Insects detect specific chemicals in the environment with olfactory receptors (ORs), which represent a novel class of ligand-gated ion channel. Insect ORs are comprised of at least one common subunit (OR83b in Drosophila) and at least one odorant-binding subunit. However, the molecular details of insect OR architecture, such as how they bind odorants, are unknown. This lack of knowledge hinders the development of compounds that may modulate OR function and potentially control insects involved in disease propagation and agricultural damage. The intent of this project is to investigate the structure and function of insect ORs. To this end, the utility of the Xenopus oocyte heterologous expression system was explored. Assay optimization, accuracy, and investigations on functional requirements were first performed using the Drosophila OR (DmOR) 35a/83b. The utility of the assay system was also demonstrated by identification of the honey bee (Apis mellifera) OR 11/2 as a receptor for the queen pheromone, 9-oxo-2-decenoic acid. A series of DmORs was cloned and expressed in Xenopus oocytes and individual receptors were selected for further study. DmOR85a/83b was shown to possess an incredibly high degree of enantioselectivity for the odorant ethyl 3-hydroxybutyrate. The receptive range of DmOR67a/83b was explored and observations were made on potential features of the odorant-binding site and a ligand odorophore. DmORs were also used to investigate the contributions of individual subunits toward the odorant-binding site and pore structure. Also, evidence for receptor antagonism by odorants was revealed. DmORs were screened with methanethiosulfonate reagents and the substituted cysteine accessibility method to identify residues 146-150 of DmOR85b as functionally important in receptor activation. This region, located at the predicted interface between transmembrane segment 3 (TMS3) and extracellular loop 2, was shown to be physically adjacent to the odorant-binding site itself. Finally, residues within the extracellular half of TMS3 in DmOR85b were implicated in odorant-induced activation by screening DmOR85b mutants for altered ligand preferences. Therefore, this project provides the first identification of insect OR subunit components involved in odorant recognition, and represents an important starting point for detailed analysis of the molecular basis for insect OR activation by odorants.

Mammalian neurons are anoxia-sensitive and rapidly undergo excitotoxic cell death when deprived of oxygen, mediated largely by calcium entry through N-methyl-D-aspartate receptors (NMDARs). This does not occur in neurons of the anoxia-tolerant western painted turtle, where a decrease in NMDAR currents is observed with anoxia. This decrease is dependent on a modest increase in cytosolic [Ca2+] during anoxia. The aim of this study was to determine if activation of the mitochondrial permeability transition pore (mPTP) decreases NMDAR currents through release of mitochondrial Ca2+. The data indicate that mPTP opening is sufficient to cause a decrease in NMDAR currents during normoxia and the anoxia-mediated rise in cytosolic [Ca2+] and depolarization of the mitochondrial membrane potential is due to opening of the mPTP. Furthermore, since a mitochondrial uncoupler releases additional calcium during anoxia we speculate that the mitochondrial membrane potential decreases in a regulated fashion to a new set-point.

Mammalian neurons are anoxia-sensitive and rapidly undergo excitotoxic cell death when deprived of oxygen, mediated largely by calcium entry through N-methyl-D-aspartate receptors (NMDARs). This does not occur in neurons of the anoxia-tolerant western painted turtle, where a decrease in NMDAR currents is observed with anoxia. This decrease is dependent on a modest increase in cytosolic [Ca2+] during anoxia. The aim of this study was to determine if activation of the mitochondrial permeability transition pore (mPTP) decreases NMDAR currents through release of mitochondrial Ca2+. The data indicate that mPTP opening is sufficient to cause a decrease in NMDAR currents during normoxia and the anoxia-mediated rise in cytosolic [Ca2+] and depolarization of the mitochondrial membrane potential is due to opening of the mPTP. Furthermore, since a mitochondrial uncoupler releases additional calcium during anoxia we speculate that the mitochondrial membrane potential decreases in a regulated fashion to a new set-point.

TLRs play a key role in initiating innate immunity and in regulating the nature of the adaptive immune response. We hypothesized that functional responsiveness to TLR stimulation differs in clinically; environmentally; ethnically distinct pediatric populations. PBMC obtained from 272 children were stimulated with a panel of TLR ligands. Levels of pro- and anti-inflammatory, Th1-, Th2-associated cytokines were quantified by ELISA. We demonstrate that use of threshold concentrations of TLR4 and TLR2 ligands reveal striking differences in cytokine responses between asthmatic and non-atopic children. Specifically, non-atopic controls produce higher levels of pro-inflammatory cytokines, whereas asthmatics exhibit increased anti-inflammatory IL-10 responses. Asthmatic children exposed to environmental tobacco smoke (ETS) demonstrated elevated levels of chemokines relative to non-ETS exposed asthmatics and controls. First Nation children favor anti-inflammatory IL-10 responses, whereas Caucasian population respond to TLR activation by production of more robust pro-inflammatory and Th1 biased cytokine and chemokine responses. / October 2007

The estrogen receptor (ER) is a ligand-activated transcription factor that regulates gene expression. The classical mechanisms of nuclear ER action include ligand-induced dimerization of ER which binds estrogen responsive elements (EREs) in promoters of target genes. In addition, non-genomic pathways of ER action have also been identified in breast cancer cells. Cdc25A is a tyrosine phosphatase that catalyzes dephosphorylation of cyclin/cyclin-dependent kinase complexes to regulate G1- to S-phase cell cycle progression. Cdc25A mRNA levels are induced by 17β-estradiol (E2) in ZR-75 breast cancer cells, and deletion analysis of the Cdc25A promoter identified the -151 to -12 region as the minimal E2-responsive sequence. Subsequent mutation/deletion analysis showed that at least three different cis-elements were involved in activation of Cdc25A by E2, namely, GC-rich Sp1 binding sites, CCAAT motifs, and E2F sites. Studies with inhibitors and dominant negative expression plasmids show that E2 activates Cdc25A expression through activation of genomic ERα/Sp1 and E2F1 and cAMP-dependent activation of NF-YA. Thus, both genomic and non-genomic pathways of estrogen action are involved in induction of Cdc25A in breast cancer cells. The PIAS family was initially identified as cytokine-induced inhibitors of STATs which contain several conserved domains involved in binding to other nuclear coactivators. In this study we have investigated coactivation of ERα by PIAS3 in breast cancer cell lines transiently cotransfected with the pERE3 constructs which contain three tandem EREs linked to a luciferase reporter gene. PIAS3 coactivated ERα-mediated transactivation in cells cotransfected with pERE3 and wild-type ERα. In contrast to many other coactivators, PIAS3 also enhanced transactivation of ERα when cells were cotransfected with the TAF1 ERα mutant. In addition, PIAS3 does not interact with activation function 2 (AF2) domain of ERα in a mammalian two-hybrid assay. These data indicate that coactivation of ERα by PIAS3 was AF2-domain independent. Analysis of several PIAS3 deletion mutants showed that the region containing amino acids 274 to 416 of PIAS3 are required for coactivation suggesting that the RING finger domain and acidic region of PIAS3 are important for interactions with wild-type ERα. These results demonstrate that PIAS3 coactivated ERα and this represents a non-classical LXXLL-independent coactivation pathway.

Huntington’s disease (HD) is caused by the expansion of a CAG repeats encoding polyglutamine (polyQ) in the first exon of Huntingtin (Htt) gene. In HD patients, polyQ contains 36-183 glutamine residues, whereas normal individuals have a polyQ of only 8-35 residues. To elucidate this threshold phenomenon of polyQ aggregation, fluorescence proteins CFP and YFP were attached to both ends of polyQ of different lengths. FRET (fluorescence resonance energy transfer) was conducted to characterize the conformation of polyQ in the pre-aggregation state. Our FRET data show that both the normal and expanded polyQ tracts reveal the same extended structure in low concentration. Longer polyQ has multiple cooperative binding sites with higher avidity. PolyQ tracts form aggregates when proteins exceed a critical concentration. The antibody MW1 Fv fragment binds to polyQ, breaks apart polyQ oligomer and stabilizes it in a more extended conformation. The addition of polyproline to the C-terminus inhibits polyQ aggregation by inducing PPII-like Helix structure. To understand how the flanking sequence affects the polyQ structure, the structure of Q10P10 peptide in complex with MW1 Fv was determined by protein crystallography and compared with Q10/Fv crystal structure. Q10P10 peptide bound to Fv has a similar extended structure as Q10 peptide when a polyproline tract adopts PPII helical structure sticking out of the complex. Toll-like receptors are transmembrane receptors on different kinds of leukocytes. They can recognize the structural conserved molecular motifs derived from microbes. On the upstream of the TLR signal pathway, TLRs recruit the adaptor protein-MyD88 through TIR/TIR domain interaction, and MyD88 recruits the downstream kinases IRAK4 and IRAK1 through death domain/death domain interaction. Pellino1, a newly identified E3 ubiquitin ligase, is also involved in TLR signaling by adding polyubiquitin chain to IRAK1 in conjugation with Ubc13/Uev1a E2 complex. TIR/TIR and DD/DD binding motifs were studied with techniques including mutagenesis, analytical gel filtration, NMR spectroscopy and crystallography. We identified a MyD88DD (E52QR62S) double-mutant that attenuates protein aggregation without interrupting the binding with IRAK4. This double mutant is a good candidate for structure determination by NMR spectroscopy. Our ubiquitination assay showed Pellino1 catalyzes polyubiquitination in the presence of Ubc13/Uev1a in vitro. Needle cluster-shaped crystals of Pellino1/Ubc13/ Uev1a protein complex were obtained by “hanging drop” method of vapor diffusion. Once the crystallization conditions are optimized, we will be able to collect X-ray diffraction data for this E2/E3 complex.

Vitamin D interacting protein 205 (DRIP205) is a mediator complex protein that anchors the complex to the estrogen receptor (ER) and other nuclear receptors (NRs). In ZR-75 breast cancer cells treated with 17?-estradiol (E2) and transfected with a construct containing three tandem estrogen responsive elements (pERE3), DRIP205 coactivates ER?-mediated transactivation. DRIP205?587-636 is a DRIP205 mutant in which both NR boxes within amino acids 587-636 have been deleted and, in parallel transfection studies, DRIP205?587-636 also coactivates ER?. Moreover, both wild-type and variant DRIP205 also colocalize with ER? in the nuclei of transfected cells. AF1 and AF2 of ER? are both required for DRIP205 coactivation. Extensive deletion analysis of DRIP205 shows that multiple domains of this protein play a role in coactivation of ER? and in interactions with ER?. On the other hand, both DRIP205 and DRIP205?587-636 coactivate E2-induced transactivation of ER?/Sp1 in cells transfected with a construct containing three GC-rich sites (pSp13). Coactivation of ER?/Sp1 by DRIP205 is dependent on AF1 of ER?. Enhancement of ER? and ER?/Sp1 by DRIP205 does not require NR boxes of DRIP205, and deletion mutants DRIP205 (1-714) and DRIP205 (516-1566) significantly coactivate ER? and ER?/Sp1. RNA interference study showed that DRIP205 coactivation of ER?/Sp was abolished in cells transfected with iSp3 and iSp4, suggesting that Sp3 and Sp4 are required for coactivation of ER?/Sp by DRIP205 in ZR-75 cells.

Estrogen Receptor ? (ER?)/Sp1 activation of GC-rich gene promoters in breast cancer cells is dependent, in part, on the activation function 1 (AF1) of ER?. This study investigates contributions of the DNA binding domain (C) and AF2 (DEF) regions of ER? on activation of ER?/Sp1. 17Beta-estradiol (E2) and the antiestrogens 4-hydroxytamoxifen and ICI 182,780 induced reporter gene activity in MCF-7 and MDA-MB-231 cells cotransfected with human or mouse ER? (hER? or MOR), but not ER? and GC-rich constructs containing three tandem Sp1 binding sites (pSp13) or other E2-responsive GC-rich promoters. Estrogen and antiestrogen activation of hER?/Sp1 was dependent on overlapping and different regions of the C, D, E, and F domains of ER?. Antiestrogen-induced activation of hER?/Sp1 was lost using hER? mutants deleted in zinc finger 1 (amino acids (aa) 185-205), zinc finger 2 (aa 218-245), and the hinge/helix 1 (aa 265-330) domains. In contrast with antiestrogens, E2-dependent activation of hER?/Sp1 required the C-terminal F domain (aa 579-595), which contains a ?-strand structural motif. Moreover, in peptide competition experiments overexpression of NR-box peptides inhibits E2-induced luciferase activity of pERE3, which contains three tandem repeats of consensus ERE sites, whereas E2-induced hER?/Sp1 action was not inhibited by NR-box peptide expression. In contrast, overexpression of a C-terminal (aa 575-595) F domain peptide specifically blocked E2-dependent activation of hER?/Sp1, but not on activation of pERE3, suggesting that F domain interactions with nuclear cofactors are specifically required for ER?/Sp1 action. Furthermore, direct physical interactions between hER? and Sp1 protein in vivo have been investigated by using Fluorescence Resonance Energy Transfer (FRET) microscopy and image analysis. Consistent with results from transient transfection assay, E2, 4OHT, and ICI enhanced hER?/Sp1 interactions in living cells and these interactions were also confirmed by coimmunoprecipitation. In addition, endogenous hER?/Sp1 action was evaluated by using si RNA for Sp1 and a significant decrease in ligand-induced hER?/Sp1 action was observed after decreased Sp1 expression.

EphB4, a known venous marker, represents a potential therapeutic target in modern vascular medicine. This study looked at the role of EphB4 as it pertains to basic cell functions in a mouse lung endothelium model (MLEC). Basic science techniques of microscopy, blotting and antibody labeling were used to evaluate and measure cellular response to EphB4 stimulation and manipulation. We found significant changes in MLEC cellular functions due to heterozygous knockout of the EphB4 receptor. These changes included decreased cellular migration and proliferation in knockout cells. We also saw increases in other cellular functions, such as tube formation and nitric oxide formation. From these data we were able to conclude that EphB4 is an active kinase in differentiated cells with a significant inhibitory effect. In EphB4 +/- knockout cell lines there was a lack of EphB4 inhibition and AKT and ERK showed increased activity. This work clearly implicates EphB4 as a major regulator of the basic cellular function of endothelia and highlights the need for further investigation into the specific pathways by which it functions.

The Notch receptor is part of a highly conserved signaling pathway shared in Drosophila, C. elegans and mammals. Extensive studies of Notch signaling have revealed its participation in the development of diverse organ systems including brain, blood cells, blood vessels, gut, and skin. Many genetic modifiers of the Notch signaling pathway have been identified, including some which act at the membrane and others in the nucleus. One such member is Deltex, an E3 ubiquitin ligase, which was originally identified as a modifier of Notch in a Drosophila genetic screen. In early lymphoid development, Deltex has been demonstrated functionally to antagonize Notch signaling but the precise molecular mechanism for this functional antagonism between Notch and Deltex is not understood. However, in Drosophila, recent data supports the formation of a trimeric complex between Deltex, Kurtz and Notch that promotes Notch ubiquitin-mediated proteosomal degradation. Beta-arrestin1 is one of the closest mammalian homologues of Kurtz and functions as an adaptor protein in a variety of cellular processes such as endocytosis, ubiquitination and nuclear shuttling. We hypothesize that a similar interaction occurs in mammalian cells between Notch, beta-arrestin1 and Deltex to negatively modulate the Notch signaling pathway. Our data reveal a physical interaction between beta-arrestin1 and the Notch receptor. We could not, however, detect an interaction between Deltex and beta-arrestin1 by co-immunoprecipitation. We also demonstrate that Notch and beta-arrestin1 physically associate with both a membrane-bound form of activated Notch, as well as the intracellular form of Notch after membrane cleavage. Using RNA interference, as well as overexpression of beta-arrestin1, we demonstrate that beta-arrestin1 negatively regulates a Notch/CSL dependant reporter assay. We also show that the presence of Deltex enhances the negative modulation of the Notch signaling pathway mediated by beta-arrestin1. Therefore, we reveal a new Notch interacting protein and a novel role for beta-arrestin1 in the Notch signaling pathway.