, which enters glycolysis to fulfill the energetic requirements of the organism. Maintaining control of blood glucose levels is critical in minimizing the debilitating effects of diabetes, making liver glycogen phosphorylase a potential therapeutic target. To support inhibitor design, we determined the crystal structures of the active and inactive forms of human liver glycogen phosphorylase a.

A crystallizable form of RIIbeta regulatory domain obtained by limited proteolysis.

The type RIIbeta regulatory subunit of protein kinase A is primarily expressed in adipose tissue and brain. Knockout mice suggest a role for RIIbeta in regulating energy balance and adipose-tissue content, thus making it a potential target for therapeutic intervention in obesity. A truncated version of the RIalpha subunit has been used in a crystallographic study and was used here to design an analogous RIIbeta construct.

Spontaneous alpha-N-6-phosphogluconoylation of a "His tag" in Escherichia coli: the cause of extra mass of 258 or 178 Da in fusion proteins.

Several proteins expressed in Escherichia coli with the N-terminus Gly-Ser-Ser-[His]6- consisted partly (up to 20%) of material with 178 Da of excess mass, sometimes accompanied by a smaller fraction with an excess 258 Da. The preponderance of unmodified material excluded mutation, and the extra masses were attributed to posttranslational modifications. As both types of modified protein were N-terminally blocked, the alpha-amino group was modified in each case.

RARalpha antagonist Ro 41-5253 inhibits proliferation and induces apoptosis in breast-cancer cell lines.

Ro 41-5253 is a RARalpha-selective antagonist that binds RARalpha but does not induce transcriptional activation and does not influence RAR/RXR heterodimerization and DNA binding. This retinoid inhibits proliferation and induces apoptosis in MCF-7 and ZR-75.1 estrogen-receptor-positive breast-carcinoma cells in a dose-dependent way.

Differential expression of CRABP II, psoriasin and cytokeratin 1 mRNA in human skin diseases.

In order to confirm and further explore the significance of the overexpression of the CRABP II (cellular retinoic acid binding protein type II) and psoriasin genes in psoriatic versus normal skin, we examined the mRNA expression levels of these two genes by in situ hybridization in skin samples from psoriatic plaques and in one case from the border between a psoriatic plaque and uninvolved skin. Both genes were markedly upregulated in lesional skin, with a shift from low to high expression in the transitional zone of the plaque. Expression of the cytokeratin 1 (K1) gene was, in contrast, high in normal skin and decreased in the transition from uninvolved skin to psoriatic plaque, Examination of mRNA levels of CRABP II and psoriasin in other hyperproliferative and inflammatory skin diseases showed high expression of psoriasin, and in some cases also of CRABP II, in atopic dermatitis, mycosis fungoides, Darier's disease and inflammatory lichen sclerosus et atrophicus.

Characterization of synthetic retinoids with selectivity for retinoic acid or retinoid X nuclear receptors.

The broad spectrum of physiological activities of retinoids is mediate d by two types of receptors, the retinoic acid receptors (RARs) and the retinoid X receptors (RXRs). Though they have 9-cis retinoic acid as a common ligand, the amino acid sequence of their ligand binding domains is only distantly related (27%). This fact makes it probable that the ligand binding pockets of RARs and RXRs differ significantly with respect to their three dimensional structure.

Phytanic acid is a retinoid X receptor ligand.

Metabolic defects in phytanic acid catabolism have been shown to be connected with a number of human diseases which can lead to lethal defects of the nervous system and other organs. These effects are probably a result of the very high accumulation of phytanic acid in tissues throughout the body, due to defects in phytanic acid oxidation, the peroxisome being a major site for this process. The nuclear hormone receptors peroxisome proliferator-activated receptor and retinoid X receptor (RXR) have been shown to function as transcription factors in the control of the peroxisomal enzyme expression.

Enhancement of HL-60 differentiation by a new class of retinoids with selective activity on retinoid X receptor.

Cellular responsiveness to retinoic acid and its metabolites is conferred through two distinct families of receptors: the retinoic acid receptors (RARs) and the retinoid X receptors (RXRs). Herein, we report on the identification and characterization of several conformationally restricted retinoids, which selectively bind and activate RX receptors. Under the influence of retinoids, HL-60 myelocytic leukemia cells differentiate into granulocytes.

In situ hybridization analysis of CRABP II expression in sebaceous follicles from 13-cis retinoic acid-treated acne patients.

The aim of this study was to investigate the effects of 13-cis retinoic acid treatment on cellular retinoic acid binding protein II (CRABP II) mRNA expression in sebaceous follicles from acne patients, using in situ hybridization. Biopsies were taken from uninvolved skin areas in close juxtaposition to inflamed comedos before therapy, and at 2-4 or 14-16 weeks of treatment. Paraffin sections were used for in situ hybridization study with riboprobes transcribed from human CRABP II cDNA.

Marked resistance of RAR gamma-deficient mice to the toxic effects of retinoic acid.

Excessive intake of retinol or of retinoic acid causes a syndrome of characteristic toxic effects known as hypervitaminosis A. To test the role of the nuclear retinoic acid receptor (RAR gamma) in this process we produced mice with a targeted disruption of the RAR gamma gene and examined toxic effects of repeated doses of retinoic acid and two other synthetic retinoids, Ro 15-1570 and Ro 40-6055. Surprisingly, homozygous mutant mice were resistant to fourfold higher doses of retinoic acid than wild-type mice as well as to elevated doses of the synthetic retinoids, indicating that RAR gamma may have a major role in mediating retinoid toxicity, a finding that possibly has practical implications for reducing the toxicity of synthetic retinoids in clinical use.

Different agonist- and antagonist-induced conformational changes in retinoic acid receptors analyzed by protease mapping.

The pleiotropic effects of retinoic acid on cell differentiation and proliferation are mediated by two subfamilies of nuclear receptors, the retinoic acid receptors (RARs) and the retinoid X receptors (RXRs). Recently the synthetic retinoid Ro 41-5253 was identified as a selective RAR alpha antagonist. As demonstrated by gel retardation assays, Ro 41-5253 and two related new RAR alpha antagonists do not influence RAR alpha/RXR alpha heterodimerization and DNA binding.

Rapid and efficient generation of PCR-derived riboprobe templates for in situ hybridization histochemistry.

In situ hybridization histochemistry (ISH) using cRNA probes (riboprobes) has become a powerful technique for the examination of gene expression in tissue sections. The construction of plasmid templates for the synthesis of riboprobes with phage RNA polymerases is often a difficult and time-consuming step. We have therefore developed a rapid, efficient, and flexible method to generate totally artificial riboprobe templates by the polymerase chain reaction (PCR).

RXR-dependent and RXR-independent transactivation by retinoic acid receptors.

The binding affinity of retinoic acid receptors (RARs) to their response elements is strongly enhanced in vitro by the formation of heterodimers with retinoid X receptors (RXRs) suggesting that heterodimerization with RXR may be a prerequisite for a RAR-mediated transcriptional response. We found that in Drosophila SL-3 cells that are devoid of endogenous RARs and RXRs the presence of RAR is sufficient to confer a response to all-trans retinoic acid (RA). The transfection of both RAR and RXR and stimulation with their respective ligands all-trans and 9-cis RA leads to a synergistic response.

A retinoic acid receptor alpha antagonist selectively counteracts retinoic acid effects.

Retinoic acid (RA) exerts its pleiotropic effects on cell growth and differentiation through the activation of a family of transcription factors-the RA receptors (RARs). Three subtypes of these receptors exist, RAR alpha, RAR beta, and RAR gamma. The receptors are differentially expressed in different cell types and stages of development, suggesting that they may regulate different sets of genes.