Publications by authors named "Que Lan"

27 Publications

  • Page 1 of 1

Specificity and putative mode of action of a mosquito larvicidal toxin from the bacterium Xenorhabdus innexi.

J Invertebr Pathol 2017 10 13;149:21-28. Epub 2017 Jul 13.

Department of Entomology, University of Wisconsin-Madison, Madison, WI 53706, USA.

Reduction of mosquito-borne diseases relies, in part, on the use of synthetic pesticides to control pest mosquitoes. This reliance has led to genetic resistance, environmental contamination and the nondiscriminatory elimination of both pest and non-pest species. To expand our options for control, we screened entomopathogenic bacteria for potential larvicidal activity. A lipopeptide from the bacterium, Xenorhabdus innexi, was discovered that displayed potent larvicidal activity. The LCs of the lipopeptide towards Aedes aegypti, Culex pipiens and Anopheles gambiae larvae were 1.81, 1.25 and 1.86 parts-per-million, respectively. No mortality was observed in other insect species tested. The putative mode of action of the lipopeptide suggested that after orally ingestion, it bound to the apical membrane of anterior midgut cells and created pores in the cellular membranes. The rapid neutralization of midgut pH suggested the pores disabled the H-V-ATPase on the basal membrane and led to epithelial cell death. Specificity and toxicity towards mosquito larvae and the unique mode of action makes this lipopeptide a potentially attractive bacterial insecticide for control of mosquitoes.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.jip.2017.07.002DOI Listing
October 2017

RNA interference of three up-regulated transcripts associated with insecticide resistance in an imidacloprid resistant population of Leptinotarsa decemlineata.

Pestic Biochem Physiol 2017 Jan 9;135:35-40. Epub 2016 Jul 9.

Department of Entomology, University of Wisconsin-Madison, Madison, WI 53706, USA. Electronic address:

The Colorado potato beetle, Leptinotarsa decemlineata (Say), is a major agricultural pest of potatoes in the Central Sands production region of Wisconsin. Previous studies have shown that populations of L. decemlineata have become resistant to many classes of insecticides, including the neonicotinoid insecticide, imidacloprid. Furthermore, L. decemlineata has multiple mechanisms of resistance to deal with a pesticide insult, including enhanced metabolic detoxification by cytochrome p450s and glutathione S-transferases. With recent advances in the transcriptomic analysis of imidacloprid susceptible and resistant L. decemlineata populations, it is possible to investigate the role of candidate genes involved in imidacloprid resistance. A recently annotated transcriptome analysis of L. decemlineata was obtained from select populations of L. decemlineata collected in the Central Sands potato production region, which revealed a subset of mRNA transcripts constitutively up-regulated in resistant populations. We hypothesize that a portion of the up-regulated transcripts encoding for genes within the resistant populations also encode for pesticide resistance and can be suppressed to re-establish a susceptible phenotype. In this study, a discrete set of three up-regulated targets were selected for RNA interference experiments using a resistant L. decemlineata population. Following the successful suppression of transcripts encoding for a cytochrome p450, a cuticular protein, and a glutathione synthetase protein in a select L. decemlineata population, we observed reductions in measured resistance to imidacloprid that strongly suggest these genes control essential steps in imidacloprid metabolism in these field populations.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.pestbp.2016.07.001DOI Listing
January 2017

Multifaceted biological insights from a draft genome sequence of the tobacco hornworm moth, Manduca sexta.

Insect Biochem Mol Biol 2016 09 12;76:118-147. Epub 2016 Aug 12.

Centre of Systems Biology, Biomedical Research Foundation, Academy of Athens, Athens, Greece.

Manduca sexta, known as the tobacco hornworm or Carolina sphinx moth, is a lepidopteran insect that is used extensively as a model system for research in insect biochemistry, physiology, neurobiology, development, and immunity. One important benefit of this species as an experimental model is its extremely large size, reaching more than 10 g in the larval stage. M. sexta larvae feed on solanaceous plants and thus must tolerate a substantial challenge from plant allelochemicals, including nicotine. We report the sequence and annotation of the M. sexta genome, and a survey of gene expression in various tissues and developmental stages. The Msex_1.0 genome assembly resulted in a total genome size of 419.4 Mbp. Repetitive sequences accounted for 25.8% of the assembled genome. The official gene set is comprised of 15,451 protein-coding genes, of which 2498 were manually curated. Extensive RNA-seq data from many tissues and developmental stages were used to improve gene models and for insights into gene expression patterns. Genome wide synteny analysis indicated a high level of macrosynteny in the Lepidoptera. Annotation and analyses were carried out for gene families involved in a wide spectrum of biological processes, including apoptosis, vacuole sorting, growth and development, structures of exoskeleton, egg shells, and muscle, vision, chemosensation, ion channels, signal transduction, neuropeptide signaling, neurotransmitter synthesis and transport, nicotine tolerance, lipid metabolism, and immunity. This genome sequence, annotation, and analysis provide an important new resource from a well-studied model insect species and will facilitate further biochemical and mechanistic experimental studies of many biological systems in insects.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.ibmb.2016.07.005DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5010457PMC
September 2016

Characterizing Molecular Mechanisms of Imidacloprid Resistance in Select Populations of Leptinotarsa decemlineata in the Central Sands Region of Wisconsin.

PLoS One 2016 28;11(1):e0147844. Epub 2016 Jan 28.

Department of Entomology, University of Wisconsin-Madison, Madison, Wisconsin, United States of America.

The Colorado potato beetle, Leptinotarsa decemlineata (Say), is a major agricultural pest in the Central Sands region of Wisconsin. Imidacloprid, a neonicotinoid insecticide, has commonly been used for control of L. decemlineata since its registration in 1995. In the last 10 years, many field populations of L. decemlineata have begun to show increasing imidacloprid resistance. We studied resistance phenotype as a phenomenon that reduces neonicotinoid efficacy and has practical consequences for potato pest management. Although we have not observed complete field failure following the use of these products, multiple studies have demonstrated that the lethal concentration to kill 50% of the test organisms (LC50) in different field populations of L. decemlineata varies greatly which may suggest that resistance of L. decemlineata is heritable and involves genetic changes. An important challenge in understanding resistance is assessing the genetic mechanisms associated with resistance and classifying up-regulated genes that may be involved in combating an insecticide insult. In this study we uncovered trends in imidacloprid phenotypic response that have developed in the region by estimating the LC50 values among different field populations against a range of imidacloprid doses. The LC50 values collected in 2008-2011, and more recently in 2013 and 2014, show that some field locations remain susceptible to imidacloprid, while nearby fields (<100km) have developed high levels of resistance. We also sought to uncover potential mechanisms of resistance at each field location. We compiled a transcriptome for populations, characterized as phenotypically 'susceptible' and 'resistant', by isolating mRNA from adult beetles and analyzing gene expression level differences. Strong differences were observed in constituently up and down-regulated genes among different field populations. Most significantly, the up-regulation of 3 cytochrome p450s and a glutathione synthetase related protein in multiple resistant populations provide a mechanistic explanation of resistance evolution in L. decemlineata.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0147844PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4731083PMC
July 2016

NMR structure and function of Helicoverpa armigera sterol carrier protein-2, an important insecticidal target from the cotton bollworm.

Sci Rep 2015 Dec 10;5:18186. Epub 2015 Dec 10.

School of Life Sciences, Central China Normal University, Wuhan, P.R.China.

The cotton bollworm, Helicoverpa armigera, has developed strong resistance to many insecticides. Sterol Carrier Protein-2 (SCP-2) is an important non-specific lipid transfer protein in insects and appears to be a potential new target. In order to elucidate the structure and function of Helicoverpa armigera SCP-2 (HaSCP-2), NMR spectroscopy, docking simulations, mutagenesis and bioassays were performed. HaSCP-2 composed of five α-helices and four stranded β-sheets. The folds of α-helices and β-sheets interacted together to form a hydrophobic cavity with putative entrance and exit openings, which served as a tunnel for accommodating and transporting of lipids. Several sterols and fatty acids could interact with HaSCP-2 via important hydrophobic sites, which could be potential targets for insecticides. Mutagenesis experiments indicated Y51, F53, F89, F110, I117 and Q131 may be the key functional sites. HaSCP-2 showed high cholesterol binding activity and SCP-2 inhibitors (SCPIs) could inhibit the biological activity of HaSCP-2. SCPI-treated larvae at young stage showed a significant decrease of cholesterol uptake in vivo. Our study describes for the first time a NMR structure of SCP-2 in lepidopteran H. armigera and reveals its important function in cholesterol uptake, which facilitates the screening of effective insecticides targeting the insect cholesterol metabolism.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/srep18186DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4674756PMC
December 2015

Sterol Carrier Protein 2, a Critical Host Factor for Dengue Virus Infection, Alters the Cholesterol Distribution in Mosquito Aag2 Cells.

J Med Entomol 2015 Sep 25;52(5):1124-34. Epub 2015 Jul 25.

Department of Entomology, University of Wisconsin-Madison, WI. Deceased.

Host factors that enable dengue virus (DENV) to propagate in the mosquito host cells are unclear. It is known that cellular cholesterol plays an important role in the life cycle of DENV in human host cells but unknown if the lipid requirements differ for mosquito versus mammalian. In mosquito Aedes aegypti, sterol carrier protein 2 (SCP-2) is critical for cellular cholesterol homeostasis. In this study, we identified SCP-2 as a critical host factor for DENV production in mosquito Aag2 cells. Treatment with a small molecule commonly referred to as SCPI-1, (N-(4-{[4-(3,4-dichlorophenyl)-1,3-thiazol-2-yl]amino}phenyl)acetamide hydrobromide, a known inhibitor of SCP-2, or knockdown of SCP-2 dramatically repressed the virus production in mosquito but not mammalian cells. We showed that the intracellular cholesterol distribution in mosquito cells was altered by SCP-2 inhibitor treatment, suggesting that SCP-2-mediated cholesterol trafficking pathway is important for DENV viral production. A comparison of the effect of SCP-2 on mosquito and human cells suggests that SCPI-1 treatment decreases cholesterol in both cell lines, but this decrease in cholesterol only leads to a decline in viral titer in mosquito host cells, perhaps, owing to a more drastic effect on perinuclear cholesterol storages in mosquito cells that was absent in human cells. SCP-2 had no inhibitory effect on another enveloped RNA virus grown in mosquito cells, suggesting that SCP-2 does not have a generalized anti-cellular or antiviral effect. Our cell culture results imply that SCP-2 may play a limiting role in mosquito-dengue vector competence.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1093/jme/tjv101DOI Listing
September 2015

Thiosemicarbazones as Aedes aegypti larvicidal.

Eur J Med Chem 2015 Jul 28;100:162-75. Epub 2015 May 28.

Quantum Theory Project, University of Florida, 2234 New Physics Building, Gainesville, PO Box 118435, Florida, USA.

A set of aryl- and phenoxymethyl-(thio)semicarbazones were synthetized, characterized and biologically evaluated against the larvae of Aedes aegypti (A. aegypti), the vector responsible for diseases like Dengue and Yellow Fever. (Q)SAR studies were useful for predicting the activities of the compounds not included to create the QSAR model as well as to predict the features of a new compound with improved activity. Docking studies corroborated experimental evidence of AeSCP-2 as a potential target able to explain the larvicidal properties of its compounds. The trend observed between the in silico Docking scores and the in vitro pLC50 (equals -log LC50, at molar concentration) data indicated that the highest larvicidal compounds, or the compounds with the highest values for pLC50, are usually those with the higher docking scores (i.e., greater in silico affinity for the AeSCP-2 target). Determination of cytotoxicity for these compounds in mammal cells demonstrated that the top larvicide compounds are non-toxic.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.ejmech.2015.04.061DOI Listing
July 2015

THAP and ATF-2 regulated sterol carrier protein-2 promoter activities in the larval midgut of the yellow fever mosquito, Aedes aegypti.

PLoS One 2012 4;7(10):e46948. Epub 2012 Oct 4.

College of Life Sciences, Central China Normal University, Wuhan, Hubei, China.

Expression of sterol carrier protein-2 (SCP-2) in Aedes aegypti shows a distinct temporal/spatial pattern throughout the life cycle. In order to identify the transcription factors responsible for the larval temporal/spatial regulation of AeSCP-2 transcription, AeSCP-2 promoter activities were studied in vivo via transient transfection of promoter/reporter gene assays. Regulatory sequences upstream -1.3 kb of the transcription start site of AeSCP-2 were found to be critical for the in vivo temporal/spatial promoter activity. Interestingly, the -1.6 kb promoter sequence efficiently drove the larval midgut-specific siRNA expression, indicating that the -1.6 kb upstream sequence is sufficient for temporal/spatial AeSCP-2 transcriptional activity. Four transcription factors were identified in the midgut nuclear extract from feeding larvae via labeled -1.6/-1.3 kb DNA probe pull-down and proteomic analysis. Co-transfection of the promoter/reporter gene with inducible siRNA expression of each transcription factor was performed to confirm the regulatory function of individual transcription factor on AeSCP-2 transcriptional activities in the larval midgut. The results indicate that two of the identified transcription factors, Thanatos-associated protein (THAP) and activating transcription factor-2 (ATF-2), antagonistically control AeSCP-2 transcriptional activity in the midgut of feeding larvae via the regulatory sequences between -1.6 to -1.3 kb 5' upstream of the transcription start site. In vivo expression knockdown of THAP and ATF-2 resulted in significant changes in developmental progression, which may be partially due to their effects on AeSCP-2 expression.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0046948PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3464256PMC
May 2013

Characterization of the sterol carrier protein-x/sterol carrier protein-2 gene in the cotton bollworm, Helicoverpa armigera.

J Insect Physiol 2012 Nov 23;58(11):1413-23. Epub 2012 Aug 23.

College of Life Sciences, Central China Normal University, 100 Luoyu Road, Wuhan, Hubei 430079, PR China.

Cholesterol is a membrane component and the precursor of ecdysteroids in insects, but insects cannot synthesize cholesterol de novo. Therefore, cholesterol uptake and transportation during the feeding larval stages are critical processes in insects. The sterol carrier protein-2 domain (SCP-2) in sterol carrier proteins-x (SCP-x) has been speculated to be involved in intracellular cholesterol transfer and metabolism in vertebrates. However, a direct association between SCP-x gene expression, cholesterol absorption and development in lepidopteran insects is poorly understood. We identified the Helicoverpa armigera sterol carrier protein-x/2 (HaSCP-x/2) gene from the larval midgut cDNAs. The HaSCP-x/2 gene is well conserved during evolution and relatively divergent in heterogenetic species. Transcripts of HaSCP-x/2 were detected by qRT-PCR at the highest level in the midgut of H. armigera during the larval stages. Expression knockdown of HaSCP-x/2 transcripts via dsRNA interference resulted in delayed larval development and decreased adult fecundity. Sterol carrier protein-2 inhibitors were lethal to young larvae and decreased fertility in adults emerged from treated elder larvae in H. armigera. The results taken together suggest that HaSCPx/2 gene is important for normal development and fertility in H. armigera.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.jinsphys.2012.08.005DOI Listing
November 2012

Mosquitocidal properties of natural product compounds isolated from Chinese herbs and synthetic analogs of curcumin.

J Med Entomol 2012 Mar;49(2):350-5

Department of Entomology, University of Wisconsin, 840 Russell Labs, 1630 Linden Drive, Madison, WI 53706, USA.

Because of resistance to current insecticides and to environmental, health, and regulatory concerns, naturally occurring compounds and their derivatives are of increasing interest for the development of new insecticidal compounds against vectors of disease-causing pathogens. Fifty-eight compounds, either extracted and purified from plants native to China or synthetic analogs of curcumin, were evaluated for both their larvicidal activity against Aedes aegypti (L.) and their ability to inhibit binding of cholesterol to Ae. aegypti sterol carrier protein-2 in vitro. Of the compounds tested, curcumin analogs seem especially promising in that of 24 compounds tested five were inhibitors of Ae. aegypti sterol carrier protein-2 with EC50 values ranging from 0.65 to 62.87 microM, and three curcumin analogs exhibited larvicidal activity against fourth instar Ae. aegypti larvae with LC50 values ranging from 17.29 to 27.90 microM. Adding to the attractiveness of synthetic curcumin analogs is the relative ease of synthesizing a large diversity of compounds; only a small fraction of such diversity has been sampled in this study.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3538819PMC
http://dx.doi.org/10.1603/me11117DOI Listing
March 2012

In vivo functional genomic studies of sterol carrier protein-2 gene in the yellow fever mosquito.

PLoS One 2011 Mar 18;6(3):e18030. Epub 2011 Mar 18.

College of Life Sciences, Central China Normal University, Wuhan, Hubei, China.

A simple and efficient DNA delivery method to introduce extrachromosomal DNA into mosquito embryos would significantly aid functional genomic studies. The conventional method for delivery of DNA into insects is to inject the DNA directly into the embryos. Taking advantage of the unique aspects of mosquito reproductive physiology during vitellogenesis and an in vivo transfection reagent that mediates DNA uptake in cells via endocytosis, we have developed a new method to introduce DNA into mosquito embryos vertically via microinjection of DNA vectors in vitellogenic females without directly manipulating the embryos. Our method was able to introduce inducible gene expression vectors transiently into F0 mosquitoes to perform functional studies in vivo without transgenic lines. The high efficiency of expression knockdown was reproducible with more than 70% of the F0 individuals showed sufficient gene expression suppression (<30% of the controls' levels). At the cohort level, AeSCP-2 expression knockdown in early instar larvae resulted in detectable phenotypes of the expression deficiency such as high mortality, lowered fertility, and distorted sex ratio after induction of AeSCP-2 siRNA expression in vivo. The results further confirmed the important role of AeSCP-2 in the development and reproduction of A. aegypti. In this study, we proved that extrachromosomal transient expression of an inducible gene from a DNA vector vertically delivered via vitellogenic females can be used to manipulate gene expression in F0 generation. This new method will be a simple and efficient tool for in vivo functional genomic studies in mosquitoes.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0018030PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3060925PMC
March 2011

Effects of mutations in Aedes aegypti sterol carrier protein-2 on the biological function of the protein.

Biochemistry 2010 Sep;49(35):7532-41

Department of Entomology, University of Wisconsin, Madison, Wisconsin 53706, USA.

Sterol carrier protein-2 (SCP-2) is a nonspecific intracellular lipid carrier protein. However, the molecular mechanism of ligand selectivity and the in vivo function of SCP-2 remain unclear. In this study, we used site-directed mutagenesis to investigate the ligand selectivity and in vivo function of the yellow fever mosquito sterol carrier protein-2 protein (AeSCP-2). Mutations to amino acids in AeSCP-2 known to interact with bound ligand also weakened NBD-cholesterol binding. Substitution of amino acids in the ligand cavity changed the ligand specificity of mutant AeSCP-2. Overexpressing wild-type AeSCP-2 in the Aedes aegypti cultured Aag-2 cells resulted in an increase in the level of incorporation of [(3)H]cholesterol. However, overexpressing mutants that were deleterious to the binding of NBD-cholesterol in AeSCP-2 showed a loss of ability to enhance uptake of [(3)H]cholesterol in cultured cells. Interestingly, when [(3)H]palmitic acid was used as the substrate for incorporation in vivo, there was no change in the levels of incorporation with overexpression of wild-type protein or mutated AeSCP-2s. The in vivo data suggest that AeSCP-2 is involved in sterol uptake, but not fatty acid uptake. This is the first report that the cholesterol binding ability may directly correlate with AeSCP-2's in vivo function in aiding the uptake of cholesterol.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1021/bi902026vDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2933382PMC
September 2010

Sterol carrier protein-x gene and effects of sterol carrier protein-2 inhibitors on lipid uptake in Manduca sexta.

Authors:
Min-Sik Kim Que Lan

BMC Physiol 2010 Jun 9;10. Epub 2010 Jun 9.

Department of Entomology, University of Wisconsin, 1630 Linden Drive, Madison, WI 53706, USA.

Background: Cholesterol uptake and transportation during the feeding larval stages are critical processes in insects because they are auxotrophic for exogenous (dietary) cholesterol. The midgut is the main site for cholesterol uptake in many insects. However, the molecular mechanism by which dietary cholesterol is digested and absorbed within the midgut and then released into the hemolymph for transportation to utilization or storage sites is poorly understood. Sterol carrier proteins (SCP), non-specific lipid transfer proteins, have been speculated to be involved in intracellular cholesterol transfer and metabolism in vertebrates. Based on the high degree of homology in the conserved sterol transfer domain to rat and human SCP-2, it is supposed that insect SCP-2 has a parallel function to vertebrate SCP-2.

Results: We identified the Manduca sexta sterol carrier protein-x and the sterol carrier protein-2 (MsSCP-x/SCP-2) gene from the larval fat body and the midgut cDNAs. The MsSCP-x/SCP-2 protein has a high degree of homology in the SCP-2 domain to other insects' SCP-2. Transcripts of MsSCP-2 were detected at high levels in the midgut and the fat body of M. sexta during the larval stages. Recombinant MsSCP-2 bound to NBD-cholesterol with high affinity, which was suppressed by sterol carrier protein-2 inhibitors.

Conclusions: The results suggest that MsSCP-2 may function as a lipid carrier protein in vivo, and targeting insect SCP-2 may be a viable approach for the development of new insecticides.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1186/1472-6793-10-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2903571PMC
June 2010

The biological activity of alpha-mangostin, a larvicidal botanic mosquito sterol carrier protein-2 inhibitor.

J Med Entomol 2010 Mar;47(2):249-57

Department of Entomology, University of Wisconsin, Madison, WI 53706, USA.

alpha-Mangostin derived from mangosteen was identified as a mosquito sterol carrier protein-2 inhibitor via high throughput insecticide screening, alpha-Mangostin was tested for its larvicidal activity against third instar larvae of six mosquito species, and the median lethal concentration values range from 0.84 to 2.90 ppm. The residual larvicidal activity of alpha-mangostin was examined under semifield conditions. The results indicated that alpha-mangostin was photolytic with a half-life of 53 min in water under full sunlight exposure. The effect of alpha-mangostin on activities of major detoxification enzymes such as P450, glutathione S-transferase, and esterase was investigated. The results showed that alpha-mangostin significantly elevated activities of P450 and glutathione S-transferase in larvae, whereas it suppressed esterase activity. Toxicity of alpha-mangostin against young rats was studied, and there was no detectable adverse effect at dosages as high as 80 mg/kg. This is the first multifaceted study of the biological activity of alpha-mangostin in mosquitoes. The results suggest that alpha-mangostin may be a lead compound for the development of a new organically based mosquito larvicide.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2855149PMC
http://dx.doi.org/10.1603/me09160DOI Listing
March 2010

Differences in the structure and dynamics of the apo- and palmitate-ligated forms of Aedes aegypti sterol carrier protein 2 (AeSCP-2).

J Biol Chem 2010 May 31;285(22):17046-53. Epub 2010 Mar 31.

Department of Biochemistry, National Magnetic Resonance Facility at Madison, Wisconsin 53706, USA.

Sterol carrier protein-2 (SCP-2) is a nonspecific lipid-binding protein expressed ubiquitously in most organisms. Knockdown of SCP-2 expression in mosquitoes has been shown to result in high mortality in developing adults and significantly lowered fertility. Thus, it is of interest to determine the structure of mosquito SCP-2 and to identify its mechanism of lipid binding. We report here high quality three-dimensional solution structures of SCP-2 from Aedes aegypti determined by NMR spectroscopy in its ligand-free state (AeSCP-2) and in complex with palmitate. Both structures have a similar mixed alpha/beta fold consisting of a five-stranded beta-sheet and four alpha-helices arranged on one side of the beta-sheet. Ligand-free AeSCP-2 exhibited regions of structural heterogeneity, as evidenced by multiple two-dimensional (15)N heteronuclear single-quantum coherence peaks for certain amino acids; this heterogeneity disappeared upon complex formation with palmitate. The binding of palmitate to AeSCP-2 was found to decrease the backbone mobility of the protein but not to alter its secondary structure. Complex formation is accompanied by chemical shift differences and a loss of mobility for residues in the loop between helix alphaI and strand betaA. The structural differences between the alphaI and betaA of the mosquito and the vertebrate SCP-2s may explain the differential specificity (insect versus vertebrate) of chemical inhibitors of the mosquito SCP-2.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1074/jbc.M110.101154DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2878086PMC
May 2010

Larvicidal activity of mosquito sterol carrier protein-2 inhibitors to the insecticide-resistant mosquito Culex quinquefasciatus (Diptera: Culicidae).

J Med Entomol 2009 Nov;46(6):1430-5

Department of Entomology and Plant Pathology, Auburn University, Auburn, AL 36849, USA.

AeSCP-2 inhibitors (SCPIs) compete with cholesterol for binding to a mosquito sterol carrier protein-2 (AeSCP-2) known to aid in the uptake of cholesterol in mosquito cells. The larvicidal activities of AeSCP-2 inhibitor-1 (SCPI-1) and inhibitor-2 (SCPI-2) against Culex quinquefasciatus Say (Diptera: Culicidae) were therefore examined in insecticide-resistant Culex mosquitoes HAmCq(G9), MAmCq(G2), and BAmCq(G0). All of the resistant Culex mosquito strains exhibited similar sensitivity to SCPI-1 and SCPI-2 inhibitors compared with a susceptible S-Lab strain. When an AeSCP-2 inhibitor was applied simultaneously with permethrin, the toxicity of permethrin to the second-instar larvae of all four strains of Culex mosquitoes increased, suggesting a synergistic effect of AeSCP-2 inhibitors on the toxicity of permethrin against Culex mosquitoes. Both SCPI-1 and SCPI-2 inhibitors caused a 2.4- to 3-fold reduction in the level of permethrin resistance in the highly resistant strain HAmCq(G9). This result suggests that the mode of action of the AeSCP-2 inhibitors, which reduces the uptake of cholesterol by inhibiting the function of AeSCP-2 in mosquito cells, may interfere with the mechanisms or ability that govern permethrin resistance in the HAmCq(G9) mosquito strain.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1603/033.046.0626DOI Listing
November 2009

Characterization of the yellow fever mosquito sterol carrier protein-2 like 3 gene and ligand-bound protein structure.

Mol Cell Biochem 2009 Jun 7;326(1-2):67-77. Epub 2009 Jan 7.

Department of Bacteriology, University of Wisconsin-Madison, Madison, WI 53706, USA.

The sterol carrier protein-2 like 3 gene (AeSCP-2L3), a new member of the SCP-2 protein family, is identified from the yellow fever mosquito, Aedes aegypti. The predicted molecular weight of AeSCP-2L3 is 13.4 kDa with a calculated pI of 4.98. AeSCP-2L3 transcription occurs in the larval feeding stages and the mRNA levels decrease in pupae and adults. The highest levels of AeSCP-2L3 gene expression are found in the body wall, and possibly originated in the fat body. This is the first report of a mosquito SCP-2-like protein with prominent expression in tissue other than the midgut. The X-ray protein crystal structure of AeSCP-2L3 reveals a bound C16 fatty acid whose acyl tail penetrates deeply into a hydrophobic cavity. Interestingly, the ligand-binding cavity is slightly larger than previously described for AeSCP-2 (Dyer et al. J Biol Chem 278:39085-39091, 2003) and AeSCP-2L2 (Dyer et al. J Lipid Res M700460-JLR200, 2007). There are also an additional 10 amino acids in SCP-2L3 that are not present in other characterized mosquito SCP-2s forming an extended loop between beta 3 and beta 4. Otherwise, the protein backbone is exceedingly similar to other SCP-2 and SCP-2-like proteins. In contrast to this observed high structural homology of members in the mosquito SCP2 family, the amino acid sequence identity between the members is less than 30%. The results from structural analysis imply that there have been evolutionary constraints that favor the SCP-2 C(alpha) backbone fold while the specificity of ligand binding can be altered.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1007/s11010-008-0007-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2682627PMC
June 2009

Larvicidal activity of sterol carrier protein-2 inhibitor in four species of mosquitoes.

J Med Entomol 2008 May;45(3):439-44

Department of Entomology, University of Wisconsin-Madison, Madison, WI 53706, USA.

A previous report has shown that mosquito sterol carrier protein-2 inhibitors (SCPIs) are larvicidal to larvae of the yellowfever mosquito, Aedes aegypti (L.) (J. Lipid Res. 46: 650-657, 2005). In the current study, we tested SCPI-1 in an additional four mosquito species for larvicidal activities: Culex pipiens pipiens, Anopheles gambiae, Culex restuans, and Aedes vexans. Cholesterol accumulation in SCPI-treated Ae. aegypti fourth instars was examined. SCPI-1 is lethal to all tested mosquito species, with the LC50 value ranging from 5.2 to 15 microM when treatments started at the first to third instar. However, LC50 values increase to from 5.2 to 38.7 microM in treatments started at first and fourth instar, respectively. The results indicate that the lethal effect of SCPI-1 decreases with the growth of larvae, which suggests that SCPI-1 is more effective before the larvae reach final growth period (the last instar). SCPI-1 suppressed cholesterol uptake in Ae. aegypti fourth instars, suggesting that one of the modes of action of SCPI-1 is via reduction in cholesterol absorption.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2670991PMC
http://dx.doi.org/10.1603/0022-2585(2008)45[439:laoscp]2.0.co;2DOI Listing
May 2008

The genome of the model beetle and pest Tribolium castaneum.

Nature 2008 Apr 23;452(7190):949-55. Epub 2008 Mar 23.

Human Genome Sequencing Center, Baylor College of Medicine, One Baylor Plaza, Houston, Texas 77030, USA.

Tribolium castaneum is a member of the most species-rich eukaryotic order, a powerful model organism for the study of generalized insect development, and an important pest of stored agricultural products. We describe its genome sequence here. This omnivorous beetle has evolved the ability to interact with a diverse chemical environment, as shown by large expansions in odorant and gustatory receptors, as well as P450 and other detoxification enzymes. Development in Tribolium is more representative of other insects than is Drosophila, a fact reflected in gene content and function. For example, Tribolium has retained more ancestral genes involved in cell-cell communication than Drosophila, some being expressed in the growth zone crucial for axial elongation in short-germ development. Systemic RNA interference in T. castaneum functions differently from that in Caenorhabditis elegans, but nevertheless offers similar power for the elucidation of gene function and identification of targets for selective insect control.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/nature06784DOI Listing
April 2008

Three-dimensional structure/function analysis of SCP-2-like2 reveals differences among SCP-2 family members.

J Lipid Res 2008 Mar 15;49(3):644-53. Epub 2007 Dec 15.

Department of Bacteriology, University of Wisconsin-Madison, Madison, WI 53706, USA.

Mosquito sterol carrier protein-2 (AeSCP-2) and sterol carrier protein-2-like2 (AeSCP-2L2) are members of the SCP-2 protein family with similar expression profiles in the mosquito life cycle. In an effort to understand how lipids can be transported by different SCP-2 proteins, the three-dimensional crystal structure of AeSCP-2L2 was solved at 1.7 A resolution. AeSCP-2L2 forms a dimer and binds three fatty acids, one of which resides in a position within the internal cavity at a right angle to the others. This first report of ligand-bound dimerized protein in the SCP-2 protein family indicates that the family has a much more divergent mode of interaction with ligands than previously reported. The potential function of AeSCP-2L2 was investigated via in vivo incorporation of [(3)H]cholesterol and [3H]palmitic acid. Overexpression of AeSCP-2L2 in mosquito cells leads to an increased uptake of free fatty acid, whereas knockdown of AeSCP-2L2 in adult females decreases the accumulation of free fatty acid in the fat body from a blood meal. In contrast, overexpression or knockdown of AeSCP-2L2 has no effect on cholesterol uptake. Our results suggest that the main function of AeSCP-2L2 is as a general intracellular fatty acid carrier, as opposed to having a dedicated role in cholesterol transport.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1194/jlr.M700460-JLR200DOI Listing
March 2008

Regulatory mechanisms of chitin biosynthesis and roles of chitin in peritrophic matrix formation in the midgut of adult Aedes aegypti.

Insect Biochem Mol Biol 2006 Jan 25;36(1):1-9. Epub 2005 Oct 25.

Department of Animal Health and Biomedical Sciences, University of Wisconsin-Madison, 1656 Linden Drive Madison, WI 53706, USA.

In mosquitoes, the peritrophic matrix is formed in response to blood feeding and can be a physical barrier when pathogens ingested with blood meal attempt to reach and transverse the midgut epithelium. The main components of the peritrophic matrix are chitin-biding-domain containing proteins, glycosylated proteins, and chitin fibrils. Chitin is synthesized from fructose-6-phosphate by a series of five enzymatic reactions. We previously found that blood feeding induces transcriptional up-regulation of glutamine: fructose-6-phosphate amidotransferase-1 (AeGfat-1) and chitin synthase (AeCs), the first and last enzymes of the biosynthetic pathway, respectively, in the midgut of Aedes aegypti. In this study, we demonstrated that formation of the peritrophic matrix is disrupted when the transcript abundance of either gene is knocked-down using RNAi methodologies. We also have shown that enzymatic activity of recombinant AeGFAT-1 is sensitive to feedback inhibition by UDP-N-acetylglucosamine, a substrate of chitin synthase. These findings demonstrate that in the midgut of adult Ae. aegypti, (1) chitin is synthesized de novo in response to blood feeding and is an essential component of the peritrophic matrix, and (2) chitin biosynthesis is negatively regulated, in part, by inhibitory sensitivity of AeGFAT-1 to UDP-N-acetylglucosamine.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.ibmb.2005.09.003DOI Listing
January 2006

Mosquito glucosamine-6-phosphate N-acetyltransferase: cDNA, gene structure and enzyme kinetics.

Insect Biochem Mol Biol 2005 Jun 27;35(6):637-46. Epub 2005 Mar 27.

Department of Animal Health and Biomedical Sciences, 1656 Linden Dr. University of Wisconsin-Madison, Madison, WI 53706, USA.

Mosquito midgut epithelial cells secrete digestive enzymes as well as components of the peritrophic matrix in response to blood-feeding. The peritrophic matrix is composed of proteins, glycoproteins and chitin fibrils in a proteoglycan matrix and may function to protect the midgut epithelium from mechanical damage and insult from pathogens and toxins. Chitin biosynthesis takes place via the hexosamine pathway converting fructose-6-phosphate to UDP-N-acetylglucosamine, which is then polymerized to chitin by chitin synthase. Glucosamine-6-phosphate N-acetyltransferase (GNA) is one of the hexosamine pathway enzymes and catalyzes the transfer of the acetyl group from acetyl-CoA to the primary amine of glucosamine-6-phosphate. We cloned and sequenced the GNA cDNA, gene (AeGna) and its putative promoter regions from Aedes aegypti. AeGna consists of five exons and four introns and lacks a TATA box near the transcription start site. The AeGna cDNA is 1.3 kb in length and the predicted protein is approximately 23.6 kDa. The amino acid sequence of AeGna has high homology to its orthologues. AeGna mRNA is constitutively expressed in all developmental stages and blood-feeding causes no obvious effect on levels of AeGna transcript in the midgut. The Km value of recombinant GNA for glucosamine-6-phosphate was 330 microM and the Km for acetyl-CoA was 500 microM.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.ibmb.2005.02.005DOI Listing
June 2005

Identification of mosquito sterol carrier protein-2 inhibitors.

J Lipid Res 2005 Apr 1;46(4):650-7. Epub 2005 Jan 1.

Department of Entomology, University of Wisconsin-Madison, Madison, Wisconsin, USA.

A mosquito sterol carrier protein-2, AeSCP-2, has been shown to aid in the uptake of cholesterol in mosquito cells. The discovery of chemical inhibitors of AeSCP-2 is reported here. AeSCP-2 inhibitors (SCPIs) belong to several chemotypes of hydrophobic compounds. Those inhibitors competed with cholesterol for AeSCP-2, binding with relatively high binding affinities. In cultured insect cells, SCPIs reduced cholesterol uptake by as much as 30% at 1-5 microM concentrations. SCPIs were potent larvicides to the yellow fever mosquito, Aedes aegypti, and to the tobacco hornworm, Manduca sexta, with 50% lethal doses (LD50s) of 5-21 microM and 0.013-15 ng/mg diet, respectively. The results indicate that sterol carrier protein-2 has functional similarity in two different insect species.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1194/jlr.M400389-JLR200DOI Listing
April 2005

Critical period for pupal commitment in the yellow fever mosquito, Aedes aegypti.

J Insect Physiol 2004 Jul;50(7):667-76

Department of Entomology, University of Wisconsin-Madison, Madison, WI 53706, USA.

Changes in ecdysteroid levels that lead to commitment of pupal and adult development were studied in the yellow fever mosquito, Aedes aegypti. Application of juvenile hormone at the time of pupal commitment usually results in delay or blockage of pupal and adult development. With methoprene, a juvenile hormone mimic, the JH sensitive period was found to be within 19 h after ecdysis to the fourth instar, at which time methoprene treatment caused a delay in pupation. Consistent with this observation, there was a small peak of ecdysteroid levels between 14 and 28 h after ecdysis to the fourth instar. Therefore, the commitment to pupal development occurs most likely between 14 and 19 h after ecdysis to the fourth instar. Levels of transcription of the ecdysone receptor gene show a small peak between 12 and 24 h, and a larger peak between 46 and 66 h after ecdysis to the fourth instar.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.jinsphys.2004.04.012DOI Listing
July 2004

Subcellular localization of the mosquito sterol carrier protein-2 and sterol carrier protein-x.

J Lipid Res 2004 Aug 16;45(8):1468-74. Epub 2004 May 16.

Department of Entomology, University of Wisconsin-Madison, Madison, WI 53706, USA.

Subcellular distribution of Aedes aegypti sterol carrier protein-2 (AeSCP-2) and AeSCP-x was studied using electron microscopy. In both cultured A. aegypti cells and in the larval midgut, AeSCP-2 was detected mostly in the cytosol, with some labeling mitochondria and nucleus, but not in membranous vesicles. The widespread distribution of AeSCP-2 in the midgut epithelium is consistent with its potential lipid transfer function in all phases of cholesterol absorption. In contrast, AeSCP-x was found mostly in the peroxisome. Differences in the subcellular distribution of AeSCP-2 and AeSCP-x suggest that these two members of the SCP-2 gene family are functionally distinct. Overexpression of AeSCP-2 in A. aegypti cells showed increased localization of AeSCP-2 to cytosol, mitochondria, and nucleus. This is the first report on the nuclear distribution of an SCP. Overexpression of AeSCP-2 resulted in increased cholesterol incorporation in cells, suggesting that AeSCP-2 enhances cholesterol uptake.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1194/jlr.M400003-JLR200DOI Listing
August 2004

The structural determination of an insect sterol carrier protein-2 with a ligand-bound C16 fatty acid at 1.35-A resolution.

J Biol Chem 2003 Oct 10;278(40):39085-91. Epub 2003 Jul 10.

Department of Biochemistry, University of Wisconsin, Madison, Wisconsin 53706, USA.

Yellow fever mosquito sterol carrier protein (SCP-2) is known to bind to cholesterol. We report here the three-dimensional structure of the complex of SCP-2 from Aedes aegypti with a C16 fatty acid to 1.35-A resolution. The protein fold is exceedingly similar to the human and rabbit proteins, which consist of a five-stranded beta-sheet that exhibits strand order 3-2-1-4-5 with an accompanying layer of four alpha-helices that cover the beta-sheet. A large cavity exists at the interface of the layer alpha-helices and the beta-sheet, which serves as the fatty acid binding site. The carboxylate moiety of the fatty acid is coordinated by a short loop that connects the first alpha-helix to the first beta-strand, whereas the acyl chain extends deep into the interior of the protein. Interestingly, the orientation of the fatty acid is opposite to the observed orientation for Triton X-100 in the SCP-2-like domain from the peroxisomal multifunctional enzyme (Haapalainen, A. M., van Aalten, D. M., Merilainen, G., Jalonen, J. E., Pirila, P., Wierenga, R. K., Hiltunen, J. K., and Glumoff, T. (2001) J. Mol. Biol. 313, 1127-1138). The present study suggests that the binding pocket in the SCP-2 family of proteins may exhibit conformational flexibility to allow coordination of a variety of lipids.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1074/jbc.M306214200DOI Listing
October 2003

Use of subtracted libraries and macroarray to isolate developmentally specific genes from the mosquito, Aedes aegypti.

Insect Biochem Mol Biol 2002 Dec;32(12):1757-67

Department of Entomology, University of Wisconsin-Madison, 53706, USA.

Subtracted cDNA libraries were screened with cDNA macroarrays to isolate larval and pupal stage-specific genes from Aedes aegypti. Of 103 partial cDNAs sequenced from the 4th instar subtracted cDNA library, 62 have counterpart genes in other organisms while 41 of them have no significant similarity to any known genes. Sequences of 116 partial cDNA clones from the pupal subtracted library revealed that 57 belong to unknown genes and 59 have homologous genes in other organisms. Results of cDNA macroarrays showed that 42-50% of randomly selected genes in the subtracted cDNA libraries were differentially expressed. Of the unknown genes, transcripts of 15-19% of the genes were detected in larval or pupal stages, respectively. The results indicate that a subtracted cDNA library in combination with a cDNA macroarray can be used effectively to identify genes expressed in a particular stage.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/s0965-1748(02)00116-9DOI Listing
December 2002