Structure of the human histamine H1 receptor complex with doxepin

The biogenic amine histamine is an important pharmacological mediator involved in pathophysiological processes such as allergies and inflammations. Histamine H(1) receptor (H(1)R) antagonists are very effective drugs alleviating the symptoms of allergic reactions. Here we show the crystal structure of the H(1)R complex with doxepin, a first-generation H(1)R antagonist. Doxepin sits deep in the ligand-binding pocket and directly interacts with Trp?428(6.48), a highly conserved key residue in G-protein-coupled-receptor activation. This well-conserved pocket with mostly hydrophobic nature contributes to the low selectivity of the first-generation compounds. The pocket is associated with an anion-binding region occupied by a phosphate ion. Docking of various second-generation H(1)R antagonists reveals that the unique carboxyl group present in this class of compounds interacts with Lys?191(5.39) and/or Lys?179(ECL2), both of which form part of the anion-binding region. This region is not conserved in other aminergic receptors, demonstrating how minor differences in receptors lead to pronounced selectivity differences with small molecules. Our study sheds light on the molecular basis of H(1)R antagonist specificity against H(1)R.     

De novo mutations in the gene encoding STXBP1 (MUNC18-1) cause early infantile epileptic encephalopathy

Early infantile epileptic encephalopathy with suppression-burst (EIEE), also known as Ohtahara syndrome, is one of the most severe and earliest forms of epilepsy. Using array-based comparative genomic hybridization, we found a de novo 2.0-Mb microdeletion at 9q33.3-q34.11 in a girl with EIEE. Mutation analysis of candidate genes mapped to the deletion revealed that four unrelated individuals with EIEE had heterozygous missense mutations in the gene encoding syntaxin binding protein 1 (STXBP1). STXBP1 (also known as MUNC18-1) is an evolutionally conserved neuronal Sec1/Munc-18 (SM) protein that is essential in synaptic vesicle release in several species. Circular dichroism melting experiments revealed that a mutant form of the protein was significantly thermolabile compared to wild type. Furthermore, binding of the mutant protein to syntaxin was impaired. These findings suggest that haploinsufficiency of STXBP1 causes EIEE.     

Microstructure evolution and reaction behavior of Cu–Ni alloy and B_4C powder system

Microstructure evolution and reaction behavior of Cu–Ni alloy and B_4C power system was studied by in-situ and static experimental investigations along with theoretical calculations. The reaction process was as follows. Firstly,B_4C decomposed into B and C atoms, and then B atoms diffused into Cu–Ni matrix, leading to the formation of Ni_2B particles. Subsequently, Ni atoms diffused into B_4C, forming a loose mixture of Ni_2B and amorphous C at the initial powder boundary. Finally, with the completion of reaction, Ni_2B particles at the powder boundary grew into a monolithic block, and then C substance was excluded out and accumulated at the edge of this monolithic Ni_2B block. It is believed that the formation of Ni_2B phase is caused by the most negative change of Gibbs free energy among all the potential reactions between Ni–B and Ni–B_4C systems.     

Haploinsufficiency of NSD1 causes Sotos syndrome

We isolated NSD1 from the 5q35 breakpoint in an individual with Sotos syndrome harboring a chromosomal translocation. We identified 1 nonsense, 3 frameshift and 20 submicroscopic deletion mutations of NSD1 among 42 individuals with sporadic cases of Sotos syndrome. The results indicate that haploinsufficiency of NSD1 is the major cause of Sotos syndrome.     

Periostin in vitreoretinal diseases

Proliferative vitreoretinal diseases such as diabetic retinopathy, proliferative vitreoretinopathy (PVR), and age-related macular degeneration are a leading cause of decreased vision and blindness in developed countries. In these diseases, retinal fibro(vascular) membrane (FVM) formation above and beneath the retina plays an important role. Gene expression profiling of human FVMs revealed significant upregulation of periostin. Subsequent analyses demonstrated increased periostin expression in the vitreous of patients with both proliferative diabetic retinopathy and PVR. Immunohistochemical analysis showed co-localization of periostin with α-SMA and M2 macrophage markers in FVMs. In vitro, periostin blockade inhibited migration and adhesion induced by PVR vitreous and transforming growth factor-β2 (TGF-β2). In vivo, a novel single-stranded RNAi agent targeting periostin showed the inhibitory effect on experimental retinal and choroidal FVM formation without affecting the viability of retinal cells. These results indicated that periostin is a pivotal molecule for FVM formation and a promising therapeutic target for these proliferative vitreoretinal diseases.     

Tbx6-dependent Sox2 regulation determines neural or mesodermal fate in axial stem cells

The classical view of neural plate development held that it arises from the ectoderm, after its separation from the mesodermal and endodermal lineages. However, recent cell-lineage-tracing experiments indicate that the caudal neural plate and paraxial mesoderm are generated from common bipotential axial stem cells originating from the caudal lateral epiblast. Tbx6 null mutant mouse embryos which produce ectopic neural tubes at the expense of paraxial mesoderm must provide a clue to the regulatory mechanism underlying this neural versus mesodermal fate choice. Here we demonstrate that Tbx6-dependent regulation of Sox2 determines the fate of axial stem cells. In wild-type embryos, enhancer N1 of the neural primordial gene Sox2 is activated in the caudal lateral epiblast, and the cells staying in the superficial layer sustain N1 activity and activate Sox2 expression in the neural plate. In contrast, the cells destined to become mesoderm activate Tbx6 and turn off enhancer N1 before migrating into the paraxial mesoderm compartment. In Tbx6 mutant embryos, however, enhancer N1 activity persists in the paraxial mesoderm compartment, eliciting ectopic Sox2 activation and transforming the paraxial mesoderm into neural tubes. An enhancer-N1-specific deletion mutation introduced into Tbx6 mutant embryos prevented this Sox2 activation in the mesodermal compartment and subsequent development of ectopic neural tubes, indicating that Tbx6 regulates Sox2 via enhancer N1. Tbx6-dependent repression of Wnt3a in the paraxial mesodermal compartment is implicated in this regulatory process. Paraxial mesoderm-specific misexpression of a Sox2 transgene in wild-type embryos resulted in ectopic neural tube development. Thus, Tbx6 represses Sox2 by inactivating enhancer N1 to inhibit neural development, and this is an essential step for the specification of paraxial mesoderm from the axial stem cells.     

Crystal structure of a human membrane protein involved in cysteinyl leukotriene biosynthesis

The cysteinyl leukotrienes, namely leukotriene (LT)C4 and its metabolites LTD4 and LTE4, the components of slow-reacting substance of anaphylaxis, are lipid mediators of smooth muscle constriction and inflammation, particularly implicated in bronchial asthma. LTC4 synthase (LTC4S), the pivotal enzyme for the biosynthesis of LTC4 (ref. 10), is an 18-kDa integral nuclear membrane protein that belongs to a superfamily of membrane-associated proteins in eicosanoid and glutathione metabolism that includes 5-lipoxygenase-activating protein, microsomal glutathione S-transferases (MGSTs), and microsomal prostaglandin E synthase 1 (ref. 13). LTC4S conjugates glutathione to LTA4, the endogenous substrate derived from arachidonic acid through the 5-lipoxygenase pathway. In contrast with MGST2 and MGST3 (refs 15, 16), LTC4S does not conjugate glutathione to xenobiotics. Here we show the atomic structure of human LTC4S in a complex with glutathione at 3.3 A resolution by X-ray crystallography and provide insights into the high substrate specificity for glutathione and LTA4 that distinguishes LTC4S from other MGSTs. The LTC4S monomer has four transmembrane alpha-helices and forms a threefold symmetric trimer as a unit with functional domains across each interface. Glutathione resides in a U-shaped conformation within an interface between adjacent monomers, and this binding is stabilized by a loop structure at the top of the interface. LTA4 would fit into the interface so that Arg 104 of one monomer activates glutathione to provide the thiolate anion that attacks C6 of LTA4 to form a thioether bond, and Arg 31 in the neighbouring monomer donates a proton to form a hydroxyl group at C5, resulting in 5(S)-hydroxy-6(R)-S-glutathionyl-7,9-trans-11,14-cis-eicosatetraenoic acid (LTC4). These findings provide a structural basis for the development of LTC4S inhibitors for a proinflammatory pathway mediated by three cysteinyl leukotriene ligands whose stability and potency are different and by multiple cysteinyl leukotriene receptors whose functions may be non-redundant.     

G-protein-coupled receptor inactivation by an allosteric inverse-agonist antibody

G-protein-coupled receptors are the largest class of cell-surface receptors, and these membrane proteins exist in equilibrium between inactive and active states. Conformational changes induced by extracellular ligands binding to G-protein-coupled receptors result in a cellular response through the activation of G proteins. The A(2A) adenosine receptor (A(2A)AR) is responsible for regulating blood flow to the cardiac muscle and is important in the regulation of glutamate and dopamine release in the brain. Here we report the raising of a mouse monoclonal antibody against human A(2A)AR that prevents agonist but not antagonist binding to the extracellular ligand-binding pocket, and describe the structure of A(2A)AR in complex with the antibody Fab fragment (Fab2838). This structure reveals that Fab2838 recognizes the intracellular surface of A(2A)AR and that its complementarity-determining region, CDR-H3, penetrates into the receptor. CDR-H3 is located in a similar position to the G-protein carboxy-terminal fragment in the active opsin structure and to CDR-3 of the nanobody in the active β(2)-adrenergic receptor structure, but locks A(2A)AR in an inactive conformation. These results suggest a new strategy to modulate the activity of G-protein-coupled receptors.