| The cholesterol pathway is important for maintaining and regulating many cellular processes such as fluidity and permeability of the cell membrane, assembly of lipid rafts, and protein sorting. Farnesyl pyrophosphate (FPP), a cholesterol pathway intermediate, and its derivative, geranylgeranyl pyrophosphate (GGPP), donate their isoprenoid moieties to the post-translational modification of many GTP-binding proteins, including RAS and Rap1. Isoprenylation of these proteins is necessary for their proper membrane targeting and function. Depletion of intracellular mevalonate, the precursor of FPP and GGPP, decreases isoprenylation and disrupts the activities of isoprenylated proteins. Farnesol (FOH) and geranylgeraniol (GGOH), products of FPP and GGPP metabolism, exhibit biological activities, including induction of apoptosis in tumor-derived cells, modulation of ion channels (FOH), and modulation of the activity of nuclear hormone receptors. FOH and GGOH also restore protein isoprenylation in mevalonate-depleted cells, thus restoring cellular functions that have been disrupted by the loss of protein isoprenylation. Kinase activity that converts FOH to FPP has been demonstrated in prokaryotic, plant and mammalian cell extracts. The activity of kinase(s) converting GGOH to GGPP has been demonstrated in bacteria and plant cells extracts. It is hypothesized that a novel salvage pathway, the Isoprenoid Shunt, exists that utilizes FOH and GGOH as metabolic precursors for isoprenoid pyrophosphates. The Isoprenoid Shunt in mouse embryo fibroblasts is characterized by the following specific aims: assessing the roles of FOH and GGOH in cholesterol synthesis; determining the effects of FOH and GGOH on protein isoprenylation in mevalonate-depleted cells; defining the role of FPP synthase in the ability of FOH and GGOH to restore protein isoprenylation; examining the effects of agents that alter cholesterol synthesis on the conversion of FOH to FPP and GGOH to GGPP; and characterizing the uptake of FOH and GGOH into cells. We demonstrate that the Isoprenoid Shunt enzymes utilizing FOH and GGOH as metabolic precursors for isoprenoid pyrophosphates are distinct and differentially regulated. They are constitutively present but not constitutively active. Cholesterol pathway intermediates both positively and negatively regulate the activities of the enzymes of the Isoprenoid Shunt. |
