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Raymond J Hohl - One of the best experts on this subject based on the ideXlab platform.
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targeting Geranylgeranylation reduces adrenal gland tumor burden in a murine model of prostate cancer metastasis
Clinical & Experimental Metastasis, 2015Co-Authors: Jacqueline E Reilly, Huaxiang Tong, Raymond J Hohl, Jeffrey D Neighbors, Michael D HenryAbstract:The isoprenoid biosynthetic pathway (IBP) is critical for providing substrates for the post-translational modification of proteins key in regulating malignant cell properties, including proliferation, invasion, and migration. Inhibitors of the IBP, including statins and nitrogenous bisphosphonates, are used clinically for the treatment of hypercholesterolemia and bone disease respectively. The statins work predominantly in the liver, while the nitrogenous bisphosphonates are highly sequestered to bone. Inhibition of the entire IBP is limited by organ specificity and side effects resulting from depletion of all isoprenoids. We have developed a novel compound, disodium [(6Z,11E,15E)-9-[bis(sodiooxy)phosphoryl]-17-hydroxy-2,6,12,16-tetramethyheptadeca-2,6,11,15-tetraen-9-yl]phosphonate (GGOHBP), which selectively targets geranylgeranyl diphosphate synthase, reducing post-translational protein Geranylgeranylation. Intracardiac injection of luciferase-expressing human-derived 22Rv1 PCa cells into SCID mice resulted in tumor development in bone (100 %), adrenal glands (72 %), mesentery (22 %), liver (17 %), and the thoracic cavity (6 %). Three weeks after tumor inoculation, daily subcutaneous (SQ) injections of 1.5 mg/kg GGOHBP or the vehicle were given for one month. Dissected tumors revealed a reduction in adrenal gland tumors corresponding to a 54 % (P < 0.005) reduction in total adrenal gland tumor weight of the treated mice as compared to vehicle-treated controls. Western blot analysis of the harvested tissues showed a reduction in Rap1A Geranylgeranylation in adrenal glands and mesenteric tumors of the treated mice while non-tumorous tissues and control mice showed no Rap1A alteration. Our findings detail a novel bisphosphonate compound capable of preferentially altering the IBP in tumor-burdened adrenal glands of a murine model of PCa metastasis.
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Bisphosphonates Induce Autophagy by Depleting
2010Co-Authors: Geranylgeranyl Diphosphate, Brian M. Wasko, Amel Dudakovic, Raymond J HohlAbstract:Multiple studies have implicated the depletion of isoprenoid biosynthetic pathway intermediates in the induction of au-tophagy. However, the exact mechanism by which isoprenoid biosynthesis inhibitors induce autophagy has not been well established. We hypothesized that inhibition of farnesyl diphos-phate synthase (FDPS) and geranylgeranyl diphosphate syn-thase (GGDPS) by bisphosphonates would induce autophagy by depleting cellular geranylgeranyl diphosphate (GGPP) and impairing protein Geranylgeranylation. Herein, we show that an inhibitor of FDPS (zoledronate) and an inhibitor of GGDPS (digeranyl bisphosphonate, DGBP) induce autophagy in PC3 prostate cancer and MDA-MB-231 breast cancer cells as measured by accumulation of the autophagic marker LC3-II. Treatment of cells with lysosomal protease inhibitors [(2S,3S)-trans-epoxysuccinyl-L-leucylamido-3-methylbutane ethyl ester (E-64d) and pepstatin A] in combination with zoledronate or digeranyl bisphosphonate further enhances the formation of LC3-II, indicating that these compounds induce autophagic flux. It is noteworthy that the addition of exogenous GGPP prevented the accumulation of LC3-II and impairment of Rab6 (a GGTase II substrate) Geranylgeranylation by isoprenoid path-way inhibitors (lovastatin, zoledronate, and DGBP). However, exogenous GGPP did not restore isoprenoid pathway inhibitor-induced impairment of Rap1a (a GGTase I substrate) gera-nylgeranylation. In addition, specific inhibitors of farnesyl trans-ferase and geranylgeranyl transferase I are unable to induce autophagy in our system. Furthermore, the addition of bafilo-mycin A1 (an inhibitor of autophagy processing) enhanced the antiproliferative effects of digeranyl bisphosphonate. These re-sults are the first to demonstrate that bisphosphonates induce autophagy. Our study suggests that induction of autophagy in PC3 cells with these agents is probably dependent upon im-pairment of Geranylgeranylation of GGTase II substrates
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pivaloyloxymethyl modified isoprenoid bisphosphonates display enhanced inhibition of cellular Geranylgeranylation
Bioorganic & Medicinal Chemistry, 2008Co-Authors: Andrew J. Wiemer, Raymond J Hohl, Brian M. Wasko, Larry W Shull, Rocky J Barney, Kimberly M Lamb, David F WiemerAbstract:Nitrogenous bisphosphonate inhibitors of farnesyl disphosphate synthase have been used clinically for treatment of bone disease. Because many of their effects may be mediated by depletion of geranylgeranyl diphosphate, our group has sought compounds that do this more directly through inhibition of geranylgeranyl diphosphate synthase and we have discovered a number of isoprenoid-containing bisphosphonates that selectively inhibit this enzyme. These compounds have a high negative charge at physiological pH which is necessary for inhibition of the enzyme but may limit their ability to enter cells. Therefore, chemical modifications that mask this charge may enhance their cellular potency. We now have synthesized novel pivaloyloxymethyl-modified isoprenoid bisphosphonates and investigated their ability to inhibit protein Geranylgeranylation within cells. We have found that addition of pivaloyloxymethyl moieties to isoprenoid bisphosphonates increases their potency towards cellular Geranylgeranylation even though this modification decreases their in vitro inhibition of geranylgeranyl diphosphate synthase. Pivaloyloxymethyl modifications more effectively increase the cellular activity of the more polar isoprenoid bisphosphonates. These results reveal structural relationships between in vitro and cellular activity which may serve as the basis for future development of more potent and/or drug-like inhibitors of geranylgeranyl diphosphate synthase.
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lovastatin suppresses erythropoietin receptor surface expression through dual inhibition of glycosylation and Geranylgeranylation
Biochemical Pharmacology, 2007Co-Authors: Sumaya N Hamadmad, Raymond J HohlAbstract:Erythropoietin (Epo) is a cytokine that is required for the survival of erythroid progenitors through interaction with its receptor on the surface of these cells. Recent studies showed that erythropoietin receptor (EpoR) is expressed on many cancer cells. The factors that govern EpoR expression on the cell surface are poorly understood. Using both biotinlyation and radiolabeled Epo binding experiments, we show here that Epo starvation of the Epo-dependent erythroleukemia cell line, ASE2, leads to a time-dependent increase in both forms of EpoR, the maturing 64 kDa and the mature 66 kDa proteins. Mevalonate depletion inhibits the formation of the highly glycosylated mature form of EpoR without affecting the other form. Treatment of cells with lovastatin, a selective inhibitor of the rate-limiting enzyme in the mevalonate pathway leads to inhibition of cell surface EpoR that is induced by Epo starvation. The effect of lovastatin appears to be the consequence of inhibition of two processes, glycosylation and Geranylgeranylation. Adding back geranylgeranyl pyrophosphate to lovastatin-treated cells completely prevents the lovastatin effect on EpoR expression. Dolichol, the sugar carrier in N-linked glycosylation that is derived from the mevalonate pathway, partially reverses lovastatin's effect. The glycosylation inhibitor tunicamycin also partially suppresses EpoR surface expression. Inhibiting protein Geranylgeranylation mimics the effect of lovastatin and inhibits EpoR surface expression in a concentration-dependent manner. Finally, lovastatin inhibits Epo's stimulatory effects on cell proliferation. These results indicate that mevalonate derivatives are required for normal EpoR expression on the cell surface through two pathways, glycosylation and Geranylgeranylation.
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synthesis of fluorescently tagged isoprenoid bisphosphonates that inhibit protein Geranylgeranylation
Bioorganic & Medicinal Chemistry, 2007Co-Authors: Mona A Maalouf, Raymond J Hohl, Andrew J. Wiemer, Craig H Kuder, David F WiemerAbstract:Geminal bisphosphonates can be used for a variety of purposes in human disease including reduction of bone resorption in osteoporosis, treatment of fractures associated with malignancies of the prostate, breast, and lung, and direct anticancer activity against bone marrow derived malignancies. Previous research led to identification of some novel isoprenoid bisphosphonates that inhibit geranylgeranyl pyrophosphate (GGPP) synthesis and diminish protein Geranylgeranylation. Described here is the synthesis of fluorescent anthranilate analogues of the most active isoprenoid bisphosphonates and examine their ability to impact post-translational processing of the small GTPases Ras, Rap1a, and Rab6. Similar to their non-fluorescent counterparts, some of these fluorescent isoprenoid bisphosphonates diminish protein Geranylgeranylation. Their biological activity and fluorescent character suggest that they may be useful in studies of bisphosphonate localization both in cultured cells and in whole organisms.
Said M Sebti - One of the best experts on this subject based on the ideXlab platform.
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targeting protein prenylation for cancer therapy
Nature Reviews Cancer, 2011Co-Authors: Norbert Berndt, Andrew D Hamilton, Said M SebtiAbstract:Protein farnesylation and Geranylgeranylation, together referred to as prenylation, are lipid post-translational modifications that are required for the transforming activity of many oncogenic proteins, including some RAS family members. This observation prompted the development of inhibitors of farnesyltransferase (FT) and geranylgeranyl-transferase 1 (GGT1) as potential anticancer drugs. In this Review, we discuss the mechanisms by which FT and GGT1 inhibitors (FTIs and GGTIs, respectively) affect signal transduction pathways, cell cycle progression, proliferation and cell survival. In contrast to their preclinical efficacy, only a small subset of patients responds to FTIs. Identifying tumours that depend on farnesylation for survival remains a challenge, and strategies to overcome this are discussed. One GGTI has recently entered the clinic, and the safety and efficacy of GGTIs await results from clinical trials.
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abstract 620 development of a dual protein farnesyltransferase geranylgeranyltransferase i inhibitor with antitumor activity against human cancer cells
Cancer Research, 2011Co-Authors: Aslamuzzaman Kazi, Andrew D Hamilton, Yunting Luo, Norbert Berndt, Steven Fletcher, Christopher G Cummings, Harshani R Lawrence, Said M SebtiAbstract:Many low molecular weight GTPases such as Ras, Ral and Rho require posttranslational farnestylation or Geranylgeranylation for mediating malignant transformation. This prompted the development of farnesyltransferase (FT) inhibitors (FTI) and geranylgeranyltransferase-I (GGT-1) inhibitors (GGTI) as potential anticancer agents. However, when cancer cells are treated with FTIs, K-Ras becomes geranylgeranylated suggesting that inhibition of both FT and GGT-1 may be required to inhibit tumors harboring K-Ras mutations. Recently our group has developed a series of dual FT and GGT-1 inhibitors based on an ethylenediamine scaffold. Among these dual inhibitors FGTI-2734 was most potent in vitro (FT IC50 = 250±190 nM and GGT-1 IC50 = 520±90 nM). In human cancer cells FGTI-2734 inhibits potently HDJ-2 farnesylation, Rap 1A Geranylgeranylation and K-Ras prenylation. Furthermore, FGTI-2734 inhibits the phosphorylation of Erk1/2, Akt and S6K, induces p27 accumulation and inhibits potently tumor cell growth. Finally, FGTI-2734 significantly reduces survivin protein levels, and reduction of survivin levels is associated with induction of apoptosis. Thus, this single molecule with dual FT and GGT-1 inhibitory activities may have distinct advantage over selective FTIs and GGTIs and warrants further advanced pre-clinical studies. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 620. doi:10.1158/1538-7445.AM2011-620
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simvastatin potentiates tumor necrosis factor α mediated apoptosis of human vascular endothelial cells via the inhibition of the Geranylgeranylation of rhoa
Life Sciences, 2006Co-Authors: Dongjiang Tang, Andrew D Hamilton, Said M Sebti, Hojin Park, Serban P Georgescu, Jonas B GalperAbstract:Abstract HMG-CoA reductase inhibitors (statins) are widely used in the treatment and prevention of atherosclerosis. Here we demonstrate that the HMG-CoA reductase inhibitor simvastatin potentiates TNFα-mediated apoptosis and TNFα signaling in human umbilical vein endothelial cells (HUVECs). While 2.5 μM simvastatin or 40 ng/ml TNFα alone had only a small effect on apoptosis in HUVECs, co-incubation with simvastatin and TNFα markedly increased apoptosis in a time- and dose-dependent manner as measured by FACS analysis of propidium iodide-stained cells. Geranylgeraniol, which serves as a substrate for the Geranylgeranylation of small GTP binding proteins such as RhoA, which is required for the function and membrane localization of Rho, reversed the effect of simvastatin on apoptosis. GGTI, an inhibitor of protein Geranylgeranylation, mimicked the effect of simvastatin on apoptosis and interfered with the membrane localization of RhoA. Furthermore, simvastatin increased the expression of the TNFα type I receptor (TNFαRI) with a dose dependence and a dependence on Geranylgeranylation similar to that demonstrated for the potentiation of TNFα-mediated apoptosis. Adenoviral expression of a dominant-negative RhoA mimicked the effect of simvastatin on the expression of TNFαRI, while adenoviral expression of a dominant-activating RhoA mutant reversed the effect of simvastatin on the expression of TNFαRI. Simvastatin also potentiated TNFα signaling as determined by increased TNFα-mediated E-selectin expression. These data support the conclusion that TNFα signaling is under the negative control of RhoA and that statins potentiate TNFα signaling at least in part via interference with RhoA inhibition of TNFα type I receptor expression.
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inhibitory effects of mevastatin and a geranylgeranyl transferase i inhibitor ggti 2166 on mononuclear osteoclast formation induced by receptor activator of nfκb ligand rankl or tumor necrosis factor α tnf α
Biochemical Pharmacology, 2005Co-Authors: Jetae Woo, Hiroshi Nakagawa, Annette M Krecic, Kazuo Nagai, Andrew D Hamilton, Said M Sebti, Paula H SternAbstract:Abstract We have previously reported that the statin mevastatin (compactin) reversibly inhibits the fusion of TRAP-positive mononuclear preosteoclasts (pOCs) into multinucleated osteoclasts and disrupts the actin ring in mature osteoclasts through the inhibition of protein prenylation. Protein Geranylgeranylation, specifically, is known to be required for pOC fusion and for the function and survival of mature osteoclasts. However, it has not been determined whether protein Geranylgeranylation is involved in early differentiation of osteoclasts (pOC formation). The current study shows that statins and the geranylgeranyl transferase I inhibitor GGTI-2166 inhibit the pOC formation induced by RANKL or TNF-α in cultures of both mouse marrow-derived macrophage-colony-stimulating factor (M-CSF) dependent monocytes (MD cells) and the mouse monocyte cell line RAW 264.7 (RAW cells). Mevastatin, 0.1–0.6 μM, inhibited the formation of pOCs induced by receptor activator of nuclear factor-κB ligand (RANKL) or tumor necrosis factor (TNF-α) in both cell cultures. The inhibitory effects of mevastatin were overcome by the addition of mevalonate, farnesyl pyrophosphate or geranylgeranyl pyrophosphate. GGTI-2166 inhibited TRAP activity induced by RANKL or TNF-α in both cell cultures and prevented the incorporation of [ 3 H]all- trans geranylgeraniol into prenylated proteins in RAW cells. However, the farnesyl transferase inhibitor FTI-2153 did not inhibit TRAP activity although FTI prevented the incorporation of [ 14 C]mevalonate into farnesylated proteins in RAW cells. Clostridium difficile cytotoxin B (toxin B) inhibited pOC formation induced by RANKL or TNF-α in both cell cultures. The inhibitory effects of statins and GGTI-2166 on pOC formation may result from the inhibition of the Geranylgeranylation of G-proteins, such as Rho or Rac, suggesting that the Geranylgeranylation of these proteins is involved in the early differentiation of progenitor cells into pOCs.
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inhibition of protein Geranylgeranylation and rhoa rhoa kinase pathway induces apoptosis in human endothelial cells
Journal of Biological Chemistry, 2002Co-Authors: Li Liu, Andrew D Hamilton, Said M Sebti, Joan C Tupper, Douglas D Bannerman, Robert K Winn, John M HarlanAbstract:Abstract Geranylgeranylation of RhoA small G-protein is essential for its localization to cell membranes and for its biological functions. Many RhoA effects are mediated by its downstream effector RhoA kinase. The role of protein Geranylgeranylation and the RhoA pathway in the regulation of endothelial cell survival has not been elucidated. The hydroxy-3-methylglutaryl (HMG)-CoA reductase inhibitor lovastatin depletes cellular pools of geranylgeranyl pyrophosphate and farnesol pyrophosphate and thereby inhibits both Geranylgeranylation and farnesylation. Human umbilical vein endothelial cells (HUVECs) were exposed to lovastatin (3 μm-30 μm) for 48 h, and cell death was quantitatively determined by cytoplasmic histone-associated DNA fragments as well as caspase-3 activity. The assays showed that lovastatin caused a dose-dependent endothelial cell death. The addition of geranylgeraniol, which restores Geranylgeranylation, rescued HUVEC from apoptosis. The geranylgeranyltransferase inhibitor GGTI-298, but not the farnesyltransferase inhibitor FTI-277, induced apoptosis in HUVEC. Cell death was also induced by a blockade of RhoA function by exoenzyme C3. In addition, treatment of HUVEC with the RhoA kinase inhibitors Y-27632 and HA-1077 caused dose-dependent cell death. Y-27632 did not inhibit other well known survival pathways, such as NF-κB, ERK, and phosphatidylinositol 3-kinase/Akt. However, there was an increase in p53 protein level concomitant with Y-27632-induced cell death. Unlike the apoptosis induced by TNF-α, which occurs only with inhibition of new protein synthesis, apoptosis induced by inhibitors of HMG-CoA reductase, geranylgeranyltransferase, or RhoA kinase was blocked by cycloheximide. Our data indicate that inhibition of protein Geranylgeranylation and RhoA pathways induce apoptosis in HUVEC and that induction of p53 or other proapoptotic proteins is required for this process.
Miguel C. Seabra - One of the best experts on this subject based on the ideXlab platform.
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Thematic review series: lipid posttranslational modifications. Geranylgeranylation of Rab GTPases.
Journal of lipid research, 2006Co-Authors: Ka Fai Leung, Rudi Baron, Miguel C. SeabraAbstract:Rab GTPases require special machinery for protein prenylation, which include Rab escort protein (REP) and Rab geranylgeranyl transferase (RGGT). The current model of Rab Geranylgeranylation proposes that REP binds Rab and presents it to RGGT. After Geranylgeranylation of Rab C-terminal cysteines, REP delivers the prenylated protein to membranes. The REP-like protein Rab GDP dissociation inhibitor (RabGDI) then recycles the prenylated Rab between the membrane and the cytosol. The recent solution of crystal structures of the Rab prenylation machinery has helped to refine this model and provided further insights. The hydrophobic prenyl binding pocket of RGGT and geranylgeranyl transferase type-I (GGT-I) differs from that of farnesyl transferase (FT). A bulky tryptophan residue in FT restricts the size of the pocket, whereas in RGGT and GGT-I, this position is occupied by smaller residues. A highly conserved phenylalanine in REP, which is absent in RabGDI, is critical for the formation of the REP:RGGT complex. Finally, a geranylgeranyl binding site conserved in REP and RabGDI has been identified within helical domain II. The postprenylation events, including the specific targeting of Rabs to target membranes and the requirement for single versus double Geranylgeranylation by different Rabs, remain obscure and should be the subject of future studies.
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Mechanism of Rab Geranylgeranylation: Formation of the Catalytic Ternary Complex†
Biochemistry, 1998Co-Authors: Janmeet S. Anant, Luc Desnoyers, Mischa Machius, Borries Demeler, Jeffrey C. Hansen, Kenneth D. Westover, Johann Deisenhofer, Miguel C. SeabraAbstract:Rab proteins are geranylgeranylated on one or two C-terminal cysteines by Rab geranylgeranyl transferase (RabGGTase). The reaction is dependent on a Rab-binding protein, termed Rab escort protein (REP). Here, we studied the role of REP in the Geranylgeranylation reaction. We first characterized the interaction between REP and ungeranylgeranylated Rab using analytical ultracentrifugation and a fluorescence-based assay. We measured an equilibrium dissociation constant of 0.2 μM for the formation of a 1:1 REP−Rab complex and showed that this interaction relies mostly on ionic bonds and does not involve the two C-terminal cysteine residues. Second, we show that REP is required for recognition of Rab by RabGGTase and therefore that the REP−Rab complex is the true substrate for RabGGTase. Third, we show that free REP inhibits the Geranylgeranylation reaction, suggesting that the complex is recognized by RabGGTase primarily via a REP-binding site. Our data suggest a model whereby REP behaves kinetically as an es...
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nucleotide dependence of rab Geranylgeranylation rab escort protein interacts preferentially with gdp bound rab
Journal of Biological Chemistry, 1996Co-Authors: Miguel C. SeabraAbstract:Abstract Geranylgeranylation of Rab GTPases is an essential post-translational modification that enables Rabs to associate with intracellular membranes where they regulate exocytic and endocytic pathways. Geranylgeranylation is initiated by formation of a stable complex between newly synthesized Rab proteins and Rab escort protein (REP). The complex is recognized by Rab geranylgeranyl (GG) transferase, which transfers two GG groups to Rabs. The geranylgeranylated Rabs regulate vesicular movement by oscillating between an inactive GDP-bound form and an active GTP-bound form. In this study, I show that the kinetics of Geranylgeranylation is influenced by the nucleotide status of nascent Rab. GDP-bound Rab is geranylgeranylated with 10–50-fold higher affinity than GTP-bound Rab (or GTP analog-bound Rab), as indicated by the apparent Km of the reaction. In vitro REP·Rab binding assays demonstrate that REP forms a stable complex only with the GDP-bound form of Rab but not the GTP-bound form, suggesting that the apparent Km effect in the prenylation reaction is due to a discrimination between the two different nucleotide-bound forms of Rab by REP. Inasmuch as Rabs are likely GTP-bound after synthesis and REP does not possess GTPase-activating protein activity, these results raise the possibility that a Rab GTPase-activating protein enhances the REP·Rab interaction prior to prenylation.
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Mechanism of diGeranylgeranylation of Rab proteins. Formation of a complex between monogeranylgeranyl-Rab and Rab escort protein.
The Journal of biological chemistry, 1996Co-Authors: Fang Shen, Miguel C. SeabraAbstract:Rab proteins are Ras-related small GTPases that are digeranylgeranylated at carboxyl-terminal cysteines, a modification essential for their action as molecular switches regulating intracellular vesicular transport. Geranylgeranylation of Rabs is a complex reaction that requires a catalytic Rab geranylgeranyl transferase (GGTase) and a Rab escort protein (REP). REP binds unprenylated Rab and presents it to Rab GGTase. After GG transfer, REP remains associated with diGG-Rab, which leads to insertion of the Rab into a specific membrane. We used recombinant Rab1a single cysteine mutants that accept only one GG group to study the mechanism of the diGeranylgeranylation reaction. Using the prenylation assay, gel filtration chromatography, and density ultracentrifugation, we show that REP, but not Rab GGTase, forms a stable complex with unprenylated, monoGG- and diGG-Rab1a. The REP.monoGG-Rab1a complex is stable in the presence of detergents or phospholipids, whereas the REP.diGG-Rab1a complex partially dissociates under these conditions. The stoichiometry of the REP.Rab complex appears to be 1:1 before prenylation. Prenylation induces a change in complex stoichiometry, with the formation of a 2:2 or 2:1 REP.Rab complex. A possible mechanism by which Rab proteins are digeranylgeranylated is suggested by the current studies. We propose that each geranylgeranyl addition is an independent reaction that leads to the production of monoGG-Rab and diGG-Rab, respectively. The stability of the REP.monoGG-Rab complex prevents monoGG-Rab from dissociating from REP prior to the second Geranylgeranylation reaction, ensuring efficient diGeranylgeranylation of Rab substrates.
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deficient Geranylgeranylation of ram rab27 in choroideremia
Journal of Biological Chemistry, 1995Co-Authors: Miguel C. Seabra, Janmeet S. AnantAbstract:Choroideremia, an X-linked form of retinal degeneration, results from defects in the Rab escort protein-1 (REP-1) gene. REP-1 and REP-2 assist in the attachment of geranylgeranyl groups to Rab GTPases, a modification essential for their action as molecular switches regulating intracellular vesicular transport. If Rabs that depend preferentially on REP-1 for prenylation exist, they will accumulate unprenylated in choroideremia cells. Using recombinant Rab geranylgeranyl transferase and REPs to label unprenylated cytosolic proteins, we identified one unprenylated protein in choroideremia lymphoblasts that was prenylated in vitro more efficiently by REP-1 than by REP-2. This protein was purified and identified as Ram (renamed Rab27), a previously cloned Rab of unknown function. Immunohistochemistry of rat retina showed that Ram/Rab27 is expressed in the pigment epithelium and choriocapillaris, the two retinal cell layers that degenerate earliest in choroideremia. These results raise the possibility that the retinal degeneration in choroideremia results from the deficient Geranylgeranylation of Ram/Rab27 or a closely related protein.
Andrew D Hamilton - One of the best experts on this subject based on the ideXlab platform.
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targeting protein prenylation for cancer therapy
Nature Reviews Cancer, 2011Co-Authors: Norbert Berndt, Andrew D Hamilton, Said M SebtiAbstract:Protein farnesylation and Geranylgeranylation, together referred to as prenylation, are lipid post-translational modifications that are required for the transforming activity of many oncogenic proteins, including some RAS family members. This observation prompted the development of inhibitors of farnesyltransferase (FT) and geranylgeranyl-transferase 1 (GGT1) as potential anticancer drugs. In this Review, we discuss the mechanisms by which FT and GGT1 inhibitors (FTIs and GGTIs, respectively) affect signal transduction pathways, cell cycle progression, proliferation and cell survival. In contrast to their preclinical efficacy, only a small subset of patients responds to FTIs. Identifying tumours that depend on farnesylation for survival remains a challenge, and strategies to overcome this are discussed. One GGTI has recently entered the clinic, and the safety and efficacy of GGTIs await results from clinical trials.
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abstract 620 development of a dual protein farnesyltransferase geranylgeranyltransferase i inhibitor with antitumor activity against human cancer cells
Cancer Research, 2011Co-Authors: Aslamuzzaman Kazi, Andrew D Hamilton, Yunting Luo, Norbert Berndt, Steven Fletcher, Christopher G Cummings, Harshani R Lawrence, Said M SebtiAbstract:Many low molecular weight GTPases such as Ras, Ral and Rho require posttranslational farnestylation or Geranylgeranylation for mediating malignant transformation. This prompted the development of farnesyltransferase (FT) inhibitors (FTI) and geranylgeranyltransferase-I (GGT-1) inhibitors (GGTI) as potential anticancer agents. However, when cancer cells are treated with FTIs, K-Ras becomes geranylgeranylated suggesting that inhibition of both FT and GGT-1 may be required to inhibit tumors harboring K-Ras mutations. Recently our group has developed a series of dual FT and GGT-1 inhibitors based on an ethylenediamine scaffold. Among these dual inhibitors FGTI-2734 was most potent in vitro (FT IC50 = 250±190 nM and GGT-1 IC50 = 520±90 nM). In human cancer cells FGTI-2734 inhibits potently HDJ-2 farnesylation, Rap 1A Geranylgeranylation and K-Ras prenylation. Furthermore, FGTI-2734 inhibits the phosphorylation of Erk1/2, Akt and S6K, induces p27 accumulation and inhibits potently tumor cell growth. Finally, FGTI-2734 significantly reduces survivin protein levels, and reduction of survivin levels is associated with induction of apoptosis. Thus, this single molecule with dual FT and GGT-1 inhibitory activities may have distinct advantage over selective FTIs and GGTIs and warrants further advanced pre-clinical studies. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 620. doi:10.1158/1538-7445.AM2011-620
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simvastatin potentiates tumor necrosis factor α mediated apoptosis of human vascular endothelial cells via the inhibition of the Geranylgeranylation of rhoa
Life Sciences, 2006Co-Authors: Dongjiang Tang, Andrew D Hamilton, Said M Sebti, Hojin Park, Serban P Georgescu, Jonas B GalperAbstract:Abstract HMG-CoA reductase inhibitors (statins) are widely used in the treatment and prevention of atherosclerosis. Here we demonstrate that the HMG-CoA reductase inhibitor simvastatin potentiates TNFα-mediated apoptosis and TNFα signaling in human umbilical vein endothelial cells (HUVECs). While 2.5 μM simvastatin or 40 ng/ml TNFα alone had only a small effect on apoptosis in HUVECs, co-incubation with simvastatin and TNFα markedly increased apoptosis in a time- and dose-dependent manner as measured by FACS analysis of propidium iodide-stained cells. Geranylgeraniol, which serves as a substrate for the Geranylgeranylation of small GTP binding proteins such as RhoA, which is required for the function and membrane localization of Rho, reversed the effect of simvastatin on apoptosis. GGTI, an inhibitor of protein Geranylgeranylation, mimicked the effect of simvastatin on apoptosis and interfered with the membrane localization of RhoA. Furthermore, simvastatin increased the expression of the TNFα type I receptor (TNFαRI) with a dose dependence and a dependence on Geranylgeranylation similar to that demonstrated for the potentiation of TNFα-mediated apoptosis. Adenoviral expression of a dominant-negative RhoA mimicked the effect of simvastatin on the expression of TNFαRI, while adenoviral expression of a dominant-activating RhoA mutant reversed the effect of simvastatin on the expression of TNFαRI. Simvastatin also potentiated TNFα signaling as determined by increased TNFα-mediated E-selectin expression. These data support the conclusion that TNFα signaling is under the negative control of RhoA and that statins potentiate TNFα signaling at least in part via interference with RhoA inhibition of TNFα type I receptor expression.
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inhibitory effects of mevastatin and a geranylgeranyl transferase i inhibitor ggti 2166 on mononuclear osteoclast formation induced by receptor activator of nfκb ligand rankl or tumor necrosis factor α tnf α
Biochemical Pharmacology, 2005Co-Authors: Jetae Woo, Hiroshi Nakagawa, Annette M Krecic, Kazuo Nagai, Andrew D Hamilton, Said M Sebti, Paula H SternAbstract:Abstract We have previously reported that the statin mevastatin (compactin) reversibly inhibits the fusion of TRAP-positive mononuclear preosteoclasts (pOCs) into multinucleated osteoclasts and disrupts the actin ring in mature osteoclasts through the inhibition of protein prenylation. Protein Geranylgeranylation, specifically, is known to be required for pOC fusion and for the function and survival of mature osteoclasts. However, it has not been determined whether protein Geranylgeranylation is involved in early differentiation of osteoclasts (pOC formation). The current study shows that statins and the geranylgeranyl transferase I inhibitor GGTI-2166 inhibit the pOC formation induced by RANKL or TNF-α in cultures of both mouse marrow-derived macrophage-colony-stimulating factor (M-CSF) dependent monocytes (MD cells) and the mouse monocyte cell line RAW 264.7 (RAW cells). Mevastatin, 0.1–0.6 μM, inhibited the formation of pOCs induced by receptor activator of nuclear factor-κB ligand (RANKL) or tumor necrosis factor (TNF-α) in both cell cultures. The inhibitory effects of mevastatin were overcome by the addition of mevalonate, farnesyl pyrophosphate or geranylgeranyl pyrophosphate. GGTI-2166 inhibited TRAP activity induced by RANKL or TNF-α in both cell cultures and prevented the incorporation of [ 3 H]all- trans geranylgeraniol into prenylated proteins in RAW cells. However, the farnesyl transferase inhibitor FTI-2153 did not inhibit TRAP activity although FTI prevented the incorporation of [ 14 C]mevalonate into farnesylated proteins in RAW cells. Clostridium difficile cytotoxin B (toxin B) inhibited pOC formation induced by RANKL or TNF-α in both cell cultures. The inhibitory effects of statins and GGTI-2166 on pOC formation may result from the inhibition of the Geranylgeranylation of G-proteins, such as Rho or Rac, suggesting that the Geranylgeranylation of these proteins is involved in the early differentiation of progenitor cells into pOCs.
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inhibition of protein Geranylgeranylation and rhoa rhoa kinase pathway induces apoptosis in human endothelial cells
Journal of Biological Chemistry, 2002Co-Authors: Li Liu, Andrew D Hamilton, Said M Sebti, Joan C Tupper, Douglas D Bannerman, Robert K Winn, John M HarlanAbstract:Abstract Geranylgeranylation of RhoA small G-protein is essential for its localization to cell membranes and for its biological functions. Many RhoA effects are mediated by its downstream effector RhoA kinase. The role of protein Geranylgeranylation and the RhoA pathway in the regulation of endothelial cell survival has not been elucidated. The hydroxy-3-methylglutaryl (HMG)-CoA reductase inhibitor lovastatin depletes cellular pools of geranylgeranyl pyrophosphate and farnesol pyrophosphate and thereby inhibits both Geranylgeranylation and farnesylation. Human umbilical vein endothelial cells (HUVECs) were exposed to lovastatin (3 μm-30 μm) for 48 h, and cell death was quantitatively determined by cytoplasmic histone-associated DNA fragments as well as caspase-3 activity. The assays showed that lovastatin caused a dose-dependent endothelial cell death. The addition of geranylgeraniol, which restores Geranylgeranylation, rescued HUVEC from apoptosis. The geranylgeranyltransferase inhibitor GGTI-298, but not the farnesyltransferase inhibitor FTI-277, induced apoptosis in HUVEC. Cell death was also induced by a blockade of RhoA function by exoenzyme C3. In addition, treatment of HUVEC with the RhoA kinase inhibitors Y-27632 and HA-1077 caused dose-dependent cell death. Y-27632 did not inhibit other well known survival pathways, such as NF-κB, ERK, and phosphatidylinositol 3-kinase/Akt. However, there was an increase in p53 protein level concomitant with Y-27632-induced cell death. Unlike the apoptosis induced by TNF-α, which occurs only with inhibition of new protein synthesis, apoptosis induced by inhibitors of HMG-CoA reductase, geranylgeranyltransferase, or RhoA kinase was blocked by cycloheximide. Our data indicate that inhibition of protein Geranylgeranylation and RhoA pathways induce apoptosis in HUVEC and that induction of p53 or other proapoptotic proteins is required for this process.
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development of an image based screening system for inhibitors of the plastidial mep pathway and of protein Geranylgeranylation
F1000Research, 2015Co-Authors: Michael Hartmann, Andrea Hemmerlin, Michel Rohmer, Elisabet Gaspascual, Thomas J BachAbstract:In a preceding study we have recently established an in vivo visualization system for the Geranylgeranylation of proteins in a stably transformed tobacco BY-2 cell line, which involves expressing a dexamethasone-inducible GFP fused to the prenylable, carboxy-terminal basic domain of the rice calmodulin CaM61, which naturally bears a CaaL Geranylgeranylation motif (GFP-BD-CVIL). By using pathway-specific inhibitors it was there demonstrated that inhibition of the methylerythritol phosphate (MEP) pathway with oxoclomazone and fosmidomycin, as well as inhibition of protein geranylgeranyl transferase type 1 (PGGT-1), shifted the localization of the GFP-BD-CVIL protein from the membrane to the nucleus. In contrast, the inhibition of the mevalonate (MVA) pathway with mevinolin did not affect this localization. Furthermore, in this initial study complementation assays with pathway-specific intermediates confirmed that the precursors for the cytosolic isoprenylation of this fusion protein are predominantly provided by the MEP pathway. In order to optimize this visualization system from a more qualitative assay to a statistically trustable medium or a high-throughput screening system, we established now new conditions that permit culture and analysis in 96-well microtiter plates, followed by fluorescence microscopy. For further refinement, the existing GFP-BD-CVIL cell line was transformed with an estradiol-inducible vector driving the expression of a RFP protein, C-terminally fused to a nuclear localization signal (NLS-RFP). We are thus able to quantify the total number of viable cells versus the number of inhibited cells after various treatments. This approach also includes a semi-automatic counting system, based on the freely available image processing software. As a result, the time of image analysis as well as the risk of user-generated bias is reduced to a minimum. Moreover, there is no cross-induction of gene expression by dexamethasone and estradiol, which is an important prerequisite for this test system.
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Development of an image-based screening system for inhibitors of the plastidial MEP pathway and of protein Geranylgeranylation [v1; ref status: indexed, http://f1000r.es/4vt]
F1000 Research Ltd, 2015Co-Authors: Michael Hartmann, Andrea Hemmerlin, Michel Rohmer, Elisabet Gas-pascual, Thomas J BachAbstract:We have recently established an in vivo visualization system for the Geranylgeranylation of proteins in a stably transformed tobacco BY-2 cell line, which involves expressing a dexamethasone-inducible GFP fused to the prenylable, carboxy-terminal basic domain of the rice calmodulin CaM61, which naturally bears a CaaL Geranylgeranylation motif (GFP-BD-CVIL). By using pathway-specific inhibitors it was demonstrated that inhibition of the methylerythritol phosphate (MEP) pathway with oxoclomazone and fosmidomycin, as well as inhibition of protein geranylgeranyl transferase type 1 (PGGT-1), shifted the localization of the GFP-BD-CVIL protein from the membrane to the nucleus. In contrast, the inhibition of the mevalonate (MVA) pathway with mevinolin did not affect this localization. Furthermore, complementation assays with pathway-specific intermediates confirmed that the precursors for the cytosolic isoprenylation of this fusion protein are predominantly provided by the MEP pathway. In order to optimize this visualization system from a more qualitative assay to a statistically trustable medium or a high-throughput screening system, we established new conditions that permit culture and analysis in 96-well microtiter plates, followed by fluorescence microscopy. For further refinement, the existing GFP-BD-CVIL cell line was transformed with an estradiol-inducible vector driving the expression of a RFP protein, C-terminally fused to a nuclear localization signal (NLS-RFP). We are thus able to quantify the total number of viable cells versus the number of inhibited cells after various treatments. This approach also includes a semi-automatic counting system, based on the freely available image processing software. As a result, the time of image analysis as well as the risk of user-generated bias is reduced to a minimum. Moreover, there is no cross-induction of gene expression by dexamethasone and estradiol, which is an important prerequisite for this test system
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Development of an image-based screening system for inhibitors of the plastidial MEP pathway and of protein Geranylgeranylation [v2; ref status: indexed, http://f1000r.es/5p9]
F1000 Research Ltd, 2015Co-Authors: Michael Hartmann, Andrea Hemmerlin, Michel Rohmer, Elisabet Gas-pascual, Thomas J BachAbstract:In a preceding study we have recently established an in vivo visualization system for the Geranylgeranylation of proteins in a stably transformed tobacco BY-2 cell line, which involves expressing a dexamethasone-inducible GFP fused to the prenylable, carboxy-terminal basic domain of the rice calmodulin CaM61, which naturally bears a CaaL Geranylgeranylation motif (GFP-BD-CVIL). By using pathway-specific inhibitors it was there demonstrated that inhibition of the methylerythritol phosphate (MEP) pathway with oxoclomazone and fosmidomycin, as well as inhibition of protein geranylgeranyl transferase type 1 (PGGT-1), shifted the localization of the GFP-BD-CVIL protein from the membrane to the nucleus. In contrast, the inhibition of the mevalonate (MVA) pathway with mevinolin did not affect this localization. Furthermore, in this initial study complementation assays with pathway-specific intermediates confirmed that the precursors for the cytosolic isoprenylation of this fusion protein are predominantly provided by the MEP pathway. In order to optimize this visualization system from a more qualitative assay to a statistically trustable medium or a high-throughput screening system, we established now new conditions that permit culture and analysis in 96-well microtiter plates, followed by fluorescence microscopy. For further refinement, the existing GFP-BD-CVIL cell line was transformed with an estradiol-inducible vector driving the expression of a RFP protein, C-terminally fused to a nuclear localization signal (NLS-RFP). We are thus able to quantify the total number of viable cells versus the number of inhibited cells after various treatments. This approach also includes a semi-automatic counting system, based on the freely available image processing software. As a result, the time of image analysis as well as the risk of user-generated bias is reduced to a minimum. Moreover, there is no cross-induction of gene expression by dexamethasone and estradiol, which is an important prerequisite for this test system
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The effect of MEP pathway and other inhibitors on the intracellular localization of a plasma membrane-targeted, isoprenylable GFP reporter protein in tobacco BY-2 cells [v2; ref status: indexed, http://f1000r.es/2af]
F1000 Research Ltd, 2013Co-Authors: Michael Hartmann, Esther Gerber, Andrea Hemmerlin, Michel Rohmer, Elisabet Gas-pascual, Denis Tritsch, Thomas J BachAbstract:We have established an in vivo visualization system for the Geranylgeranylation of proteins in a stably transformed tobacco BY-2 cell line, based on the expression of a dexamethasone-inducible GFP fused to the carboxy-terminal basic domain of the rice calmodulin CaM61, which naturally bears a CaaL Geranylgeranylation motif (GFP-BD-CVIL). By using pathway-specific inhibitors it was demonstrated that inhibition of the methylerythritol phosphate (MEP) pathway with known inhibitors like oxoclomazone and fosmidomycin, as well as inhibition of the protein geranylgeranyltransferase type 1 (PGGT-1), shifted the localization of the GFP-BD-CVIL protein from the membrane to the nucleus. In contrast, the inhibition of the mevalonate (MVA) pathway with mevinolin did not affect the localization. During the present work, this test system has been used to examine the effect of newly designed inhibitors of the MEP pathway and inhibitors of sterol biosynthesis such as squalestatin, terbinafine and Ro48-8071. In addition, we also studied the impact of different post-prenylation inhibitors or those suspected to affect the transport of proteins to the plasma membrane on the localization of the geranylgeranylable fusion protein GFP-BD-CVIL
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the plastidial 2 c methyl d erythritol 4 phosphate pathway provides the isoprenyl moiety for protein Geranylgeranylation in tobacco by 2 cells
The Plant Cell, 2009Co-Authors: Esther Gerber, Andrea Hemmerlin, Michael Hartmann, Dimitri Heintz, Marieandree Hartmann, Jerome Mutterer, Manuel Rodriguezconcepcion, Albert Boronat, Alain Van Dorsselaer, Michel RohmerAbstract:Protein farnesylation and Geranylgeranylation are important posttranslational modifications in eukaryotic cells. We visualized in transformed Nicotiana tabacum Bright Yellow-2 (BY-2) cells the Geranylgeranylation and plasma membrane localization of GFP-BD-CVIL, which consists of green fluorescent protein (GFP) fused to the C-terminal polybasic domain (BD) and CVIL isoprenylation motif from the Oryza sativa calmodulin, CaM61. Treatment with fosmidomycin (Fos) or oxoclomazone (OC), inhibitors of the plastidial 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway, caused mislocalization of the protein to the nucleus, whereas treatment with mevinolin, an inhibitor of the cytosolic mevalonate pathway, did not. The nuclear localization of GFP-BD-CVIL in the presence of MEP pathway inhibitors was completely reversed by all-transgeranylgeraniol (GGol). Furthermore, 1-deoxy-D-xylulose (DX) reversed the effects of OC, but not Fos, consistent with the hypothesis that OC blocks 1-deoxy-D-xylulose 5-phosphate synthesis, whereas Fos inhibits its conversion to 2-C-methyl-Derythritol 4-phosphate. By contrast, GGol and DX did not rescue the nuclear mislocalization of GFP-BD-CVIL in the presence of a protein geranylgeranyltransferase type 1 inhibitor. Thus, the MEP pathway has an essential role in geranylgeranyl diphosphate (GGPP) biosynthesis and protein Geranylgeranylation in BY-2 cells. GFP-BD-CVIL is a versatile tool for identifying pharmaceuticals and herbicides that interfere either with GGPP biosynthesis or with protein Geranylgeranylation.