Source: UNIV OF WISCONSIN submitted to NRP
FUNCTIONAL ANALYSIS OF STEROL CARRIER PROTEIN-2 IN INSECT MODEL SYSTEM
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
COMPLETE
Funding Source
HATCH
Reporting Frequency
Annual
Accession No.
0198118
Grant No.
(N/A)
Cumulative Award Amt.
(N/A)
Proposal No.
(N/A)
Multistate No.
(N/A)
Project Start Date
Oct 1, 2003
Project End Date
Sep 30, 2005
Grant Year
(N/A)
Program Code
[(N/A)]- (N/A)
Recipient Organization
UNIV OF WISCONSIN
21 N PARK ST STE 6401
MADISON,WI 53715-1218
Performing Department
ENTOMOLOGY
Non Technical Summary
Insects do not synthesis cholesterol de novo and depend on dietary cholesterol and cholesterol analogs for their biological needs. Despite its importance, the mechanisms of intracellular transfer of cholesterol have not been reported in insects. The information derived from this research will be an essential part of the overall scheme for rational design of new pesticides for insect population control.
Animal Health Component
(N/A)
Research Effort Categories
Basic
100%
Applied
(N/A)
Developmental
(N/A)
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
2113110104060%
7213110104040%
Knowledge Area
211 - Insects, Mites, and Other Arthropods Affecting Plants; 721 - Insects and Other Pests Affecting Humans;

Subject Of Investigation
3110 - Insects;

Field Of Science
1040 - Molecular biology;
Goals / Objectives
Cholesterol absorption and translocation is an Achilles' heel of insects, because insects depend on dietary cholesterol to survive. In insects, cholesterol is needed for cellular membranes and ecdysteroid biosynthesis. Many studies have determined that the precursor of ecdysteroid biosynthesis is cholesterol, demonstrating that [3H]cholesterol was converted to [3H]ecdysteroids (Borovsky et al., 1986; Grieneisen et al., 1991; Jouni et al., 2002). ). Yet, insects do not synthesize cholesterol de novo and depend on dietary and/or symbiotic microbes to provide cholesterol for their physiological needs (Clayton, 1964; Noda et al., 1979; Ritter and Nes, 1981; Dwivedy and Shukla, 1982; Nes et al., 1997). Targeting cholesterol metabolism for the development of new insect growth regulators for insect population control has been proposed before (Svoboda and Chitwood, 1992; Svoboda, 1994). However, only a few targeting molecules involved in cholesterol transport/metabolism are known, such as the hemolymph lipophorin (Soulages and Brenner, 1991; Jouni et al., 2002) and lipoprotein (Bianchi and Capurro, 1991). Nothing is known about the molecules involved in sterol transfer/conversion in the midgut of insects. To understand how the midgut absorbs/transfer dietary sterols, we need to identify intracellular transporters of cholesterol. We have discovered a probable intracellular sterol carrier protein, AeSCP-2, from the Yellow Fever mosquito.We also found that polyclonal anti-AeSCP-2 antibody cross-reacts with sterol carrier proteins (SCPs) of tobacco hornworm, Manduca sexta (M. sexta). We propose to use a comparative approach to study the function SCP-2 from the Yellow Fever mosquito, Ae. aegypti, and tobacco hornworm, M. sexta. Do insects have a cholesterol intracellular trafficking pathway parallel to that of vertebrates? How does this pathway vary between insects and vertebrates, given the known differences in cholesterol metabolism? This study proposes to answer these questions. Our goal is to understand the regulation of cholesterol absorption and intracellular transfer in insects in general. Such knowledge will provide the basic information to allow the rational design of new insect growth regulators for mosquitoes and insects in general.
Project Methods
We propose to use anti-AeSCP-2 antibody screening for M. sexta SCP-2 gene in fat body cDNA library. An expression cDNA library (lambda-ZAP vector) will be constructed using mRNA from the fat body of Day 1 5th instar. We expect many cDNAs of SCP family will be isolated because Manduca fat body had multiple proteins cross-reacting with anti-AeSCP-2 antibody (Fig. 2A, lane 6). It is highly possible that some of the anti-AeSCP-2 antibody cross-reacting proteins (Fig. 2A, lane 6) are not SCP gene products; but share some common epitopes with the AeSCP-2. Sequences of isolated Manduca SCPs will be obtained and compared to AeSCP-2 gene. The results will shed light on divergence of SCP-2 gene between Ae. aegytpi and M. sexta. Insect fat body is equal to vertebrate liver in term of function. In M. sexta, fat body tissue can be easily isolated cleanly from other tissues, which makes it a good model for the study of cholesterol metabolism. In M. sexta larvae, cholesterol is absorbed in the midgut and stored in fat body as free cholesterol. However, in adults, most of the cholesterol is esterified in fat body (Jouni et al., 2002) Because cholesterol is most likely stored in the lipid droplets in the fat body, translocation of cholesterol from lipid droplets to the sites of cholesterol metabolizing such as peroxisome, cytoplasma and mitochondria. Whether increasing cholesterol ester formation involves SCP-2 is unknown. After isolation of the SCP-2 gene from M. sexta, we will study the expression patterns of MsSC-2 gene from larvae to adult stages. The results will indicate whether SCP-2 activities are correlated with the physiological changes in cholesterol metabolism. We propose to generate mutant AeSCP-2 based on information obtained from the protein structure. Regions and amino acids that may be important for AeSCP-2 function such as cholesterol binding and membrane interaction domain(s) will be our primary focus. Those mutants AeSCP-2 will be used to study the interaction of AeSCP-2 with cellular organelles in Ae. aegypti cells. Expression plasmid (pIE1rh/PA, Lan et al., 1999) of the wild type and mutants of AeSCP-2 will be constructed by PCR based method. Expression constructs will be transiently transfected into Ae. aegypti using described methods (Shotkoski et.al., 1996). Aag-2 cells had extremely low level of AeSCP-2 expression (Fig. 2B, lane 8). Therefore, Aag-2 is ideal for study AeSCP-2 function in vivo because there is very low endogenous protein to interfere with the activity of transfected AeSCP-2.

Progress 10/01/03 to 09/30/05

Outputs
This research proposal is directed towards understanding the regulation of cholesterol absorption and intracellular transfer in insects. Insects do not synthesis cholesterol de novo and depend on dietary cholesterol and cholesterol analogs for their biological needs. Despite its importance, the mechanisms of intracellular transfer of cholesterol have not been reported in insects. We have discovered a putative sterol carrier protein-2 (SCP-2) gene, AeSCP-2, from the Yellow Fever mosquito, Aedes aegypti (Ae. aegypti). In vertebrates, SCP-2 is critical for intracellular transportation of cholesterol between organelles and the plasma membrane and it is an intricate part of steroid biosynthesis. The expression pattern and tissue specificity of AeSCP-2 is consistent with the idea that dietary cholesterol is absorbed in the midgut in larvae. We propose to use a comparative approach to study the function of SCP-2 in two different insect model systems. Insect growth has already been targeted for disruption by hormone analogs and insect growth regulators. Cholesterol absorption in insects can provide a target site for the development of a new insecticide. The information derived from this research will be an essential part of the overall scheme for rational design of new pesticides for insect population control. Aedes aegypti SCP-2 (AeSCP-2) is a potential biological target for inhibiting cholesterol uptake and transport in mosquitoes. AeSCP-2 plays a critical role in mosquito adult development. Knockdown of AeSCP-2 expression in the 4th instars results in a high mortality rate in developing adults. Silencing of AeSCP-2 in adults female reduced fertility (viable eggs) by more than 70%. We anticipate identifying inhibitors of AeSCP-2 that will block cholesterol uptake and transport in mosquitoes, which can be used as insecticides to control mosquito population. Inhibitors of AeSCP-2 will be molecular targeted chemicals that may replace traditional insecticides. To search for inhibitors of AeSCP-2 as insecticides, we have developed a 384-well microplate format for screening small molecular chemical libraries using high throughput technology. In the preliminary screen of the 16,340 compounds in the Chembridge DIVERSet small molecular chemical library (ChemBridge Corporation, San Diego, CA), AeSCP-2 and AeSCP-x had 0.54% and 0.35% hits, respectively. A hit in the high throughout screen is a candidate chemical that inhibited cholesterol binding in these sterol carrier proteins. From the secondary screening, we have confirmed that 10 of the compounds have inhibitory effect on cholesterol uptake in both mosquito and Manduca cultured cells. Those compounds were tested in the yellow fever mosquito, Aedes aegypti larvae and Tobacco hornworm, Manduca sexta, and Gypsy Moth, Lymantria dispar, larvae for insecticidal activities. Five compounds showed high larvicidal activity in Aedes aegypti, and four compounds were toxic to Manduca sexta larvae. Surprisingly, tested SCPIs were not toxic to Lymantria dispar larvae, indicating the newly identified SCPIs have species-specificities.

Impacts
This research targets unique aspects of insect physiology for the development of novel insecticides. The results from our study have proven that blocking cholesterol uptake pathway in insects is a possible approach for insect population control.

Publications

  • Blitzer, E.J., Vyazunova, I., Lan, Q. 2005. Functional analysis of AeSCP-2 using gene expression knockdown in the yellow fever mosquito, Aedes aegypti. Insect Mol. Biol. 14(3):301-7.
  • Kim, M-S., Wessely, V., Lan, Q. 2005. Identification of mosquito sterol carrier protein-2 inhibitors. J. Lipid Res. 46(4):650-7.


Progress 01/01/04 to 12/31/04

Outputs
This research is directed towards understanding the regulation of cholesterol absorption and intracellular transfer in insects. Insects do not synthesize cholesterol de novo and depend on dietary cholesterol and cholesterol analogs for their biological needs. Despite its importance, the mechanisms of intracellular transfer of cholesterol have not been reported in insects. We have discovered a putative sterol carrier protein-2 (SCP-2) gene, AeSCP-2, from the Yellow Fever mosquito, Aedes aegypti (Ae. aegypti). In vertebrates, SCP-2 is critical for intracellular transportation of cholesterol between organelles and the plasma membrane and it is an intricate part of steroid biosynthesis. The expression pattern and tissue specificity of AeSCP-2 is consistent with the idea that dietary cholesterol is absorbed in the midgut in larvae. We prepare a comparative approach to study the function of SCP-2 in two different insect model systems. Insect growth has already been targeted for disruption by hormone analogs and insect growth regulators. Cholesterol absorption in insects can provide a target site for the development of a new insecticide. The information derived from this research will be an essential part of the overall scheme for rational design of new pesticides for insect population control. Aedes aegypti SCP-2 (AeSCP-2) is a potential biological target for inhibiting cholesterol uptake and transport in mosquitoes. AeSCP-2 plays a critical role in mosquito adult development. Knockdown of AeSCP-2 expression in the 4th instars results in a high mortality rate in developing adults. Silencing of AeSCP-2 in adults female reduced fertility (viable eggs) by more than 70%. We anticipate identifying inhibitors of AeSCP-2 that will block cholesterol uptake and transport in mosquitoes, which can be used as insecticides to control mosquito population. Inhibitors of AeSCP-2 will be molecular targeted chemicals that may replace traditional insecticides. To search for inhibitors of AeSCP-2 as insecticides, we have developed a 384-well microplate format for screening small molecular chemical libraries using high throughput technology. In the preliminary screen of the 16,340 compounds in the Chembridge DIVERSet small molecular chemical library (ChemBridge Corporation, San Diego, CA), AeSCP-2 and AeSCP-x had 0.54% and 0.35% hits, respectively. A hit in the high throughout screen is a candidate chemical that inhibited cholesterol binding in these sterol carrier proteins. From the secondary screening, we have confirmed that 10 of the compounds have inhibitory effect on cholesterol uptake in both mosquito and Manduca cultured cells. Those compounds were tested in the yellow fever mosquito, Aedes aegypti larvae and Tobacco hornworm, Manduca sexta, larvae for insecticidal activities. Five compounds showed high larvicidal activity in Aedes aegypti, and four compounds were toxic to Manduca sexta larvae.

Impacts
This research targets unique aspects of insect physiology for the development of novel insecticides. The results from our study have proven that blocking cholesterol uptake pathway in insects is a possible approach for insect population control.

Publications

  • Lan, Q and Massey, R.J. 2004. Subcellular localization of mosquito sterol carrier protein-2 and sterol carrier protein-x. J. Lipid Res. 45(8):1468-74.
  • Blitzer, E.J., Vyazunova, I., Lan, Q. 2004. Functional analysis of AeSCP-2 using gene expression knockdown in the yellow fever mosquito, Aedes aegypti. Insect Mol. Biol. In press.
  • Kim, M-S., Wessely, V., Lan, Q. 2004. Identification of mosquito sterol carrier protein-2 inhibitors. J. Lipid Res. In press.


Progress 01/01/03 to 12/31/03

Outputs
This research proposal is directed towards understanding the regulation of cholesterol absorption and intracellular transfer in insects. Insects do not synthesize cholesterol de novo and depend on dietary cholesterol and cholesterol analogs for their biological needs. Despite its importance, the mechanisms of intracellular transfer of cholesterol have not been reported in insects. We have discovered a putative sterol carrier protein-2 (SCP-2) gene, AeSCP-2, from the Yellow Fever mosquito, Aedes aegypti (Ae. aegypti). In vertebrates, SCP-2 is critical for intracellular transportation of cholesterol between organelles and the plasma membrane and it is an intricate part of steroid biosynthesis. The expression pattern and tissue specificity of AeSCP-2 is consistent with the idea that dietary cholesterol is absorbed in the midgut in larvae. We propose to use a comparative approach to study the function of SCP-2 in two different insect model systems. Insect growth has already been targeted for disruption by hormone analogs and insect growth regulators. Cholesterol absorption in insects can provide a target site for the development of a new insecticide. The information derived from this research will be an essential part of the overall scheme for rational design of new pesticides for insect population control. Aedes aegypti SCP-2 (AeSCP-2) is a potential biological target for inhibiting cholesterol uptake and transport in mosquitoes. AeSCP-2 plays a critical role in mosquito adult development. Knockdown of AeSCP-2 expression in the 4th instars results in a high mortality rate in developing adults. We anticipate identifying inhibitors of AeSCP-2 that will block cholesterol uptake and transport in mosquitoes, which can be used as insecticides to control mosquito population. Inhibitors of AeSCP-2 will be molecular targeted chemicals that may replace traditional insecticides. To search for inhibitors of AeSCP-2 as insecticides, we have developed a 384-well microplate format for screening small molecular chemical libraries using high throughput technology. In the preliminary screen of the 16,340 compounds in the Chembridge DIVERSet small molecular chemical library (ChemBridge Corporation, San Diego, CA), AeSCP-2 and AeSCP-x had 0.54% and 0.35% hits, respectively. A hit in the high throughput screen is a candidate chemical that inhibited cholesterol binding in these sterol carrier proteins. Interestingly, 63% of preliminary hits were the same compounds that inhibited cholesterol binding in both AeSCP-2 and AeSCP-x. On the other hand, 37% of preliminary hits were inhibitors unique only to AeSCP-2. The results suggest that finding protein-specific inhibitors of cholesterol binding for AeSCP-2 is highly possible using high throughput screen technology.

Impacts
This research targets unique aspects of insect physiology for the development of new insecticides.

Publications

  • No publications reported this period