Geranylgeranyltransferase Type Research Articles

Silencing geranylgeranyltransferase I inhibits the migration and invasion of salivary adenoid cystic carcinoma through RhoA/ROCK1/MLC signaling and suppresses proliferation through cell cycle regulation.

Geranylgeranyltransferase type I (GGTase-I) significantly affects Rho proteins, such that the malignant progression of several cancers may be induced. Nevertheless, the effect and underlying mechanism of GGTase-I in the malignant progression of salivary adenoid cystic carcinoma (SACC) remain unclear. This study primarily aimed to investigate the role and mechanism of GGTase-I in mediating the malignant progression of SACC. The level of GGTase-I gene in cells was stably knocked down by short hairpin RNA-EGFP-lentivirus. The effects of GGTase-I silencing on the migration, invasion, and spread of cells were examined, the messenger RNA levels of GGTase-I and RhoA genes of SACC cells after GGTase-I knockdown were determined, and the protein levels of RhoA and RhoA membrane of SACC cells were analyzed. Moreover, the potential underlying mechanism of silencing GGTase-I on the above-mentioned aspects in SACC cells was assessed by examining the protein expression of ROCK1, MLC, p-MLC, E-cadherin, Vimentin, MMP2, and MMP9. Furthermore, the underlying mechanism of SACC cells proliferation was investigated through the analysis of the expression of cyclinD1, MYC, E2F1, and p21CIP1/WAF1 . Besides, the change of RhoA level in SACC tissues compared with normal paracancer tissues was demonstrated through quantitative reverse-transcription polymerase chain reaction and western blot experiments. Next, the effect after GGTase-I silencing was assessed through the subcutaneous tumorigenicity assay. As indicated by the result of this study, the silencing of GGTase-I significantly reduced the malignant progression of tumors in vivo while decreasing the migration, invasion, and proliferation of SACC cells and RhoA membrane, Vimentin, ROCK1, p-MLC, MMP2, MMP9, MYC, E2F1, and CyclinD1 expression. However, the protein expression of E-cadherin and p21CIP1/WAF1 was notably upregulated. Subsequently, no significant transform of RhoA and MLC proteins was identified. Furthermore, RhoA expression in SACC tissues was significantly higher than that in paracancerous tissues. As revealed by the results of this study, GGTase-I shows a correlation with the proliferation of SACC through the regulation of cell cycle and may take on vital significance in the migration and invasion of SACC by regulating RhoA/ROCK1/MLC signaling pathway. GGTase-I is expected to serve as a novel exploration site of SACC.

Rho-GTPase dependent leukocyte interaction generates pro-inflammatory thymic Tregs and causes arthritis

Conditional mutation of protein geranylgeranyltransferase type I (GGTase-I) in macrophages (GLC) activates Rho-GTPases and causes arthritis in mice. Knocking out Rag1 in GLC mice alleviates arthritis which indicates that lymphocytes are required for arthritis development in those mice. To study GLC dependent changes in the adaptive immunity, we isolated CD4+ T cells from GLC mice (CD4+GLCs). Spleen and joint draining lymph nodes (dLN) CD4+GLCs exhibited high expression of Cdc42 and Rac1, which repressed the caudal HOXA proteins and activated the mechanosensory complex to facilitate migration. These CDC42/RAC1 rich CD4+GLCs presented a complete signature of GARP+NRP1+IKZF2+FOXP3+ regulatory T cells (Tregs) of thymic origin. Activation of the β-catenin/Lef1 axis promoted a pro-inflammatory Th1 phenotype of Tregs, which was strongly associated with arthritis severity. Knockout of Cdc42 in macrophages of GLC mice affected CD4+ cell biology and triggered development of non-thymic Tregs. Knockout of Rac1 and RhoA had no such effects on CD4+ cells although it alleviated arthritis in GLC mice. Disrupting macrophage and T cell interaction with CTLA4 fusion protein reduced the Th1-driven inflammation and enrichment of thymic Tregs into dLNs. Antigen challenge reinforced the CD4+GLC phenotype in non-arthritic heterozygote GLC mice and increased accumulation of Rho-GTPase expressing thymic Tregs in dLNs. Our study demonstrates an unexpected role of macrophages in stimulating the development of pro-inflammatory thymic Tregs and reveal activation of Rho-GTPases behind their arthritogenic phenotype.

Protein Prenyltransferases and Their Inhibitors: Structural and Functional Characterization.

Protein prenylation is a post-translational modification controlling the localization, activity, and protein–protein interactions of small GTPases, including the Ras superfamily. This covalent attachment of either a farnesyl (15 carbon) or a geranylgeranyl (20 carbon) isoprenoid group is catalyzed by four prenyltransferases, namely farnesyltransferase (FTase), geranylgeranyltransferase type I (GGTase-I), Rab geranylgeranyltransferase (GGTase-II), and recently discovered geranylgeranyltransferase type III (GGTase-III). Blocking small GTPase activity, namely inhibiting prenyltransferases, has been proposed as a potential disease treatment method. Inhibitors of prenyltransferase have resulted in substantial therapeutic benefits in various diseases, such as cancer, neurological disorders, and viral and parasitic infections. In this review, we overview the structure of FTase, GGTase-I, GGTase-II, and GGTase-III and summarize the current status of research on their inhibitors.

Molecular and Pharmacological Characterization of the Interaction between Human Geranylgeranyltransferase Type I and Ras-Related Protein Rap1B.

Geranylgeranyltransferase type-I (GGTase-I) represents an important drug target since it contributes to the function of many proteins that are involved in tumor development and metastasis. This led to the development of GGTase-I inhibitors as anti-cancer drugs blocking the protein function and membrane association of e.g., Rap subfamilies that are involved in cell differentiation and cell growth. In the present study, we developed a new NanoBiT assay to monitor the interaction of human GGTase-I and its substrate Rap1B. Different Rap1B prenylation-deficient mutants (C181G, C181S, and ΔCQLL) were designed and investigated for their interaction with GGTase-I. While the Rap1B mutants C181G and C181S still exhibited interaction with human GGTase-I, mutant ΔCQLL, lacking the entire CAAX motif (defined by a cysteine residue, two aliphatic residues, and the C-terminal residue), showed reduced interaction. Moreover, a specific, peptidomimetic and competitive CAAX inhibitor was able to block the interaction of Rap1B with GGTase-I. Furthermore, activation of both Gαs-coupled human adenosine receptors, A2A (A2AAR) and A2B (A2BAR), increased the interaction between GGTase-I and Rap1B, probably representing a way to modulate prenylation and function of Rap1B. Thus, A2AAR and A2BAR antagonists might be promising candidates for therapeutic intervention for different types of cancer that overexpress Rap1B. Finally, the NanoBiT assay provides a tool to investigate the pharmacology of GGTase-I inhibitors.

Scaffold association factor B (SAFB) is required for expression of prenyltransferases and RAS membrane association

Inhibiting membrane association of RAS has long been considered a rational approach to anticancer therapy, which led to the development of farnesyltransferase inhibitors (FTIs). However, FTIs proved ineffective against KRAS-driven tumors. To reveal alternative therapeutic strategies, we carried out a genome-wide CRISPR-Cas9 screen designed to identify genes required for KRAS4B membrane association. We identified five enzymes in the prenylation pathway and SAFB, a nuclear protein with both DNA and RNA binding domains. Silencing SAFB led to marked mislocalization of all RAS isoforms as well as RAP1A but not RAB7A, a pattern that phenocopied silencing FNTA, the prenyltransferase α subunit shared by farnesyltransferase and geranylgeranyltransferase type I. We found that SAFB promoted RAS membrane association by controlling FNTA expression. SAFB knockdown decreased GTP loading of RAS, abrogated alternative prenylation, and sensitized RAS-mutant cells to growth inhibition by FTI. Our work establishes the prenylation pathway as paramount in KRAS membrane association, reveals a regulator of prenyltransferase expression, and suggests that reduction in FNTA expression may enhance the efficacy of FTIs.

Neuronal Protein Farnesylation Regulates Hippocampal Synaptic Plasticity and Cognitive Function.

Protein prenylation is a post-translational lipid modification that governs a variety of important cellular signaling pathways, including those regulating synaptic functions and cognition in the nervous system. Two enzymes, farnesyltransferase (FT) and geranylgeranyltransferase type I (GGT), are essential for the prenylation process. Genetic reduction of FT or GGT ameliorates neuropathology but only FT haplodeficiency rescues cognitive function in transgenic mice of Alzheimer's disease. A follow-up study showed that systemic or forebrain neuron-specific deficiency of GGT leads to synaptic and cognitive deficits under physiological conditions. Whether FT plays different roles in shaping neuronal functions and cognition remains elusive. This study shows that in contrast to the detrimental effects of GGT reduction, systemic haplodeficiency of FT has little to no impact on hippocampal synaptic plasticity and cognition. However, forebrain neuron-specific FT deletion also leads to reduced synaptic plasticity, memory retention, and hippocampal dendritic spine density. Furthermore, a novel prenylomic analysis identifies distinct pools of prenylated proteins that are affected in the brain of forebrain neuron-specific FT and GGT knockout mice, respectively. Taken together, this study uncovers that physiological levels of FT and GGT in neurons are essential for normal synaptic/cognitive functions and that the prenylation status of specific signaling molecules regulates neuronal functions.

Protein Farnesyltransferase Catalyzes Unanticipated Farnesylation and Geranylgeranylation of Shortened Target Sequences.

Protein prenylation is a posttranslational modification involving the attachment of a C15 or C20 isoprenoid group to a cysteine residue near the C-terminus of the target substrate by protein farnesyltransferase (FTase) or protein geranylgeranyltransferase type I (GGTase-I), respectively. Both of these protein prenyltransferases recognize a C-terminal "CaaX" sequence in their protein substrates, but recent studies in yeast- and mammalian-based systems have demonstrated FTase can also accept sequences that diverge in length from the canonical four-amino acid motif, such as the recently reported five-amino acid C(x)3X motif. In this work, we further expand the substrate scope of FTase by demonstrating sequence-dependent farnesylation of shorter three-amino acid "Cxx" C-terminal sequences using both genetic and biochemical assays. Strikingly, biochemical assays utilizing purified mammalian FTase and Cxx substrates reveal prenyl donor promiscuity leading to both farnesylation and geranylgeranylation of these sequences. These findings expand the substrate pool of sequences that can be potentially prenylated, further refine our understanding of substrate recognition by FTase and GGTase-I, and suggest the possibility of a new class of prenylated proteins within proteomes.

Inhibiting Fungal Echinocandin Resistance by Small-Molecule Disruption of Geranylgeranyltransferase Type I Activity.

Echinocandin resistance in Candida is a great concern, as the echinocandin drugs are recommended as first-line therapy for patients with invasive candidiasis. However, therapeutic efforts to thwart echinocandin resistance have been hampered by a lack of fungal specific drug targets. Here, we show that deleting CDC43, the β subunit of geranylgeranyltransferase type I (GGTase I), confers hypersensitivity to echinocandins, which renders GGTase I a tractable target in combatting echinocandin resistance. The membrane localization of Rho1, which is critical for (1,3)-β-d-glucan synthase Fks1 activation, is disrupted in the cdc43 mutant, resulting in decreased amounts of glucans in the cell wall, thereby exacerbating the cell wall stress upon caspofungin addition. Guided by this insight, we found that selective chemical inhibition of GGTase I by L-269289 potentiates echinocandin activity and renders echinocandin-resistant Candida albicans responsive to treatment in vitro and in animal models for disseminated infection. Furthermore, L-269289 and echinocandins also act in a synergistic manner for the treatment of Candida tropicalis and Candida parapsilosis Importantly, deletion of CDC43 is lethal in Candida glabrata L-269289 is active on its own to kill C. glabrata, and its fungicidal activity is enhanced when combined with caspofungin. Thus, targeting GGTase I has therapeutic potential to address the clinical challenge of echinocandin-resistant candidiasis.

Protein prenylation restrains innate immunity by inhibiting Rac1 effector interactions

Rho family proteins are prenylated by geranylgeranyltransferase type I (GGTase-I), which normally target proteins to membranes for GTP-loading. However, conditional deletion of GGTase-I in mouse macrophages increases GTP-loading of Rho proteins, leading to enhanced inflammatory responses and severe rheumatoid arthritis. Here we show that heterozygous deletion of the Rho family gene Rac1, but not Rhoa and Cdc42, reverses inflammation and arthritis in GGTase-I-deficient mice. Non-prenylated Rac1 has a high affinity for the adaptor protein Ras GTPase-activating-like protein 1 (Iqgap1), which facilitates both GTP exchange and ubiquitination-mediated degradation of Rac1. Consistently, inactivating Iqgap1 normalizes Rac1 GTP-loading, and reduces inflammation and arthritis in GGTase-I-deficient mice, as well as prevents statins from increasing Rac1 GTP-loading and cytokine production in macrophages. We conclude that blocking prenylation stimulates Rac1 effector interactions and unleashes proinflammatory signaling. Our results thus suggest that prenylation normally restrains innate immune responses by preventing Rac1 effector interactions.

Inhibiting PGGT1B Disrupts Function of RHOA, Resulting in T-cell Expression of Integrin α4β7 and Development of Colitis in Mice

It is not clear how regulation of T-cell function is altered during development of inflammatory bowel diseases (IBD). We studied the mechanisms by which geranylgeranyltransferase-mediated prenylation controls T-cell localization to the intestine and chronic inflammation. We generated mice with T-cell-specific disruption of the geranylgeranyltransferase type I, beta subunit gene (Pggt1b), called Pggt1bΔCD4 mice, or the ras homolog family member A gene (Rhoa), called RhoaΔCD4 mice. We also studied mice with knockout of CDC42 or RAC1 and wild-type mice (controls). Intestinal tissues were analyzed by histology, multiphoton and confocal microscopy, and real-time polymerase chain reaction. Activation of CDC42, RAC1, and RHOA were measured with G-LISA, cell fractionation, and immunoblots. T cells and lamina propria mononuclear cells from mice were analyzed by flow cytometry or transferred to Rag1-/- mice. Mice were given injections of antibodies against integrin alpha4beta7 or gavaged with the RORC antagonist GSK805. We obtained peripheral blood and intestinal tissue samples from patients with and without IBD and analyzed them by flow cytometry. Pggt1bΔCD4 mice developed spontaneous colitis, characterized by thickening of the intestinal wall, edema, fibrosis, accumulation of T cells in the colon, and increased expression of inflammatory cytokines. Compared with control CD4+ T cells, PGGT1B-deficient CD4+ T cells expressed significantly higher levels of integrin alpha4beta7, which regulates their localization to the intestine. Inflammation induced by transfer of PGGT1B-deficient CD4+ T cells to Rag1-/- mice was blocked by injection of an antibody against integrin alpha4beta7. Lamina propria of Pggt1bΔCD4 mice had increased numbers of CD4+ T cells that expressed RORC and higher levels of cytokines produced by T-helper 17 cells (granulocyte-macrophage colony-stimulating factor, interleukin [IL]17A, IL17F, IL22, and tumor necrosis factor [TNF]). The RORC inverse agonist GSK805, but not antibodies against IL17A or IL17F, prevented colitis in Pggt1bΔCD4 mice. PGGT1B-deficient CD4+ T cells had decreased activation of RHOA. RhoAΔCD4 mice had a similar phenotype to Pggt1bΔCD4 mice, including development of colitis, increased numbers of CD4+ T cells in colon, increased expression of integrin alpha4beta7 by CD4+ T cells, and increased levels of IL17A and other inflammatory cytokines in lamina propria. T cells isolated from intestinal tissues from patients with IBD had significantly lower levels ofPGGT1B than tissues from individuals without IBD. Loss of PGGT1B from T cells in mice impairs RHOA function, increasing CD4+ T-cell expression of integrin alpha4beta7 and localization to colon, resulting in increased expression of inflammatory cytokines and colitis. T cells isolated from gut tissues from patients with IBD have lower levels of PGGT1B than tissues from patients without IBD.

Comparative transcriptomics approach in elucidation of carotenoid biosynthesis regulation in grains of rice (Oryza sativa L.)

Estimation of phytoene, lycopene, β-carotene, lutein, and zeaxanthin in grains of white, brown and purple cultivars of rice revealed marked differences in the levels of these carotenoid intermediates amongst the cultivars. Grains of white rice did not show any significant accumulation of carotenoid intermediates at any stage of development. On the other hand, grains of the purple cultivar accumulated 49.16 ± 5 µg of β-carotene, 28.89 ± 3.2 µg of lutein and 34.65 ± 4.6 µg of zeaxanthin per gm of grain fresh weight. In addition to PSY1, higher expression of βLCY than εLCY appears to be an important factor in determining the flux of pathway towards synthesis of β-β branch carotenoids in purple rice. This cultivar showed a higher fold change in carotenoid precursors during transition from milky to doughing stages and an enhanced flux of lycopene towards β-carotene during grain maturation. Our results indicate that higher level of carotenoids in purple rice is a consequence of higher expression of genes involved in pyruvate metabolism as well as those involved in carotenoid biosynthesis such as PSY1, PDS and β-LCY. Co-expression networking revealed a strong positive relationship between the expression profiles of genes involved in carotenoid biosynthesis and genes coding for geranylgeranyl transferase type II, glutathione S-transferase, DnaJ and SET domain containing proteins as well as MADS26 and R2R3MYB family of transcription factors.

32.1 MECHANISMS OF ABNORMAL POSTTRANSLATIONAL PROTEIN PROCESSING IN SCHIZOPHRENIA BRAIN

32.1 MECHANISMS OF ABNORMAL POSTTRANSLATIONAL PROTEIN PROCESSING IN SCHIZOPHRENIA BRAIN

Efficient farnesylation of an extended C-terminal C(x)3X sequence motif expands the scope of the prenylated proteome

Protein prenylation is a post-translational modification that has been most commonly associated with enabling protein trafficking to and interaction with cellular membranes. In this process, an isoprenoid group is attached to a cysteine near the C terminus of a substrate protein by protein farnesyltransferase (FTase) or protein geranylgeranyltransferase type I or II (GGTase-I and GGTase-II). FTase and GGTase-I have long been proposed to specifically recognize a four-amino acid CAAX C-terminal sequence within their substrates. Surprisingly, genetic screening reveals that yeast FTase can modify sequences longer than the canonical CAAX sequence, specifically C(x)3X sequences with four amino acids downstream of the cysteine. Biochemical and cell-based studies using both peptide and protein substrates reveal that mammalian FTase orthologs can also prenylate C(x)3X sequences. As the search to identify physiologically relevant C(x)3X proteins begins, this new prenylation motif nearly doubles the number of proteins within the yeast and human proteomes that can be explored as potential FTase substrates. This work expands our understanding of prenylation's impact within the proteome, establishes the biologically relevant reactivity possible with this new motif, and opens new frontiers in determining the impact of non-canonically prenylated proteins on cell function.

Expression of key enzymes in the mevalonate pathway are altered in monocrotaline-induced pulmonary arterial hypertension in rats.

Pulmonary arterial hypertension (PAH) is a serious pulmonary vascular disease. The changes in the structure, function and metabolism of endothelial cells are some of the important features of PAH. Previous studies have demonstrated that the mevalonate pathway is important in cardiovascular remodeling. However, whether the mevalonate pathway is involved in the development of PAH remains to be elucidated. The present study aimed to investigate the expression pattern of mevalonate pathway-related enzymes in monocrotaline (MCT)-induced PAH. F344 rats were randomly divided into two groups (n=6/group): Control group rats were injected with a single dose of saline, and MCT group rats were injected with a single dose of MCT (60mg/kg). After 4weeks, the right ventricular systolic pressure (RVSP) was measured, and lung and pulmonary artery tissue samples were collected. It was demonstrated that the RVSP increased and pulmonary vascular remodeling was detected in the PAH group. The expression levels of the enzymes farnesyldiphosphate synthase farnesyltransferase α and geranylgeranyltransferase typeI increased in the PAH group, which suggested that the mevalonate pathway may be involved in the pathological development of PAH.

Inhibiting HDAC1 Enhances the Anti-Cancer Effects of Statins through Downregulation of GGTase-Iβ Expression.

Hydroxy-methyl-glutaryl-coenzyme A (HMG-CoA) reductase inhibitors, namely statins, are potential anti-tumor agents. Previously, we showed that a pan-histone deacetylase (HDAC) inhibitor enhances the anti-tumor effects of the HMG-CoA inhibitor. However, the underlying mechanisms were not fully understood. Cancer cell lines (CAL-27 and SACC-83) were exposed to pan-HDAC inhibitor, or HDAC1 inhibitor, or geranylgeranyl transferase type I (GGTase-I) inhibitor alone or in combination with statin. Cell viability, apoptosis, migration, and invasion were assessed by Cell Count Kit-8, 4′,6-diamidino-2-phenylindole staining, and transwell assay, respectively. A xenograft model was used for assessing tumor growth in vivo. Western blot and real-time PCR were used to assess the expression of genes. We observed that inhibiting HDAC1 could enhance the anti-tumor effects of statins both in vitro and in vivo. Inhibiting HDAC1 blocked the statin-induced upregulation of geranylgeranyl transferase type Iβ subunit (GGTase-Iβ), resulting in an enhancement of the anti-cancer effects of statin. Overexpression of GGTase-Iβ or constitutively active RhoA abolished the enhancement by inhibiting HDAC1 on anti-tumor effects of statins. The HDAC1 inhibitor failed to enhance cytotoxicity in non-tumor primary cells treated with statin. Inhibiting HDAC1 enhanced the anti-cancer effects of statins through downregulation of GGTase-Iβ expression, and thus further inactivation of RhoA. A combination of statin with HDAC1 or GGTase-I inhibitor would be a new strategy for cancer chemotherapy.

Protein Geranylgeranyltransferase Type 1 as a Target in Cancer.

The process of protein prenylation involves the covalent linkage of either farnesyl (15-carbon) or geranylgeranyl (20-carbon) isoprenoid lipds to conserved cysteine residues in the carboxyl-terminus of proteins. Protein geranylgeranyltransferase I (GGTase-I) is the enzyme that catalyzes the addition of the geranylgeranyl moiety from geranylgeranyl pyrophosphate to the target protein, which contains a Cterminal consensus sequence termed a CaaX motif. Geranylgeranylation is important to the function of a number of proteins, including the majority of Rho GTPases, G protein gamma subunits, and several other regulatory proteins. Studies over the past two decades have revealed that many of these proteins contribute to tumor development and metastasis. Blocking Rho GTPase activity through inhibition of GGTase-I in particular has been advanced as a potential strategy for disease therapy. This review will provide an overview of the CaaX prenyltransferases, the rationale for targeting GGTase-I in cancer in particular, and the current status of GGTase-I inhibitor (GGTI) development.

Simultaneous Site-Specific Dual Protein Labeling Using Protein Prenyltransferases.

Site-specific protein labeling is an important technique in protein chemistry and is used for diverse applications ranging from creating protein conjugates to protein immobilization. Enzymatic reactions, including protein prenylation, have been widely exploited as methods to accomplish site-specific labeling. Enzymatic prenylation is catalyzed by prenyltransferases, including protein farnesyltransferase (PFTase) and geranylgeranyltransferase type I (GGTase-I), both of which recognize C-terminal CaaX motifs with different specificities and transfer prenyl groups from isoprenoid diphosphates to their respective target proteins. A number of isoprenoid analogues containing bioorthogonal functional groups have been used to label proteins of interest via PFTase-catalyzed reaction. In this study, we sought to expand the scope of prenyltransferase-mediated protein labeling by exploring the utility of rat GGTase-I (rGGTase-I). First, the isoprenoid specificity of rGGTase-I was evaluated by screening eight different analogues and it was found that those with bulky moieties and longer backbone length were recognized by rGGTase-I more efficiently. Taking advantage of the different substrate specificities of rat PFTase (rPFTase) and rGGTase-I, we then developed a simultaneous dual labeling method to selectively label two different proteins by using isoprenoid analogue and CaaX substrate pairs that were specific to only one of the prenyltransferases. Using two model proteins, green fluorescent protein with a C-terminal CVLL sequence (GFP-CVLL) and red fluorescent protein with a C-terminal CVIA sequence (RFP-CVIA), we demonstrated that when incubated together with both prenyltransferases and the selected isoprenoid analogues, GFP-CVLL was specifically modified with a ketone-functionalized analogue by rGGTase-I and RFP-CVIA was selectively labeled with an alkyne-containing analogue by rPFTase. By switching the ketone-containing analogue to an azide-containing analogue, it was possible to create protein tail-to-tail dimers in a one-pot procedure through the copper(I)-catalyzed alkyne-azide cycloaddition (CuAAC) reaction. Overall, with the flexibility of using different isoprenoid analogues, this system greatly extends the utility of protein labeling using prenyltransferases.

Development of an image-based screening system for inhibitors of the plastidial MEP pathway and of protein geranylgeranylation.

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.

Development of an image-based screening system for inhibitors of the plastidial MEP pathway and of protein geranylgeranylation.

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.

Prenylation is required for polar cell elongation, cell adhesion, and differentiation in Physcomitrella patens

Protein prenylation is required for a variety of growth and developmental processes in flowering plants. Here we report the consequences of loss of function of all known prenylation subunits in the moss Physcomitrella patens. As in Arabidopsis, protein farnesyltransferase and protein geranylgeranyltransferase type I are not required for viability. However, protein geranylgeranyltransferase type I activity is required for cell adhesion, polar cell elongation, and cell differentiation. Loss of protein geranylgeranyltransferase activity results in colonies of round, single-celled organisms that resemble unicellular algae. The loss of protein farnesylation is not as severe but also results in polar cell elongation and differentiation defects. The complete loss of Rab geranylgeranyltransferase activity appears to be lethal in P.patens. Labeling with antibodies to cell wall components support the lack of polarity establishment and the undifferentiated state of geranylgeranyltransferase type I mutant plants. Our results show that prenylated proteins play key roles in P.patens development and differentiation processes.