Identification of mosquito sterol carrier protein-2 inhibitors
2005; Elsevier BV; Volume: 46; Issue: 4 Linguagem: Inglês
10.1194/jlr.m400389-jlr200
ISSN1539-7262
AutoresMin-sik Kim, Vilena Wessely, Que Lan,
Tópico(s)Invertebrate Immune Response Mechanisms
ResumoA 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 μM 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 μM and 0.013–15 ng/mg diet, respectively.The results indicate that sterol carrier protein-2 has functional similarity in two different insect species. 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 μM 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 μM and 0.013–15 ng/mg diet, respectively. The results indicate that sterol carrier protein-2 has functional similarity in two different insect species. Sterol carrier protein-2 (SCP-2) belongs to a family of proteins containing a sterol binding domain (SCP-2 domain). The SCP-2 domain is found in the following vertebrate proteins: SCP-2, SCP-x, 17β-hydroxysteroid dehydrogenase type IV (HSD17B4), and stomatin (1Gallegos A.M. Atshaves B.P. Storey S.M. Starodub O. Petrescu A.D. Huang H. McIntosh A.L. Martin G.G. Chao H. Kier A.B. Schroeder F.F. Gene structure, intracellular localization, and functional roles of sterol carrier protein-2.Prog. Lipid Res. 2001; 40: 498-563Crossref PubMed Scopus (194) Google Scholar, 2Moller G. Luders J. Markus M. Husen B. Van Veldhoven P.P. Adamski J.J. Peroxisome targeting of porcine 17beta-hydroxysteroid dehydrogenase type IV/D-specific multifunctional protein 2 is mediated by its C-terminal tripeptide AKI.J. Cell. Biochem. 1999; 73: 70-78Crossref PubMed Scopus (23) Google Scholar, 3Fricke B. Stewart G.W. Treharne K.J. Mehta A. Knopfle G. Friedrichs N. Muller K.M. von During M.M. Stomatin immunoreactivity in ciliated cells of the human airway epithelium.Anat. Embryol. (Berl.). 2003; 207: 1-7Crossref PubMed Scopus (17) Google Scholar). The vertebrate SCP-2, SCP-x, and HSD17B4 have a peroxisome localization sequence in the C terminus, targeting these proteins to the peroxisome (1Gallegos A.M. Atshaves B.P. Storey S.M. Starodub O. Petrescu A.D. Huang H. McIntosh A.L. Martin G.G. Chao H. Kier A.B. Schroeder F.F. Gene structure, intracellular localization, and functional roles of sterol carrier protein-2.Prog. Lipid Res. 2001; 40: 498-563Crossref PubMed Scopus (194) Google Scholar, 2Moller G. Luders J. Markus M. Husen B. Van Veldhoven P.P. Adamski J.J. Peroxisome targeting of porcine 17beta-hydroxysteroid dehydrogenase type IV/D-specific multifunctional protein 2 is mediated by its C-terminal tripeptide AKI.J. Cell. Biochem. 1999; 73: 70-78Crossref PubMed Scopus (23) Google Scholar, 4Starodub O. Jolly C.A. Atshaves B.P. Roths J.B. Murphy E.J. Kier A.B. Schroeder F.F. Sterol carrier protein-2 localization in endoplasmic reticulum and role in phospholipid formation.Am. J. Physiol. Cell Physiol. 2000; 279: C1259-C1269Crossref PubMed Google Scholar). The mosquito SCP-2 (AeSCP-2) appears to represent a unique nonperoxisomal and low-molecular-weight protein in the SCP-2 gene family (5Krebs K.C. Lan Q. Isolation and expression of a sterol carrier protein-2 gene from the yellow fever mosquito, Aedes aegypti.Insect Mol. Biol. 2003; 12: 51-60Crossref PubMed Scopus (59) Google Scholar, 6Lan Q. Massey R.J. Subcellular localization of the mosquito sterol carrier protein-2 and sterol carrier protein-x.J. Lipid Res. 2004; 45: 1468-1474Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar).The vertebrate SCP-2 is characterized as a nonspecific lipid carrier protein, which has affinity for different ligands in the order cholesterol >> straight-chain fatty acid > kinked-chain fatty acid (7Schroeder F. Frolov A. Starodub O. Atshaves B.B. Russell W. Petrescu A. Huang H. Gallegos A.M. McIntosh A. Tahotna D. Russell D.H. Billheimer J.T. Baum C.L. Kier A.B.B. Pro-sterol carrier protein-2: role of the N-terminal presequence in structure, function, and peroxisomal targeting.J. Biol. Chem. 2000; 275: 25547-25555Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar). Like the vertebrate SCP-2, AeSCP-2 binds cholesterol (5Krebs K.C. Lan Q. Isolation and expression of a sterol carrier protein-2 gene from the yellow fever mosquito, Aedes aegypti.Insect Mol. Biol. 2003; 12: 51-60Crossref PubMed Scopus (59) Google Scholar) and fatty acid (8Dyer D.H. Lovell S. Thoden J.B. Holden H.M. Rayment I. Lan Q.Q. 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; 278: 39085-39091Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar). Similarly, both the vertebrate and AeSCP-2 increase cholesterol uptake in SCP-2-overexpressing cells (5Krebs K.C. Lan Q. Isolation and expression of a sterol carrier protein-2 gene from the yellow fever mosquito, Aedes aegypti.Insect Mol. Biol. 2003; 12: 51-60Crossref PubMed Scopus (59) Google Scholar, 9Moncecchi D. Murphy E.J. Prows D.R. Schroeder F. Sterol carrier protein-2 expression in mouse L-cell fibroblasts alters cholesterol uptake.Biochim. Biophys. Acta. 1996; 1302: 110-116Crossref PubMed Scopus (77) Google Scholar). However, AeSCP-2 differs from the vertebrate SCP-2 in several aspects. In both cultured Aedes aegypti cells and in the larval midgut, AeSCP-2 localizes mostly in the cytosol, which is consistent with the fact that AeSCP-2 lacks the C-terminal peroxisome targeting sequence (6Lan Q. Massey R.J. Subcellular localization of the mosquito sterol carrier protein-2 and sterol carrier protein-x.J. Lipid Res. 2004; 45: 1468-1474Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar). The coordination site for a ligand in AeSCP-2 is different from the vertebrate SCP-2, in which the hydrophobic moieties of these ligands are oriented at opposite ends of the protein (8Dyer D.H. Lovell S. Thoden J.B. Holden H.M. Rayment I. Lan Q.Q. 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; 278: 39085-39091Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar). AeSCP-2 seems to be a vital gene for the survival and development of mosquitoes, whereas the vertebrate SCP-2 is not critical for survival and fertility (10Kannenberg F. Ellinghaus P. Assmann G. Seedorf U. Aberrant oxidation of the cholesterol side chain in bile acid synthesis of sterol carrier protein-2/sterol carrier protein-x knockout mice.J. Biol. Chem. 1999; 274: 35455-35460Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar, 11Fuchs M. Hafer A. Munch C. Kannenberg F. Teichmann S. Scheibner J. Stange E.F. Seedorf U.U. Disruption of the sterol carrier protein 2 gene in mice impairs biliary lipid and hepatic cholesterol metabolism.J. Biol. Chem. 2001; 276: 48058-48065Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar). Knockdown of AeSCP-2 expression in larvae led to a high mortality rate in developing adults, and silencing of AeSCP-2 in adults lowered fertility (Q. Lan, unpublished observations). AeSCP-2 expression is high in the midgut during feeding stages in larvae (5Krebs K.C. Lan Q. Isolation and expression of a sterol carrier protein-2 gene from the yellow fever mosquito, Aedes aegypti.Insect Mol. Biol. 2003; 12: 51-60Crossref PubMed Scopus (59) Google Scholar), when cholesterol and sterols are absorbed (12Svoboda J.A. Thompson M.J. Herbert Jr., E.W. Shortino T.J. Szczepanik-Vanleeuwen P.A.A. Utilization and metabolism of dietary sterols in the honey bee and the yellow fever mosquito.Lipids. 1982; 17: 220-225Crossref PubMed Scopus (39) Google Scholar). Targeting cholesterol metabolism for the development of new insect growth regulators for insect population control is one of the goals of this study. Inhibitors are useful tools for elucidating the mode of action and molecular mechanism of a functional protein. To search for inhibitors of AeSCP-2, we have developed a 384-well microplate format for screening small molecular chemical libraries using high-throughput technology.Several AeSCP-2 inhibitors (SCPIs) were identified. SCPIs belong to several chemotypes of hydrophobic compounds. Based on the inhibitory effect of SCPIs on AeSCP-2 cholesterol binding in vitro and on cholesterol uptake in cultured insect cells, it is assumed that SCPIs might reduce cholesterol absorption in vivo. SCPIs showed high larvicidal activities in the yellow fever mosquito, Aedes aegypti, and in the tobacco hornworm, Manduca sexta, even though SCPIs had very low cytotoxicity in cultured mouse cells. We report here the first successful attempt at targeting the cholesterol transport pathway in insects for the development of specific inhibitors.MATERIALS AND METHODSChemicalsChemicals and reagents were purchased from Sigma (St. Louis, MO), Fisher Scientific (Pittsburgh, PA), and ICN (Costa Mesa, CA) if their origins are not mentioned in the text. NBD cholesterol was purchased from Molecular Probes (Eugene, OR). ChemBridge Corporation (San Diego, CA) provided the Chembridge DIVERSet small molecular chemical library, and individual compounds with at least 90% purity were purchased from ChemBridge Corporation.MosquitoesThe yellow fever mosquito, Aedes aegypti, is an inbred laboratory strain (Rockefeller). The mosquitoes were maintained as described (5Krebs K.C. Lan Q. Isolation and expression of a sterol carrier protein-2 gene from the yellow fever mosquito, Aedes aegypti.Insect Mol. Biol. 2003; 12: 51-60Crossref PubMed Scopus (59) Google Scholar).The tobacco hornworm, Manduca sextaManduca sexta eggs were a gift from Dr. Walter G. Goodman, University of Wisconsin–Madison. Larvae were fed commercial gypsy moth wheat germ diet (ICN Biomedicals, Irvine, CA) and reared at 25°C and 60% relative humidity under a 16:8 light/dark cycle. Fresh food was provided every other day.Insect cell linesThe A. aegypti cell line Aag-2 was maintained in Eagle's medium (Invitrogen, Carlsbad, CA) supplemented with 5% fetal bovine serum (FBS) at 28°C under a 5% CO2 atmosphere as described (13Lan Q. Gerenday A. Fallon A.M. Cultured Aedes albopictus mosquito cells synthesize hormone-inducible proteins.In Vitro Cell. Dev. Biol. Anim. 1993; 29A: 813-818Crossref PubMed Scopus (22) Google Scholar). Cells were passed every 7 days using a 1:4 dilution of cells.The M. sexta cell line GV1 was maintained in modified Grace's medium (Invitrogen) supplemented with 10% FBS at 28°C as described (14Lan Q. Wu Z-N. Riddiford L.M. Regulation of the ecdysone receptor, USP, E75, and MHR3 genes by 20-hydroxyecdysone in the GV1 cell line of the tobacco hornworm, Manduca sexta.Insect Mol. Biol. 1997; 6: 3-10Crossref PubMed Scopus (35) Google Scholar). Cells were passed every 7 days with a 1:4 dilution of cells.Chemical library screeningNBD cholesterol (Molecular Probes, Eugene, OR) is a fluorescent cholesterol analog that is essentially nonfluorescent in water. However, upon binding to cholesterol or lipid binding protein, NBD cholesterol emits fluorescence at a distinct wavelength (excitation/emission = 470/530). AeSCP-2 was purified as described (8Dyer D.H. Lovell S. Thoden J.B. Holden H.M. Rayment I. Lan Q.Q. 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; 278: 39085-39091Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar). Stock solution of NBD cholesterol was made in 100% ethanol at 200 μM concentration, and the working solution of NBD cholesterol was made by diluting the stock solution in double-distilled water to desired concentrations. Compounds in the chemical library were dissolved in 100% DMSO at 0.1 mM concentration.Laboratory robotics for high-throughput screening were performed using a Biomek FX (Beckman-Coulter, Inc., Fullerton, CA) liquid handler, a uFill reagent dispenser (Bio-Tek Instruments, Inc., Winooski, VT), and an EnVision plate reader (Perkin Elmer, Inc., Wellesley, MA). The positive control was AeSCP-2 with NBD cholesterol and 3.3% DMSO (as solvent). Negative controls were purified bacterial glutathione S-transferase (GST) with NBD cholesterol and DMSO, and NBD cholesterol with 3.3% DMSO. Compounds that blocked cholesterol binding in AeSCP-2 would have a much lower NBD cholesterol fluorescent intensity.For the full library screening, 15 μl of 5 uM AeSCP-2 was mixed with 1 μl of 0.1 mM compound (final concentration of DMSO in the reaction, 3.3%) and 15 μl of 1.25 μM NBD cholesterol (final concentration of ethanol in the reaction, 0.3%) in 10 mM KHPO4 buffer (pH 7.4). The mixture was incubated at room temperature for 3 min and the fluorescence intensity of the NBD cholesterol-protein complex was measured using a fluorescence microplate reader (Molecular Devices, Sunnyvale, CA). A potential SCPI in the high-throughput screen was a candidate chemical that reduced NBD cholesterol fluorescence intensity in the presence of AeSCP-2 at less than three times standard deviation from the average of the entire library (15Zhang J.H. Chung T.D. Oldenburg K.R. A simple statistical parameter for use in evaluation and validation of high throughput screening assays.J. Biomol. Screen. 1999; 4: 67-73Crossref PubMed Scopus (5235) Google Scholar).SCPI-AeSCP-2 binding assaysThe inhibitory effect of SCPIs on cholesterol binding to AeSCP-2 was measured using NBD cholesterol competition assays. In a 50 μl reaction solution in each well of a 96-well plate, purified recombinant AeSCP-2 (5 μM) was incubated with NBD cholesterol (1.25 μM) in the presence of increased concentrations of an SCPI (0–5 μM) in 10 mM KHPO4 buffer (pH 7.4) for 5 min. The fluorescence intensity (excitation/emission = 470/530) of NBD cholesterol was measured using a fluorescence microplate reader (Molecular Devices). Background control was NDB alone. A separate set of tests was performed using NBD cholesterol with increasing concentration of an SCPI to assess whether an SCPI interfered with NBD cholesterol fluorescence. If an SCPI interfered with NBD cholesterol fluorescence, the background control was NBD cholesterol along with the SCPI. The net change in NBD cholesterol fluorescence intensity was calculated by subtracting the fluorescence of background controls from the NDB cholesterol-AeSCP-2 complex in the absence or presence of an inhibitor. The data were plotted with the NBD cholesterol intensity (bound NBD cholesterol) as the y-axis and molarity of inhibitor as the x-axis. Bacterial GST protein was used as a negative control for the assays. It showed that the presence of a nonbinding protein did not affect the NBD cholesterol fluorescence (Fig. 1). The 50% inhibition concentration (IC50) for NBD cholesterol binding to AeSCP-2 was calculated for each SCPI. A separate test was set up to use cholesterol competing with NBD cholesterol for AeSCP-2 binding, assessing the relative binding affinity (RBA) of cholesterol to AeSCP-2 under the same experimental conditions. The RBA of each SCPI to AeSCP-2 was estimated by dividing the IC50 of cholesterol by the IC50 of the individual SCPI and was expressed as a percent (RBA of cholesterol = 100%).Purified recombinant SCP-2 domain from the AeSCP-x (16Lan Q. Wessely V. Expression of a sterol carrier protein-x gene in the yellow fever mosquito, Aedes aegypti.Insect Mol. Biol. 2004; 13: 519-529Crossref PubMed Scopus (24) Google Scholar) was also used for SCPI binding assays to verify whether SCPIs interact with other sterol carrier proteins (SCPs). The assays were conducted as described above.Cell-based biological assaysAeSCP-2-overexpressing Aag-2 cells (6Lan Q. Massey R.J. Subcellular localization of the mosquito sterol carrier protein-2 and sterol carrier protein-x.J. Lipid Res. 2004; 45: 1468-1474Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar) were used to assess the biological activity of a potential SCPI. All experiments were performed in triplicate and repeated at least two times. Aag-2 cells were seeded at 2 ml of 5 × 105 cells/ml in 35 × 10 mm culture dishes. Transfection of AeSCP-2 expression vector was conducted as described (6Lan Q. Massey R.J. Subcellular localization of the mosquito sterol carrier protein-2 and sterol carrier protein-x.J. Lipid Res. 2004; 45: 1468-1474Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar). AeSCP-2-overexpressing cells were dislodged, diluted 2-fold (5 × 104cells/ml), and dispensed at 0.5 ml/well into 24-well culture dishes. The cells were allowed to grow in 24-well dishes for an additional 24 h, and the medium was changed to sterol-free medium (13Lan Q. Gerenday A. Fallon A.M. Cultured Aedes albopictus mosquito cells synthesize hormone-inducible proteins.In Vitro Cell. Dev. Biol. Anim. 1993; 29A: 813-818Crossref PubMed Scopus (22) Google Scholar). After overnight culture in sterol-free medium, the medium was replaced with 250 μl of fresh sterol-free medium containing 0.33 μCi [3H]cholesterol/ml (40 Ci/mM) and a potential SCPI at various concentrations (diluted from a 10 mM DMSO stock solution). Control cells were incubated in 250 μl of fresh sterol-free medium containing [3H]cholesterol and a correspondingly diluted DMSO solution. After 16 h labeling with [3H]cholesterol, cells were washed twice with 2 ml cold PBS and the total cellular lipids were extracted and determined as described (6Lan Q. Massey R.J. Subcellular localization of the mosquito sterol carrier protein-2 and sterol carrier protein-x.J. Lipid Res. 2004; 45: 1468-1474Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar). The relative [3H]cholesterol incorporation was calculated by normalizing compound-treated to [3H]cholesterol of DMSO-treated cells, which was set as 100%. The IC50 (the concentration of compound that inhibits [3H]cholesterol incorporation by 50%) was determined using standard methods.Manduca sexta GV1 cells were seeded at 0.2 ml of 5 × 104cell/ml in each well of a 96-well culture plate and incubated overnight, and the medium was changed to sterol-free medium. After overnight culture in sterol-free medium, the medium was replaced with 50 μl of fresh sterol-free medium containing 0.33 μCi [3H]cholesterol/ml (40 Ci/mM) and a potential SCPI at various concentrations (diluted from a 10 mM DMSO stock solution). Control cells were incubated in 50 μl of fresh sterol-free medium containing [3H]cholesterol and a correspondingly diluted DMSO solution. After 24 h labeling with [3H]cholesterol, cells were washed and collected, and cellular [3H]cholesterol was extracted as described above for Aag-2 cells.Cytotoxicity assays in mammalian cellsNormal mouse breast epithelial NmuMG cells were used to assess cellular toxicity of SCPI to vertebrate cells using the CellTiter-Glo Luminescent Cell Viability Assay (Promega, Madison, WI). NmuMG cells were maintained in DMEM medium from CellGro (Herndon, VA) supplemented with 10% FBS from ICN and 10 μg/ml insulin (Sigma). Cells were harvested by trypsinization using 0.25% trypsin and 0.1% EDTA (CellGro) and counted in a hemacytometer. Cells were plated at a density of 8,000 cells/well of each 96-well microtiter plate. Cells were grown for 4 h at 37°C with 5% CO2 in a humidified incubator to allow cell attachment to occur before compound addition.SCPIs were diluted in series with 100% DMSO to obtain 100× stock solutions of the dosages tested. Compound stocks were diluted 10-fold with DMEM medium with 10% FBS and penicillin-streptomycin to ensure full mixing of DMSO stocks with culture media using the FX liquid handler (Beckman). Ten microliters of each compound stock was added to duplicate wells in microtiter plates using the FX liquid handler. The final concentration of DMSO in all wells was 1%. Cells were incubated with the test compounds for 72 h before the assay was conducted. Test plates were removed from the incubator and allowed to equilibrate to room temperature for 30 min. Seventy microliters of culture medium was removed from each well, leaving 30 μl remaining in the well. An equal volume of 30 μl of room temperature CellTiter-Glo assay reagent was added to each well and incubated for 10 min at room temperature. The plates were read on the EnVision plate reader for luminescence counts.For each compound tested, the percentage of inhibition of cell proliferation was calculated at each concentration of compound tested using the formula % inhibition = 100 – (100 × (relative light units or absorbency of sample)/(relative light units or absorbency of negative control)). The average percent inhibition at each concentration was calculated by taking the average of the percent inhibition calculated for each replicate. The IC50 (the concentration of compound that inhibits the growth of cells by 50%) was determined.Biological assays in mosquito larvaeA. aegypti larvae were treated with different SCPIs. Each SCPI was diluted from a stock solution (50 or 100 mM in DMSO) to various concentrations in double-distilled water. Starting from first larval stadium, larvae (50–60 larvae/sample) were reared in 60 ml distilled water in the presence of an SCPI (0.4–25 μM) and fed 250 mg pellets of rabbit food. Control larvae were reared in correspondingly diluted DMSO (solvent for stock solutions of each compound). Dead larvae and pupae were recorded until day 12, when >95% of the adults had eclosed. The 50% lethal dose (LD50) value was determined by converting the percent mortality to the probit unit, then plotting the percent mortality (probit unit) on the y-axis and the log (concentration) on the x-axis.Biological assays in M. sexta larvaeNewly hatched larvae (30/treatment) were placed on a block of diet (954 mg) containing 20 μl of various concentrations of an SCPI. Control larvae were reared on diet containing 20 μl of ethanol (solvent for the inhibitor). Fresh diet was provided every other day. Larval development and mortality were monitored through pupation. The LD50 value was determined as described above.RESULTSIdentification of SCPIsCholesterol competes with NBC cholesterol for AeSCP-2 binding in a dose-dependent fashion (Fig. 1), which indicates that NBD cholesterol interacts with AeSCP-2 much like cholesterol itself. Using NBC cholesterol as a ligand and purified recombinant AeSCP-2, a 384-well microplate format was developed for screening chemical inhibitors using high-throughput technology. Of the 16,000 compounds of the Chembridge DIVERSet small molecular chemical library (ChemBridge Corporation, San Diego, CA), 396 interfered with NBD cholesterol-AeSCP-2 binding. To narrow down the number of candidate compounds, NBD cholesterol fluorescence intensity SCPI-2 > SCPI-4 > SCPI-3 > SCPI-1 (Table 1).AeSCP-x, another member of the mosquito SCP-2 gene family (16Lan Q. Wessely V. Expression of a sterol carrier protein-x gene in the yellow fever mosquito, Aedes aegypti.Insect Mol. Biol. 2004; 13: 519-529Crossref PubMed Scopus (24) Google Scholar), had much lower binding affinity to cholesterol in the in vitro assays. The IC50 of cholesterol for AeSCP-x was 2.47 μM, which was >21-fold higher than AeSCP-2 (Table 1). Because the IC50s of SCPIs for AeSCP-x were much higher than those for AeSCP-2, SCPIs had a much higher affinity to AeSCP-2, especially in the cases of SCPI-2, -4, and -5 (Table 1). The results indicate that SCPI-2, -4, and -5 had higher specificity in inhibiting cholesterol binding in AeSCP-2.Effects of SCPIs on cholesterol incorporation in insect cellsFifty-seven potential SCPIs were first tested for their inhibitory effect on cholesterol incorporation in AeSCP-2-overexpressing Aag-2 cells, and 12 of the SCPIs showed varied inhibitory effects on cholesterol uptake. Five SCPIs that had >20% inhibition of cholesterol incorporation in Aag-2 cells were chosen as biologically active SCPIs. SCPI-5 had the highest RBA to AeSCP-2 in the in vitro assays (Table 1); however, SCPI-5 was less effective in inhibiting cholesterol uptake in AeSCP-2-overexpressing Aag-2 cells (Fig. 2Aand Table 2). Based on the IC50 values, the effectiveness in inhibiting cholesterol uptake in Aag-2 cells was in the order SCPI-3 > SCPI-2 > SCPI-4 > SCPI-1 > SCPI-5. The inhibitory effect of SCPIs on cholesterol uptake was dose dependent (Fig. 2A). At 1 μM, SCPI-1, -2, and -3 inhibited >25% of cholesterol uptake, compared with controls (Fig. 2A), consistent with the level of decreased cholesterol uptake in AeSCP-2-silenced Aag-2 cells (6Lan Q. Massey R.J. Subcellular localization of the mosquito sterol carrier protein-2 and sterol carrier protein-x.J. Lipid Res. 2004; 45: 1468-1474Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar).Fig. 2Effect of SCPIs on cholesterol uptake and cellular protein content in cultured insect cells. A: Cholesterol incorporation in Aedes aegypti Aag-2 cells. Aag-2 cells were transfected to overexpress AeSCP-2. Relative cholesterol incorporation = levels of cellular [3H]cholesterol incorporation normalized to untreated control cells arbitrarily set at 100%. B: Cholesterol incorporation in Manduca sexta GV1 cells. Bar = mean ± SD (N = 3).View Large Image Figure ViewerDownload Hi-res image Download (PPT)TABLE 2Biological activities of SCPIsSCPI-1SCPI-2SCPI-3SCPI-4SCPI-5Aedes aegypti LD50 (μM)aLD50 = mean ± SD (N = 4–6).4.8 ± 1.310.6 ± 5.812.3 ± 9.821.8 ± 28.919.4 ± 15.6IC50 (μM) for cholesterol uptake in Aag-2 cellsbIC50 = mean ± SD (N = 2).5.53 ± 5.853.97 ± 3.223.09 ± 2.064.36 ± 0.916.92 ± 3.18Manduca sexta LD50 (ng/mg diet)cLD50 = mean ± SD (N = 2).0.220.013 ± 0.00115ND15IC50 (μM) for cholesterol uptake in GV1 cells3.33.42.32.72.0IC50 (μM) for cell viability in NmuMG cellsdIC50 = mean ± SD (N = 2).57.3 ± 5.255.3 ± 22 214 ± 2.4256.9 ± 129.5452.5 ± 190.2LD50, 50% lethal dose.a LD50 = mean ± SD (N = 4–6).b IC50 = mean ± SD (N = 2).c LD50 = mean ± SD (N = 2).d IC50 = mean ± SD (N = 2). Open table in a new tab To test whether the effect of SCPIs on cellular cholesterol uptake was specific to mosquito cells or whether they had broader specificity to other insect species, the M. sexta GV1 cells were treated with various concentrations of individual SCPIs. M. sexta GV1 cells express low levels of SCP-2 (immunoreactive to affinity-purified anti-AeSCP-2 antibody; data not shown). SCPIs showed similar inhibitory effects on cholesterol uptake in GV1 cells (Fig. 2B and Table 2), and the order of the effectiveness of SCPI in GV1 cells was SCPI5-5 > SCPI-3 > SCPI-4 > SCPI-1 > SCPI-2. SCPI-5 inhibited cholesterol uptake much more effectively in GV1 cells than in Aag-2 cells (Fig. 2A, B; Table 2).Cytotoxicity of SCPIs in cultured mouse cellsThe vertebrate SCP-2 has 46% identity and 69% similarity to the mosquito AeSCP-2 in the sterol binding domain (5Krebs K.C. Lan Q. Isolation and expression of a sterol carrier protein-2 gene from the yellow fever mosquito, Aedes aegypti.Insect Mol. Biol. 2003; 12: 51-60Crossref PubMed Scopus (59) Google Scholar). It is speculated that SCPIs may inhibit the function of the vertebrate SCPs, although the vertebrate SCP-2/SCP-x gene is not vital for the survival (10Kannenberg F. Ellinghaus P. Assmann G. Seedorf U. Aberrant oxidation of the cholesterol side chain in bile acid synthesis of sterol carrier protein-2/sterol carrier protein-x knockout mice.J. Biol. Chem. 1999; 274: 35455-35460Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar, 11Fuchs M. Hafer A. Munch C. Kannenberg F. Teichmann S. Scheibner J. Stange E.F. Seedorf U.U. Disruption of the sterol carrier protein 2 gene in mice impairs biliary lipid and hepatic cholesterol metabolism.J. Biol. Chem. 2001; 276: 48058-48065Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar). Because SCPIs selected for inhibiting AeSCP-2 function were lethal to cultured insect cells
Referência(s)