The use of in vitro peptide binding profiles and in silico ligand-receptor interaction profiles to describe ligand-induced conformations of the retinoid X receptor alpha ligand-binding domain

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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. To identify mechanisms regulating basal cell fate, we developed a screenable 3D culture system of airway epithelial morphogenesis. We performed a high-throughput screen using a collection of secreted proteins and identified inflammatory cytokines that specifically biased basal cell differentiation toward a goblet cell fate, culminating in enhanced mucus production. We also demonstrate a specific requirement for Notch2 in cytokine-induced goblet cell metaplasia in vitro and in vivo. We conclude that inhibition of Notch2 prevents goblet cell metaplasia induced by a broad range of stimuli and propose Notch2 neutralization as a therapeutic strategy for preventing goblet cell metaplasia in airway diseases

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 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. We determined the structural basis for receptor inhibition by obtaining the first co-crystal structure of a NOTCH3 antibody with the NRR protein and defined two distinct epitopes for NRR antibodies. The antibodies exhibit potent anti-leukemic activity in cell lines and tumor xenografts harboring NOTCH3 activating mutations. Screening of primary T-ALL samples reveals that two of 40 tumors examined show active NOTCH3 signaling. We also identified evidence of NOTCH3 activation in 12 of 24 patient-derived orthotopic xenograft models, two of which exhibit activation of NOTCH3 without activation of NOTCH1. Our studies provide additional insights into NOTCH3 activation and offer a path forward for identification of cancers that are likely to respond to therapy with NOTCH3 selective inhibitory antibodies

The 2.0 Å crystal structure of the ERα 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 α at a resolution of 2.0 Å. As with other SERMs, lasofoxifene diverts the receptor from its agonist-bound conformation by displacing the C-terminal AF-2 helix into the site at which the LXXLL motif of coactivator proteins would otherwise be able to bind. Lasofoxifene achieves this effect by occupying the space normally filled by residue Leu 540, as well as by modulating the conformation of residues of helix 11 (His 524, Leu 525). A well-defined salt bridge between lasofoxifene and Asp 351 suggests that charge neutralization in this region of the receptor may explain the some of the antiestrogenic effects of lasofoxifene. The results suggest general features of ERα/SERM recognition, and add a new dimension to efforts to rationalize differences between the biological activity profiles exhibited by these important pharmacological agents

Ultrastructure of skin from Refsum disease with emphasis on epidermal lamellar bodies and stratum corneum barrier lipid organization

Classic Refsum disease (RD) is a rare, autosomal recessively-inherited disorder of peroxisome metabolism due to a defect in the initial step in the alpha oxidation of phytanic acid (PA), a C 16 saturated fatty acid with four methyl side groups, which accumulates in plasma and lipid enriched tissues (please see van den Brink, et al. 2006). It has been proposed that the disease complex in RD is in part due to the high affinity of phytanic acid for retinoid X receptors and peroxisome proliferator-activated receptors. Structurally, epidermal hyperplasia, increased numbers of cornified cell layers, presence of cells with lipid droplets in stratum basale and reduction of granular layer to a single layer have been reported by Blanchet-Bardon et al (1978). However, lamellar body (LB) density and secretion were reportedly normal. We recently examined biopsies from 4 unrelated patients, using both OsO(4) and RuO(4) post-fixation to evaluate the barrier lipid structural organization. Although lamellar body density appeared normal, individual organelles often had distorted shape, or had non-lamellar domains interspersed with lamellar structures. Some of the organelles seemed to lack lamellar contents altogether, showing instead uniformly electron-dense contents. In addition, we also observed mitochondrial abnormalities in the nucleated epidermis. Stratum granulosum-stratum corneum junctions also showed co-existence of non-lamellar and lamellar domains, indicative of lipid phase separation. Also, partial detachment or complete absence of corneocyte lipid envelopes (CLE) was seen in the stratum corneum of all RD patients. In conclusion, abnormal LB contents, resulting in defective lamellar bilayers, as well as reduced CLEs, likely lead to impaired barrier function in RD