Mevalonic acid (MVA) is the unique precursor for metabolically essential compounds terms """"""""isopentenoids"""""""" (e.g., ubiquinones, dolichols, cytochromse a3, juvenile hormones, prenylated proteins, etc.). Since many isopentenoids cannot be obtained from dietary sources, continuous MVA synthesis is required for life. The regulation of MVA synthesis at the molecular level is poorly understood in eukaryotes in general and insects specifically. Moreover, insects cannot synthesize the sterol nucleus, therefore, in contrast to sterologenic organisms, their regulation of MVA synthesis is signaled exclusively by nonsterol isopentenoid availability. Thus, it is possible that a firm understanding of insect nonsterol isopentenoid-mediated regulation of MVA synthesis might provide unique insight into this process in other eukaryotes. We propose to use immortalized Dipteran Kc and secondarily Aedes albopictus C7-10 and Aedes aegypti ATCC-125 cells) as models to define nonsterol-mediated regulation of MVA synthesis. 3-Hydroxy-3-methylglutaryl coenzymeA reductase (HMGR) is the enzyme which catalyzes MVA synthesis from HMG-CoA. In addition, Drosophila Kc cell HMGR Vmax activities are congruent with absolute in vivo MVA synthesis rater. Therefore, an understanding of nonsterol isopentenoid-mediated control of Kc cell HMGR metabolism should also define the regulation of MVA synthesis. Molecular probes for Drosophila HMGR protein and mRNA will be used in conert with detailed radio-metabolic flux analyses to delineate Kc cell nonsterol-mediated regulation of MVA synthesis. Three hypotheses will be addressed: (1) nonsterol isopentenoid-mediated regulation of immortalized Dipteran cellular HMGR metabolism is primarily posttranslational. (2) C15, C20-polyprenols and/or C15, C20-polyprenl-1-pyrophosphates signal MVA-mediated down regulation of Kc cell HMGR, and (3) regulatory isopentenoids modulate Kc cell HMGR function by altering the phosphorylation status of HMGR and/or ancillary proteins. This effort will be facilitated by the use of both intact and perforated Kc cells. The proposed studies will generate new fundamental information about nonsterol isopentenoid-mediated regulation of Kc cell MVA synthesis/HMGR metabolism. Lastly, MVA-derived products are required for insect development, oogenesis, and othe ress Therefore, an understanding of different Dipterans' MVA metabolism and regulation might reveal species specific targets for new chemical/biological insecticides.
