geranylgeranyl-pyrophosphate and epoxomicin

Endogenous prenylation with sesquiterpene or diterpene isoprenoids facilitates membrane localization and functional activation of small monomeric GTP-binding proteins. A direct effect of isoprenoids on regulation of gene expression and protein stability has also been proposed. In this study, we determined the role of sesquiterpene or diterpene isoprenoids on the regulation of Rho G-protein expression, activation, and stability in human trabecular meshwork (TM) cells. In both primary and transformed human TM cells, limiting endogenous isoprenoid synthesis with lovastatin, a potent HMG-CoA reductase inhibitor, elicited marked increases in RhoA and RhoB mRNA and protein content. The effect of lovastatin was dose-dependent with newly synthesized inactive protein accumulating in the cytosol. Supplementation with geranylgeranyl pyrophosphate (GGPP) prevented, while inhibition of geranylgeranyl transferase-I mimicked, the effects of lovastatin on RhoA and RhoB protein content. Similarly, lovastatin-dependent increases in RhoA and RhoB mRNA expression were mimicked by geranylgeranyl transferase-I inhibition. Interestingly, GGPP supplementation selectively promoted the degradation of newly synthesized Rho proteins which was mediated, in part, through the 20S proteasome. Functionally, GGPP supplementation prevented lovastatin-dependent decreases in actin stress fiber organization while selectively facilitating the subcellular redistribution of accumulated Rho proteins from the cytosol to the membrane and increasing RhoA activation. Post-translational prenylation with geranylgeranyl diterpenes selectively facilitates the expression, membrane translocation, functional activation, and turnover of newly synthesized Rho proteins. Geranylgeranyl prenylation represents a novel mechanism by which active Rho proteins are targeted to the 20S proteasome for degradation in human TM cells.

Topics: Alkyl and Aryl Transferases; Cytosol; Gene Expression Regulation; Humans; Lovastatin; Male; Oligopeptides; Polyisoprenyl Phosphates; Prenylation; Proteasome Endopeptidase Complex; Protein Stability; Protein Synthesis Inhibitors; Proteolysis; rho GTP-Binding Proteins; rhoB GTP-Binding Protein; RNA, Messenger; Stress Fibers; Trabecular Meshwork

To determine the role of posttranslational isoprenylation in regulating Rho G-protein activation and stability in human trabecular meshwork (TM) cells.. Transformed human TM cells (GTM3) were incubated for 24 hours in the presence of activated lovastatin (10 μM) to enhance the endogenous synthesis of latent Rho proteins. Medium was replaced, cycloheximide (CHX) was added to inhibit synthesis of new proteins, and lovastatin-pretreated cells were subsequently incubated (0-24 hours) in the absence (control) or presence of farnesyl pyrophosphate (10 μM) or geranylgeranyl pyrophosphate (10 μM). Relative changes in the content of total and GTP-bound Rho G-proteins were quantified by Western immunoblot and GTP-binding ELISA, respectively. Changes in filamentous actin stress fiber organization were visualized with AlexaFluor488-conjugated phalloidin.. GTM3 cells cultured in the presence of lovastatin exhibited a loss of actin stress fiber organization concomitant with a marked accumulation of cytosolic inactive (GDP-bound) Rho G-proteins. Addition of geranylgeranyl pyrophosphate to the culture medium restored actin stress fiber organization while selectively facilitating the subcellular redistribution of accumulated Rho proteins from cytosol to membrane and increasing RhoA activation. Geranylgeranyl pyrophosphate selectively enhanced the degradation of newly synthesized Rho proteins. Epoxomicin, a potent and selective inhibitor of the 20S proteasome, prevented geranylgeranyl-enhanced degradation of Rho proteins.. Posttranslational geranylgeranylation selectively alters the lifecycle of newly synthesized Rho proteins by facilitating their membrane translocation, functional activation, and turnover. Geranylgeranylation represents a novel mechanism by which active Rho proteins are targeted to the proteasome for degradation in human TM cells.

Topics: Actins; Blotting, Western; Cell Survival; Cells, Cultured; Cytosol; Enzyme-Linked Immunosorbent Assay; Humans; Lovastatin; Male; Oligopeptides; Polyisoprenyl Phosphates; Prenylation; Proteasome Endopeptidase Complex; Protein Processing, Post-Translational; Protein Transport; rho GTP-Binding Proteins; Sesquiterpenes; Trabecular Meshwork