Novel RNAi-mediated approach to G protein-coupled receptor deorphanization: proof of principle and characterization of a planarian 5-HT receptor

G protein-coupled receptors (GPCRs) represent the largest known superfamily of membrane proteins extending throughout the Metazoa. There exists ample motivation to elucidate the functional properties of GPCRs given their role in signal transduction and their prominence as drug targets. In many target organisms, these efforts are hampered by the unreliable nature of heterologous receptor expression platforms. We validate and describe an alternative loss-of-function approach for ascertaining the ligand and G protein coupling properties of GPCRs in their native cell membrane environment. Our efforts are focused on the phylum Platyhelminthes, given the heavy health burden exacted by pathogenic flatworms, as well as the role of free-living flatworms as model organisms for the study of developmental biology. RNA interference (RNAi) was used in conjunction with a biochemical endpoint assay to monitor cAMP modulation in response to the translational suppression of individual receptors. As proof of principle, this approach was used to confirm the neuropeptide GYIRFamide as the cognate ligand for the planarian neuropeptide receptor GtNPR-1, while revealing its endogenous coupling to Gα(i/o). The method was then extended to deorphanize a novel Gα(s)-coupled planarian serotonin receptor, DtSER-1. A bioinformatics protocol guided the selection of receptor candidates mediating 5-HT-evoked responses. These results provide functional data on a neurotransmitter central to flatworm biology, while establishing the great potential of an RNAi-based deorphanization protocol. Future work can help optimize and adapt this protocol for higher-throughput platforms as well as other phyla.     

Detailed interrogation of trypanosome cell biology via differential organelle staining and automated image analysis

Background  

Many trypanosomatid protozoa are important human or animal pathogens. The well defined morphology and precisely choreographed division of trypanosomatid cells makes morphological analysis a powerful tool for analyzing the effect of mutations, chemical insults and changes between lifecycle stages. High-throughput image analysis of micrographs has the potential to accelerate collection of quantitative morphological data. Trypanosomatid cells have two large DNA-containing organelles, the kinetoplast (mitochondrial DNA) and nucleus, which provide useful markers for morphometric analysis; however they need to be accurately identified and often lie in close proximity. This presents a technical challenge. Accurate identification and quantitation of the DNA content of these organelles is a central requirement of any automated analysis method.     

Dominant-negative effect of truncated mannose 6-phosphate/insulin-like growth factor II receptor species in cancer

Oligomerization of the mannose 6-phosphate/insulin-like growth factor?II receptor (M6P/IGF2R) is important for optimal ligand binding and internalization. M6P/IGF2R is a tumor suppressor gene that exhibits loss of heterozygosity and is mutated in several cancers. We tested the potential dominant-negative effects of two cancer-associated mutations that truncate M6P/IGF2R in ectodomain repeats 9 and 14. Our hypothesis was that co-expression of the truncated receptors with the wild-type/endogenous full-length M6P/IGF2R would interfere with M6P/IGF2R function by heterodimer interference. Immunoprecipitation confirmed formation of heterodimeric complexes between full-length M6P/IGF2Rs and the truncated receptors, termed Rep9F and Rep14F. Remarkably, increasing expression of either Rep9F or Rep14F provoked decreased levels of full-length M6P/IGF2Rs in both cell lysates and plasma membranes, indicating a dominant-negative effect on receptor availability. Loss of full-length M6P/IGF2R was not due to increased proteasomal or lysosomal degradation, but instead arose from increased proteolytic cleavage of cell-surface M6P/IGF2Rs, resulting in ectodomain release, by a mechanism that was inhibited by metal ion chelators. These data suggest that M6P/IGF2R truncation mutants may contribute to the cancer phenotype by decreasing the availability of full-length M6P/IGF2Rs to perform tumor-suppressive functions such as binding/internalization of receptor ligands such as insulin-like growth factor II.     

Characterization of four esterase genes and esterase activity from the gut of the termite Reticulitermes flavipes

Four esterase genes and general esterase activity were investigated in the gut of the termite Reticulitermes flavipes. Two genes (RfEst1 and RfEst2) share significant translated identity with a number of insect JH esterases. The two remaining genes (RfEst3 and RfEst4) apparently code for much shorter proteins with similarity to fungal phenolic acid esterases involved in hemicellulose solubilization. All four genes showed consistently high midgut expression. This result was further supported by colorimetric activity assays and Native polyacrylamide gel electrophoresis, which showed significant esterase activity and a number of isoforms in the midgut. The greatest esterase activity and isoform composition were detected when α‐naphthyl propionate was used as a substrate. Moreover, esterase activity and diverse isoforms were present in gut mitochondrial, microsomal, and cytosolic sub‐cellular protein fractions, as well as in the hindgut lumen. These findings reveal an agreement between gut esterase gene expression and activity distributions, and support the idea that R. flavipes gut esterase activity is host (not symbiont)‐derived. In addition, these findings support the hypotheses that termite gut esterases may play important roles in lignocellulose digestion and caste differentiation. This study provides important baseline data that will assist ongoing functional‐genomic efforts to identify novel genes with roles in semiochemical, hormone, and lignocellulose processing in the termite gut. © 2009 Wiley Periodicals, Inc.     

Caterpillars' compensatory feeding response to diluted nutrients leads to toxic allelochemical dose

Many herbivores increase their consumption rate as dietary nutrient concentration declines. This compensatory response can mitigate the fitness-lowering impact of reduced food quality, but little is known about its costs. In this study we tested the hypothesis that one cost to a faster consumption rate can be the ingestion of a toxic dose of an allelochemical occurring in the food. We fed velvetbean caterpillars a diet with progressively diluted nutrient levels but containing the same concentration (% fresh mass, fm) of caffeine, a methylxanthine alkaloid. Larvae compensated for the reduced nutrient level, with those fed the most diluted diet increasing their biomass-relative consumption rate (fm) 2.6-fold over larvae fed the undiluted diet. Consequently, their rate of caffeine ingestion increased to a pharmacologically effective dose, interfering with food utilization, slowing growth, reducing subsequent feeding and lowering survival. These results suggest that greater allelochemical ingestion can be one cost of an increased consumption rate, although additional studies with other allelochemicals and species are necessary to more broadly evaluate whether insects can adaptively balance their intake of nutrients and allelochemicals through adjustments in consumption rate. In addition, these results highlight the importance of measuring consumption rates of allelochemicals and other ingested biocides, not just their dietary concentration, when assessing efficacy against herbivores.     

BIOCHEMICAL EFFECTS OF VICTORIN ON OAT TISSUES AND MITOCHONDRIA

Victorin, the pathotoxin produced by the plant pathogenic fungus Helminthosporium victoriae, causes changes in respiration and permeability which are typical of diseased plant tissues. To provide information on the site and mode of action of this toxin, the effects of victorin on mitochondria were studied and the nature and quantities of materials released from victorin-treated tissues were determined. Victorin added to isolated mitochondria had no effect on release of electrolytes or on oxidative-phosphorylative capacity. Hence the high respiratory rate found in victorin-treated tissues does not appear to be the result of a direct effect of the toxin on the respiratory centers. With mitochondria extracted from tissue pretreated with victorin, electrolyte release was unaffected but oxygen uptake was slightly higher and phosphate esterification considerably lower than controls. Thus the toxin produces indirect effects on mitochondrial activity, but the relation of these effects to tissue respiration remains undetermined. Victorin-treated tissue lost much larger quantities of certain organic and inorganic materials than control tissue with the most striking difference in the amount of potassium released.