Topology and function of nyctalopin in yeast and in-vitro translation systems.

Congenital stationary night blindness 1 (CSNBl) is a genetic disorder characterized in humans by night blindness, low visual acuity and myopia. CSNB 1 is caused by defects in genes that are involved in signaling between photoreceptors and depolarizing bipolar cells (DBCs). DBCs utilize a metabotropic glutamate receptor-6 (Grm6) cascade that modulates the activity of a non-specific cation channel. CSNBI is diagnosed by a reduced b-wave in the electroretinogram (ERG). A b-wave indicates that DBCs are depolarized in response to a flash of light. In the dark, there is a tonic release of glutamate from the photoreceptors into the synaptic cleft. This glutamate binds to the Grm6 receptor, activating a G-protein signal transduction cascade that closes a nonselective cation channel. The Gregg laboratory has identified this channel as the transient receptor potential melastatin l(Trpml) channel. The focus of my research is to determine how another protein, nyctalopin, which also lacks b-wave causes a loss of the channel\u27s activity. Nyctalopin is an integral membrane protein with the entire leucine rich repeat in the extracellular space. Nyctalopin interacts directly with extracellular loops of Trpml. Although nyctalopin alone is not able to gate or traffic the Trpml channel to the membrane, gene expression profiling and membrane split ubiquitin yeast two hybrid screen suggest that a complex of proteins including nyctalopin are involved in either assembling or trafficking of the Trpm 1 channel to the plasma membrane

Topological Analysis of Small Leucine-Rich Repeat Proteoglycan Nyctalopin

Nyctalopin is a small leucine rich repeat proteoglycan (SLRP) whose function is critical for normal vision. The absence of nyctalopin results in the complete form of congenital stationary night blindness. Normally, glutamate released by photoreceptors binds to the metabotropic glutamate receptor type 6 (GRM6), which through a G-protein cascade closes the non-specific cation channel, TRPM1, on the dendritic tips of depolarizing bipolar cells (DBCs) in the retina. Nyctalopin has been shown to interact with TRPM1 and expression of TRPM1 on the dendritic tips of the DBCs is dependent on nyctalopin expression. In the current study, we used yeast two hybrid and biochemical approaches to investigate whether murine nyctalopin was membrane bound, and if so by what mechanism, and also whether the functional form was as a homodimer. Our results show that murine nyctalopin is anchored to the plasma membrane by a single transmembrane domain, such that the LRR domain is located in the extracellular space

Insecticides From Wild Tomato: Trichome Counts and Contents

Wild species of plants contain numerous non-nutritive, bioactive compounds known as “phytochemicals”. Many of these compounds cause the leaf to be less suitable for insect growth and may influence leaf palatability. A significant positive correlation was found between the intensity of wild tomato leaf trichomes (leaf hairs) and mortality of many vegetable insects. Type-IV and type-VI glandular trichomes on the leaves of three accessions of Lycopersicon hirsutum f. typicum; six accessions of Lycopersicon hirsutum f. glabratum; two accessions of Lycopersicon pennellii; and one accession of Lycopersicon pimpinellifolium were counted monthly (January to December, 2001). Crude extracts prepared from the leaves of each species were also prepared in n-hexane and chloroform, separated, purified, and quantified using GC/MSD for biochemical composition. Monthly variations in concentration of methyl ketones, sesquiterpene hydrocarbons, and sugar esters (glycolipids) were determined. Considerable variations in biochemical constituents among accessions were detected

Insecticides From Wild Tomato: Trichome Counts and Contents

Wild species of plants contain numerous non-nutritive, bioactive compounds known as “phytochemicals”. Many of these compounds cause the leaf to be less suitable for insect growth and may influence leaf palatability. A significant positive correlation was found between the intensity of wild tomato leaf trichomes (leaf hairs) and mortality of many vegetable insects. Type-IV and type-VI glandular trichomes on the leaves of three accessions of Lycopersicon hirsutum f. typicum; six accessions of Lycopersicon hirsutum f. glabratum; two accessions of Lycopersicon pennellii; and one accession of Lycopersicon pimpinellifolium were counted monthly (January to December, 2001). Crude extracts prepared from the leaves of each species were also prepared in n-hexane and chloroform, separated, purified, and quantified using GC/MSD for biochemical composition. Monthly variations in concentration of methyl ketones, sesquiterpene hydrocarbons, and sugar esters (glycolipids) were determined. Considerable variations in biochemical constituents among accessions were detected

Epigenetic reactivation of LINE-1 retrotransposon disrupts NuRD corepressor functions and induces oncogenic transformation in human bronchial epithelial cells

Long interspersed nuclear element-1 (LINE-1 or L1) reactivation is linked to poor prognosis in non-small-cell lung carcinoma (NSCLC), but the molecular bases of this response remain largely unknown. In this report, we show that challenge of human bronchial epithelial cells (HBECs) with the lung carcinogen, benzo(a)pyrene (BaP), shifted the L1 promoter from a heterochromatic to euchromatic state through disassembly of the nucleosomal and remodeling deacetylase (NuRD) complex. Carcinogen challenge was also associated with partial displacement of constituent proteins from the nuclear to the cytoplasmic compartment. Disruption of NuRD corepression by genetic ablation or carcinogen treatment correlated with accumulation of L1 mRNA and proteins. Mi2 bound directly to the L1 promoter to effect retroelement silencing, and this response required the DNA- and ATPase-binding domains of Mi2. Sustained expression of L1 in HBECs was tumorigenic in a human-SCID mouse xenograft model, giving rise to tumors that regressed over time. Together, these results show that functional modulation of the NuRD constituent proteins is a critical molecular event in the activation of L1 retrotransposon. L1 expression creates a microenvironment in HBECs that is conducive to neoplasia and malignant transformation.University of Arizona Health Sciences [P30 CA023074]Open access journal.This item from the UA Faculty Publications collection is made available by the University of Arizona with support from the University of Arizona Libraries. If you have questions, please contact us at [email protected]

Epigenetic reactivation of LINE‐1 retrotransposon disrupts NuRD corepressor functions and induces oncogenic transformation in human bronchial epithelial cells

Long interspersed nuclear element‐1 (LINE‐1 or L1) reactivation is linked to poor prognosis in non‐small‐cell lung carcinoma (NSCLC), but the molecular bases of this response remain largely unknown. In this report, we show that challenge of human bronchial epithelial cells (HBECs) with the lung carcinogen, benzo(a)pyrene (BaP), shifted the L1 promoter from a heterochromatic to euchromatic state through disassembly of the nucleosomal and remodeling deacetylase (NuRD) complex. Carcinogen challenge was also associated with partial displacement of constituent proteins from the nuclear to the cytoplasmic compartment. Disruption of NuRD corepression by genetic ablation or carcinogen treatment correlated with accumulation of L1 mRNA and proteins. Mi2β bound directly to the L1 promoter to effect retroelement silencing, and this response required the DNA‐ and ATPase‐binding domains of Mi2β. Sustained expression of L1 in HBECs was tumorigenic in a human–SCID mouse xenograft model, giving rise to tumors that regressed over time. Together, these results show that functional modulation of the NuRD constituent proteins is a critical molecular event in the activation of L1 retrotransposon. L1 expression creates a microenvironment in HBECs that is conducive to neoplasia and malignant transformation

The N-terminus of nyctalopin is in the lumen of the ER.

<p>A. Schematic of constructs used to determine the orientation of nyctalopin in the membrane of the endoplasmic reticulum. SLucNyc (113 kDa) has luciferase (yellow rectangle) inserted after the murine nyctalopin signal sequence (SS). SNycLuc has luciferase attached to the C-terminus of full length nyctalopin. The arrows indicate the two thrombin (Thr) cleavage sites. Thrombin cleavage will generate 72.8 kDa and 34.4 kDa peptides, however, only the 72.8 kDa peptide will be labeled with biotinylated lysine and detected on a western blot. B. Western blot of <i>in vitro</i> transcription/translation reaction showing that without canine microsomal membranes (CMM), nyctalopin is not expressed. These data indicate co-translational processing and membrane insertion of nyctalopin in the ER. C. Expression of either SLucNyx or SNycLuc and treatment with proteinase K in the presence or absence of CHAPS. Lanes 1–4 indicate robust expression of full length nyctalopin. Lanes 5–8 show that SNycLuc but not SLucNyc is degraded by proteinase K, indicating the N-terminus of nyctalopin is in the ER lumen and therefore protected from degradation. Addition of CHAPS (0.5%) (lanes 9–12) disrupts the membranes and results in proteinase K digestion of both nyctalopin fusion proteins. D. Western blot of lysates from <i>in vitro</i> translation reactions, control (lanes 1–2), or when SLucNyc was included and after thrombin digestion alone (lanes 3–5) or thrombin digestion in the presence of 0.5% CHAPS (Lanes 6–8). Disruption of microsomal membranes with CHAPS allows cleavage of the fusion protein demonstrating that the N-terminus of nyctalopin is protected, and is therefore located in the lumen of the ER. Note the disappearance of the 113 kDa bands and the appearance of the 72 kDa band. E. Schematic showing a model of the orientation of nyctalopin in the ER and it’s subsequent disposition on the plasma membrane.</p

Nyctalopin does not form homo-dimers in yeast

<p>. A. Schematic of bait and prey constructs used. The components of nyctalopin are as described in Fig. 1 and 2. Alg5, Asparagine-linked glycosylation 5, is a yeast ER membrane bound protein with both the C-terminus and N-terminus in the cytoplasm. Syp-Cub and Syp-NubG are bait and prey constructs with synaptophysin, which is known to dimerize, and is used as positive control. B. Growth indicating incorporation of both bait and prey plasmids into yeast. Interaction was determined by growth on SD/-LWHA media and expression of β-galactosidase. These data show that nyctalopin does not form dimers in this system.</p