Uncoordinated (UNC)119: Coordinating the trafficking of myristoylated proteins

pre-printThe mechanism by which myristoylated proteins are targeted to specific subcellular membrane compartments is poorly understood. Two novel acyl-binding proteins, UNC119A and UNC119B, have been shown recently to function as chaperones/co-factors in the transport of myristoylated G protein a-subunits and src-type tyrosine kinases. UNC119 polypeptides feature an immunoglobulin-like b-sandwich fold that forms a hydrophobic pocket capable of binding lauroyl (C12) and myristoyl (C14) side chains. UNC119A in rod photoreceptors facilitates the transfer of transducin a subunits (Ta) from inner segment to outer segment membranes by forming an intermediate diffusible UNC119-Ta complex. Similar complexes are formed in other sensory neurons, as the G proteins ODR-3 and GPA-13 in Caenorhabditis elegans unc-119 mutants traffic inappropriately. UNC119B knockdown in IMCD3 cells prevents trafficking of myristoylated nephrocystin-3 (NPHP3), a protein associated with nephronophthisis, to cilia. Further, UNC119A was shown to transport myristoylated src-type tyrosine kinases to cell membranes and to affect T-cell receptor (TCR) and interleukin-5 receptor (IL-5R) activities. These interactions establish UNC119 polypeptides as novel lipid-binding chaperones with specificity for a diverse subset of myristoylated protein

RNA interference gene therapy in dominant retinitis pigmentosa and cone-rod dystrophy mouse models caused by GCAP1 mutations

pre-printRNA interference (RNAi) knockdown is an efficacious therapeutic strategy for silencing genes causative for dominant retinal dystrophies. To test this, we used self-complementry (sc) AAV2/8 vector to develop an RNAi-based therapy in two dominant retinal degeneration mouse models. The allele-specific model expresses transgenic bovine GCAP1(Y99C) establishing a rapid RP-like phenotype, whereas the nonallele-specific model expressed mouse GCAP1(L151F) producing a slowly progressing cone-rod dystrophy (CORD). The late onset GCAP1(151F)-CORD mimics the dystrophy observed in human GCAP1-CORD patients. Subretinal injection of scAAV2/8 carrying shRNA expression cassettes specific for bovine or mouse guanylate cyclase-activating protein 1 (GCAP1) showed strong expression at 1 week post-injection. In both allele-specific [GCAP1(Y99C)-RP] and nonallele-specific [GCAP1(L151F)-CORD] models of dominant retinal dystrophy, RNAi-mediated gene silencing enhanced photoreceptor survival, delayed onset of degeneration and improved visual function. Such results provide a "proof of concept" toward effective RNAi-based gene therapy mediated by scAAV2/8 for dominant retinal disease based on GCAPi mutation. Further, nonallele-specific RNAi knockdown of GCApi may prove generally applicable toward the rescue of any human GCApi-based dominant cone-rod dystrophy

Centrin 2 is required for mouse olfactory ciliary trafficking and development of ependymal cilia planar polarity

pre-printCentrins are ancient calmodulin-related Ca2-binding proteins associated with basal bodies. In lower eukaryotes, Centrin2 (CETN2) is required for basal body replication and positioning, although its function in mammals is undefined. We generated a germline CETN2 knock-out (KO) mouse presenting with syndromic ciliopathy including dysosmia and hydrocephalus. Absence of CETN2 leads to olfactory cilia loss, impaired ciliary trafficking of olfactory signaling proteins, adenylate cyclase III (ACIII), and cyclic nucleotide-gated (CNG) channel, as well as disrupted basal body apical migration in postnatal olfactory sensory neurons (OSNs). In mutant OSNs, cilia baseanchoring of intraflagellar transport components IFT88, the kinesin-II subunit KIF3A, and cytoplasmic dynein 2 appeared compromised. Although the densities of mutant ependymal and respiratory cilia were largely normal, the planar polarity of mutant ependymal cilia was disrupted, resulting in uncoordinated flow of CSF. Transgenic expression of GFP-CETN2 rescued the Cetn2-deficiency phenotype. These results indicate that mammalian basal body replication and ciliogenesis occur independently of CETN2; however, mouse CETN2 regulates protein trafficking of olfactory cilia and participates in specifying planar polarity of ependymal cilia

Inactivity of human β,β-carotene-9′, 10-′dioxygenase (BCO2) underlies retinal accumulation of the human macular carotenoid pigment

pre-printThe macula of the primate retina uniquely concentrates high amounts of the xanthophyll carotenoids lutein, zeaxanthin, and meso-zeaxanthin, but the underlying biochemical mechanisms for this spatial- and species-specific localization have not been fully elucidated. For example, despite abundant retinal levels in mice and primates of a binding protein for zeaxanthin and mesozeaxanthin, the pi-isoform of glutathione S-transferase (GSTP1), only human and monkey retinas naturally contain detectable levels of these carotenoids. We therefore investigated whether or not differences in expression, localization, and activity between mouse and primate carotenoid metabolic enzymes could account for this species-specific difference in retinal accumulation. We focused on β,β-carotene-9',10'-dioxygenase (BCO2; also known as BCDO2), the only known mammalian xanthophyll cleavage enzyme. RT-PCR, western blot analysis, and immunohistochemistry confirmed that BCO2 is expressed in both mouse and primate retinas. Cotransfection of expression plasmids of human or mouse BCO2 into E. coli strains engineered to produce zeaxanthin demonstrated that only mouse BCO2 is an active zeaxanthin cleavage enzyme. Surface plasmon resonance (SPR) binding studies showed that the binding affinities between human BCO2 and lutein, zeaxanthin, and meso-zeaxanthin are 10- to 40-fold weaker than those for mouse BCO2, implying that ineffective capture of carotenoids by human BCO2 prevents cleavage of xanthophyll carotenoids. Moreover, BCO2 knockout mice, unlike wild-type mice, accumulate zeaxanthin in their retinas. Our results provide a novel explanation for how primates uniquely concentrate xanthophyll carotenoids at high levels in retinal tissue

The prenyl-binding protein PrBP/δ: A chaperone participating in intracellular trafficking

pre-printExpressed ubiquitously, PrBP/d functions as chaperone/co-factor in the transport of a subset of prenylated proteins. PrBP/d features an immunoglobulin-like b-sandwich fold for lipid binding, and interacts with diverse partners. PrBP/d binds both C-terminal C15 and C20 prenyl side chains of phototransduction polypeptides and small GTP-binding (G) proteins of the Ras superfamily. PrBP/d also interacts with the small GTPases, ARL2 and ARL3, which act as release factors (GDFs) for prenylated cargo. Targeted deletion of the mouse Pde6d gene encoding PrBP/d resulted in impeded trafficking to the outer segments of GRK1 and cone PDE6 which are predicted to be farnesylated and geranylgeranylated, respectively. Rod and cone transducin trafficking was largely unaffected. These trafficking defects produce progressive cone-rod dystrophy in the Pde6d/ mouse

Identification and characterization of a Pi isoform of glutathione S-transferase (GSTP1) as a zeaxanthin-binding protein in the macula of the human eye

Journal ArticleUptake, metabolism, and stabilization of xanthophyll carotenoids in the retina are thought to be mediated by specific xanthophyll-binding proteins (XBPs). A membrane-associated XBP was purified from human macula using ion-exchange chromatography followed by gel-exclusion chromatography. Two-dimensional gel electrophoresis showed a prominent spot of 23 kDa and an isoelectric point of 5.7. Using mass spectral sequencing methods and the public NCBI database, it was identified as a Pi isoform of human glutathione S-transferase (GSTP1). Dietary (3R,3'R)-zeaxanthin displayed the highest affinity with an apparent Kd of 0.33 microm, followed by (3R,3'S-meso)-zeaxanthin with an apparent Kd of 0.52 microm. (3R,3'R,6'R)-Lutein did not display any high-affinity binding to GSTP1. Other human recombinant glutathione S-transferase (GST) proteins, GSTA1 and GSTM1, exhibited only low affinity binding of xanthophylls. (3R,3'S-meso)-Zeaxanthin, an optically inactive nondietary xanthophyll carotenoid present in the human macula, exhibited a strong induced CD spectrum in association with human macular XBP that was nearly identical to the CD spectrum induced by GSTP1. Like-wise, dietary (3R,3'R)-zeaxanthin displayed alterations in its CD spectrum in association with GSTP1 and XBP. Other mammalian xanthophyll carrier proteins such as tubulin, high-density lipoprotein, low-density lipoprotein, albumin, and beta-lactoglobulin did not bind zeaxanthins with high affinity, and they failed to induce or alter xanthophyll CD spectra to any significant extent. Immunocytochemistry with an antibody to GSTP1 on human macula sections showed highest labeling in the outer and inner plexiform layers. These results indicate that GSTP1 is a specific XBP in human macula that interacts with (3R,3'S-meso)-zeaxanthin and dietary (3R,3'R)-zeaxanthin in contrast to apparently weaker interactions with (3R,3'R,6'R)-lutein

Neurotransmitter properties of the newborn human retina.

Journal ArticleHuman retinal tissue from a newborn was examined autoradiographically for the presence of highaffinity uptake and localization of the following putative neurotransmitters: dopamine, glycine, GAB A, aspartate, and glutamate. In addition, the dopamine content of this newborn retina was measured by high pressure liquid chromatography. Our study reveals that specific uptake mechanisms for 3Hglycine, 3H-dopamine, and 3H-GABA are present at birth. However, the number and distribution of cells labeled with each of these 3H-transmitters are not identical to those observed in adult human retinas. Furthermore, the amount of endogenous dopamine in the newborn retina is approximately V20 the adult level. Photoreceptor-specific uptake of 3H-glutamate and 3H-aspartate are not observed. These findings indicate that, while some neurotransmitter-specific properties are present at birth, significant maturation of neurotransmitter systems occurs postnatally

Receptor for advanced glycation end products and age-related macular degeneration.

Journal ArticleAdvanced glycation end products (AGE) exacerbate disease progression through two general mechanisms: modifying molecules and forming nondegradable aggregates, thus impairing normal cellular/tissue functions, and altering cellular function directly through receptor-mediated activation. In the present study receptor for AGE (RAGE)-mediated cellular activation was evaluated in the etiology of human retinal aging and disease. METHODS: The maculas of human donor retinas from normal eyes and eyes with early age-related macular degeneration (AMD) and advanced AMD with geographic atrophy (GA) were assayed for AGE and RAGE by immunocytochemistry. Cultured ARPE-19 cells were challenged with known ligands for RAGE, AGE, and S100B, to test for activation capacity. Immunocytochemistry, real-time RT-PCR, immunoblot analysis, and the TUNEL assay were used to determine the consequences of RPE cellular activation. RESULTS: Little to no immunolabeling for AGE or RAGE was found in photoreceptor and RPE cell layers in normal retinas. However, when small drusen were present, AGE and RAGE were identified in the RPE or both the RPE and photoreceptors. In early AMD and GA, the RPE and remnant photoreceptor cells showed intense AGE and RAGE immunolabeling. Both AGE and S100B activated cultured RPE cells, as revealed by upregulated expression of RAGE, NFkappaB nuclear translocation, and apoptotic cell death. CONCLUSIONS: Immunolocalization of RAGE in RPE and photoreceptors coincided with AGE deposits and macular disease in aged, early AMD, and GA retinas. Further, AGE stimulated RAGE-mediated activation of cultured ARPE-19 cells in a dose-dependent fashion. AGE accumulation, as occurs with normal aging and in disease, may induce receptor-mediated activation of RPE/photoreceptor cells, contributing to disease progression in the aging human retinas

Inhibitors of metalloendoprotease activity prevent K+-stimulated neurotransmitter release from the retina of Xenopus laevis.

Journal ArticleMetalloendoprotease activity was identified in retinal homogenates using a synthetic fluorogenic metalloendoprotease substrate and specific metalloendoprotease inhibitors. The requirement of metalloendoprotease activity in neurotransmitter release was examined during the depolarization-induced release of [3H]glycine from the retina of Xenopus laevis. Neurons with high affinity uptake and calcium-dependent, K+-stimulated release of glycine have been described previously in this retina. When isolated retinas preloaded with [3H]glycine are depolarized by 22 mM K+, the usual efflux of [3H]glycine is completely abolished by the metalloendoprotease inhibitor, 1,10-phenanthroline (100 micrograms/ml). The inhibition of [3H]glycine release by 1,10-phenanthroline is dose dependent; furthermore, 1,10-phenanthroline blocks release by chelating metal and not Ca2+, since addition of equimolar calcium does not alter the inhibition. The metalloendoprotease inhibitor, carbobenzoxy (CBZ)-L-phenylalanine, also prevents release, whereas the amino acid, L-phenylalanine, has no effect. Synthetic carbobenzoxy dipeptide amides which are metalloendoprotease substrates (e.g., CBZ-Gly-Leu-amide, CBZ-Ser-Leu-amide, and CBZ-Gly-Phe-amide) also prevent [3H]glycine release in a dose-dependent and reversible manner. The synthetic dipeptide CBZ-Gly-Gly-amide, however, is not a metalloendoprotease substrate and has no effect on release. The ability of synthetic dipeptides to inhibit neurotransmitter release is amino acid specific, dose dependent, reversible, and matches their ability to interact with characterized metalloendoproteases. Depolarization-stimulated transmitter release may therefore require the activity of a metalloendoprotease

Bt Corn Farmer Compliance with Insect Resistance Management Requirements: Results from the 2002 Minnesota and Wisconsin Farm Polls

The U.S. Environmental Protection Agency (EPA) reregistered Bt corn in 2001 with mandatory Insect Resistance Management (IRM) requirements in order to promote sustainable use by farmers. Since then studies report IRM compliance rates ranging from 80 to 90 percent. Using survey data from Minnesota and Wisconsin, we show that previous compliance rate estimates are likely too high because they do not use a comprehensive measure for compliance. With a more comprehensive measure, we find compliance rates ranging between 60 to 75 percent.Bt corn, compliance, Insect Resistance Management, refuge, Crop Production/Industries,