guanosine-triphosphate and farnesyl-pyrophosphate

Small GTPases (guanosine triphosphate, GTP) are involved in many critical cellular processes, including inflammation, proliferation, and migration. GTP loading and isoprenylation are two important post-translational modifications of small GTPases, and are critical for their normal function. In this study, we investigated the role of post-translational modifications of small GTPases in regulating endothelial cell inflammatory responses and junctional integrity.. Confluent human umbilical vein endothelial cell (HUVECs ) treated with atorvastatin demonstrated significantly decreased lipopolysaccharide (LPS)-mediated IL-6 and IL-8 generation. The inhibitory effect of atorvastatin (Atorva) was attenuated by co-treatment with 100 µM mevalonate (MVA) or 10 µM geranylgeranyl pyrophosphate (GGPP), but not by 10 µM farnesyl pyrophosphate (FPP). Atorvastatin treatment of HUVECs produced a time-dependent increase in GTP loading of all Rho GTPases, and induced the translocation of small Rho GTPases from the cellular membrane to the cytosol, which was reversed by 100 µM MVA and 10 µM GGPP, but not by 10 µM FPP. Atorvastatin significantly attenuated thrombin-induced HUVECs permeability, increased VE-cadherin targeting to cell junctions, and preserved junction integrity. These effects were partially reversed by GGPP but not by FPP, indicating that geranylgeranylation of small GTPases plays a major role in regulating endothelial junction integrity. Silencing of small GTPases showed that Rho and Rac, but not Cdc42, play central role in HUVECs junction integrity.. In conclusion, our studies show that post-translational modification of small GTPases plays a vital role in regulating endothelial inflammatory response and endothelial junction integrity. Atorvastatin increased GTP loading and inhibited isoprenylation of small GTPases, accompanied by reduced inflammatory response and preserved cellular junction integrity.

Topics: Antigens, CD; Atorvastatin; Cadherins; cdc42 GTP-Binding Protein; Guanosine Triphosphate; Heptanoic Acids; Human Umbilical Vein Endothelial Cells; Humans; Hydroxymethylglutaryl-CoA Reductase Inhibitors; Intercellular Junctions; Interleukin-6; Interleukin-8; Lipopolysaccharides; Mevalonic Acid; Polyisoprenyl Phosphates; Prenylation; Protein Processing, Post-Translational; Proto-Oncogene Proteins c-akt; Pyrroles; rho-Associated Kinases; Sesquiterpenes; Thrombin

A subset of nonsteroidal anti-inflammatory drugs (NSAIDs) has been shown to preferentially reduce the secretion of the highly amyloidogenic, 42-residue amyloid-beta peptide Abeta42. We found that Rho and its effector, Rho-associated kinase, preferentially regulated the amount of Abeta42 produced in vitro and that only those NSAIDs effective as Rho inhibitors lowered Abeta42. Administration of Y-27632, a selective Rock inhibitor, also preferentially lowered brain levels of Abeta42 in a transgenic mouse model of Alzheimer's disease. Thus, the Rho-Rock pathway may regulate amyloid precursor protein processing, and a subset of NSAIDs can reduce Abeta42 through inhibition of Rho activity.

Topics: Amides; Amyloid beta-Peptides; Amyloid Precursor Protein Secretases; Animals; Anti-Inflammatory Agents, Non-Steroidal; Aspartic Acid Endopeptidases; Brain; Cell Line, Tumor; Endopeptidases; Enzyme Inhibitors; Guanosine Triphosphate; Humans; Ibuprofen; Intracellular Signaling Peptides and Proteins; Mice; Mice, Transgenic; Peptide Fragments; Polyisoprenyl Phosphates; Protein Serine-Threonine Kinases; Pyridines; rho GTP-Binding Proteins; rho-Associated Kinases; rhoA GTP-Binding Protein; Sesquiterpenes; Signal Transduction; Sulindac; Transfection

BALB/3T3 cells were transformed by transfection with DNA encoding the mutated ras(Q(61)K) from shrimp Penaeus japonicus (Huang et al., 2000). The GTPase-activating protein (GAP) in the cytosol fraction was significantly expressed and degraded, compared to untransformed cells on the western blot. To understand this in more detail, the interaction of the bacterially expressed shrimp Ras (S-Ras) with GAP was investigated using GAP purified from mouse brains. SDS-polyacrylamide gel electrophoresis revealed the monomers of the purified GAP to have a relative mass of 65,000. Since the purified GAP was bound to the Ras conjugated affinity sepharose column with high affinity and its GTP hydolysis activity upon binding with tubulin was suppressed, the purified enzyme was concluded to be neurofibromin-like. The purified GAP enhanced the intrinsic GTPase activity of the S-Ras, to convert it into the inactive GDP-bound form, in agreement with findings for GTP-bound K(B)-Ras in vitro. To compare the effects between isoprenoids and GAP on the GTP-hydrolysis of Ras, we applied the GTP-locked shrimp mutant S-Ras(Q(61)K) and GTP-locked rat mutant K(B)-ras(Q(61)K). Radioassay studies showed that geranylgeranyl pyrophosphate at microg level catalyzed the GTP hydrolysis of S-Ras(Q(61)K) and K(B)-ras(Q(61)K) competently, but not farnesyl pyrophosphate or the purified GAP. The present study provides the view that the geranylgeranyl pyrophosphate at carboxyl terminal CAAX assists GTP hydrolysis to Ras proteins probably in a manner similar to the substrate assisted catalysis in GTPase mechanism.

Topics: Animals; Blotting, Western; Cell Culture Techniques; Decapoda; Genes, ras; GTP Phosphohydrolases; GTPase-Activating Proteins; Guanosine Triphosphate; Hydrolysis; Mammals; Polyisoprenyl Phosphates; Sesquiterpenes; Transfection

Prostate cancer cells derived from transgenic mice with adenocarcinoma of the prostate (TRAMP cells) were treated with the HMG-CoA reductase inhibitor, lovastatin. This caused inactivation of the small GTPase RhoA, actin stress fiber disassembly, cell rounding, growth arrest in the G1 phase of the cell cycle, cell detachment and apoptosis. Addition of geranylgeraniol (GGOL) in the presence of lovastatin, to stimulate protein geranylgeranylation, prevented lovastatin's effects. That is, RhoA was activated, actin stress fibers were assembled, the cells assumed a flat morphology and cell growth resumed. The following observations support an essential role for RhoA in TRAMP cell growth: (1) TRAMP cells expressing dominant-negative RhoA (T19N) mutant protein displayed few actin stress fibers and grew at a slower rate than controls (35 h doubling time for cells expressing RhoA (T19N) vs 20 h for untransfected cells); (2) TRAMP cells expressing constitutively active RhoA (Q63L) mutant protein displayed a contractile phenotype and grew faster than controls (13 h doubling time). Interestingly, addition of farnesol (FOL) with lovastatin, to stimulate protein farnesylation, prevented lovastatin-induced cell rounding, cell detachment and apoptosis, and stimulated cell spreading to a spindle shaped morphology. However, RhoA remained inactive and growth arrest persisted. The morphological effects of FOL addition were prevented in TRAMP cells expressing dominant-negative H-Ras (T17N) mutant protein. Thus, it appears that H-Ras is capable of inducing cell spreading, but incapable of supporting cell proliferation, in the absence of geranylgeranylated proteins like RhoA.

Topics: Actin Cytoskeleton; Adenocarcinoma; Alkyl and Aryl Transferases; Animals; Antineoplastic Agents; Apoptosis; Cell Adhesion; Cell Division; Cell Size; Diterpenes; Drug Interactions; Enzyme Activation; Farnesol; G1 Phase; Genes, ras; GTP-Binding Proteins; Guanosine Triphosphate; Hydroxymethylglutaryl-CoA Reductase Inhibitors; Lovastatin; Male; Mevalonic Acid; Mice; Mice, Transgenic; Polyisoprenyl Phosphates; Prostatic Neoplasms; Protein Prenylation; Protein Processing, Post-Translational; Proto-Oncogene Proteins p21(ras); rac GTP-Binding Proteins; rhoA GTP-Binding Protein; Sesquiterpenes; Tumor Cells, Cultured

The mechanism by which 3-hydroxy-3-methylglutaryl (HMG)-CoA reductase inhibitors increase endothelial nitric oxide synthase (eNOS) expression is unknown. To determine whether changes in isoprenoid synthesis affects eNOS expression, human endothelial cells were treated with the HMG-CoA reductase inhibitor, mevastatin (1-10 microM), in the presence of L-mevalonate (200 microM), geranylgeranylpyrophosphate (GGPP, 1-10 microM), farnesylpyrophosphate (FPP, 5-10 microM), or low density lipoprotein (LDL, 1 mg/ml). Mevastatin increased eNOS mRNA and protein levels by 305 +/- 15% and 180 +/- 11%, respectively. Co-treatment with L-mevalonate or GGPP, but not FPP or LDL, reversed mevastatin's effects. Because Rho GTPases undergo geranylgeranyl modification, we investigated whether Rho regulates eNOS expression. Immunoblot analyses and [35S]GTPgammaS-binding assays revealed that mevastatin inhibited Rho membrane translocation and GTP binding activity by 60 +/- 5% and 78 +/- 6%, both of which were reversed by co-treatment with GGPP but not FPP. Furthermore, inhibition of Rho by Clostridium botulinum C3 transferase (50 microg/ml) or by overexpression of a dominant-negative N19RhoA mutant increased eNOS expression. In contrast, activation of Rho by Escherichia coli cytotoxic necrotizing factor-1 (200 ng/ml) decreased eNOS expression. These findings indicate that Rho negatively regulates eNOS expression and that HMG-CoA reductase inhibitors up-regulate eNOS expression by blocking Rho geranylgeranylation, which is necessary for its membrane-associated activity.

Topics: ADP Ribose Transferases; Bacterial Toxins; Botulinum Toxins; Cells, Cultured; Cytosol; Cytotoxins; Endothelium, Vascular; Escherichia coli Proteins; Gene Expression Regulation, Enzymologic; GTP Phosphohydrolases; GTP-Binding Proteins; Guanosine 5'-O-(3-Thiotriphosphate); Guanosine Triphosphate; Humans; Hydroxymethylglutaryl-CoA Reductase Inhibitors; Kinetics; Lipoproteins, LDL; Lovastatin; Membrane Proteins; Mevalonic Acid; Nitric Oxide Synthase; Nitric Oxide Synthase Type III; Polyisoprenyl Phosphates; ras Proteins; rhoA GTP-Binding Protein; rhoB GTP-Binding Protein; RNA Processing, Post-Transcriptional; RNA, Messenger; Sesquiterpenes