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Journal of Bone and Mineral Research :... May 2006N-BPs, which inhibit bone resorption by preventing prenylation of small GTPases, unexpectedly cause the accumulation of GTP-bound, unprenylated Rho family GTPases in...
UNLABELLED
N-BPs, which inhibit bone resorption by preventing prenylation of small GTPases, unexpectedly cause the accumulation of GTP-bound, unprenylated Rho family GTPases in macrophages and osteoclasts. In macrophages, this also leads to sustained, Rac-mediated activation of p38. The antiresorptive activity of N-BPs may therefore be caused at least in part, by the accumulation of unprenylated small GTPases, causing inappropriate activation of downstream signaling pathways.
INTRODUCTION
Nitrogen-containing bisphosphonates (N-BPs) are potent inhibitors of bone resorption that act by inhibiting farnesyl diphosphate synthase, thereby indirectly preventing the prenylation of Rho family GTPases that are required for the function and survival of bone-resorbing osteoclasts. However, the effect that these drugs have on the activity of Rho family GTPases has not been determined.
MATERIALS AND METHODS
The effect of N-BPs on the activity of Rho family GTPases in J774 macrophages and osteoclasts was measured using a pull-down assay to isolate the GTP-bound forms. The effect of N-BPs, or decreasing Rac expression using siRNA, on downstream p38 activity was evaluated by Western blotting and apoptosis assessed by measurement of caspase 3/7 activity.
RESULTS
Rather than inhibiting GTPase function, loss of prenylation after treatment with N-BPs caused an increase in the GTP-bound form of Rac, Cdc42, and Rho in J774 cells and osteoclast-like cells, which paralleled the rate of accumulation of unprenylated small GTPases. Activation of Rac also occurred with other inhibitors of prenylation of Rho-family proteins, such as mevastatin and the geranylgeranyl transferase I inhibitor GGTI-298. The Rac-GTP that increased after N-BP treatment was newly translated, cytoplasmic unprenylated protein, because it was not labeled with [(14)C] mevalonate, and the increase in Rac-GTP was prevented by cycloheximide. Furthermore, this unprenylated Rac-GTP retained at least part of its functional activity in J774 cells, because it mediated N-BP-induced activation of p38. Paradoxically, although risedronate induces apoptosis of J774 macrophages by inhibiting protein prenylation, the p38 inhibitor SB203580 enhanced N-BP-induced apoptosis, suggesting that Rac-induced p38 activation partially suppresses the pro-apoptotic effect of N-BPs in these cells.
CONCLUSIONS
N-BP drugs may disrupt the function of osteoclasts in vivo and affect other cell types in vitro by inhibiting protein prenylation, thereby causing inappropriate and sustained activation, rather than inhibition, of some small GTPases and their downstream signaling pathways.
Topics: Animals; Blotting, Western; Cell Line; Diphosphonates; Enzyme Activation; Macrophages; Male; Mice; Mice, Inbred C57BL; Protein Prenylation; cdc42 GTP-Binding Protein; p38 Mitogen-Activated Protein Kinases; rac GTP-Binding Proteins
PubMed: 16734383
DOI: 10.1359/jbmr.060118 -
The Journal of Biological Chemistry Feb 2018Protein prenylation is a post-translational modification that has been most commonly associated with enabling protein trafficking to and interaction with cellular...
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 C C-terminal sequence within their substrates. Surprisingly, genetic screening reveals that yeast FTase can modify sequences longer than the canonical C sequence, specifically C() 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() sequences. As the search to identify physiologically relevant C() 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.
Topics: Alkyl and Aryl Transferases; Amino Acid Motifs; Animals; Databases, Protein; Enzyme Inhibitors; Genes, Reporter; Green Fluorescent Proteins; HEK293 Cells; Humans; Microscopy, Fluorescence; Models, Molecular; Protein Prenylation; Protein Subunits; Proteomics; Rats; Recombinant Fusion Proteins; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins; Substrate Specificity
PubMed: 29282289
DOI: 10.1074/jbc.M117.805770 -
Nature Protocols Oct 2011The importance of the post-translational lipid modifications farnesylation and geranylgeranylation in protein localization and function coupled with the critical role of...
The importance of the post-translational lipid modifications farnesylation and geranylgeranylation in protein localization and function coupled with the critical role of prenylated proteins in malignant transformation has prompted interest in their biology and the development of farnesyl transferase and geranylgeranyl transferase inhibitors (FTIs and GGTIs) as chemical probes and anticancer agents. The ability to measure protein prenylation before and after FTI and GGTI treatment is important to understanding and interpreting the effects of these agents on signal transduction pathways and cellular phenotypes, as well as to the use of prenylation as a biomarker. Here we describe protocols to measure the degree of protein prenylation by farnesyl transferase or geranylgeranyl transferase in vitro, in cultured cells and in tumors from animals and humans. The assays use [(3)H]farnesyl diphosphate and [(3)H]geranylgeranyl diphosphate, electrophoretic mobility shift, membrane association using subcellular fractionation or immunofluorescence of intact cells, [(3)H]mevalonic acid labeling, followed by immunoprecipitation and SDS-PAGE, and in vitro transcription, translation and prenylation in reticulocyte lysates. These protocols require from 1 d (enzyme assays) to up to 3 months (autoradiography of [(3)H]-labeled proteins).
Topics: Animals; Biopsy; Cells, Cultured; Farnesyltranstransferase; Geranylgeranyl-Diphosphate Geranylgeranyltransferase; Humans; Lipid Metabolism; Mammals; Protein Prenylation; Protein Transport
PubMed: 22036881
DOI: 10.1038/nprot.2011.387 -
The Journal of Biological Chemistry Feb 1997Protein geranylgeranyltransferase-I (PGGT-I) and protein farnesyltransferase (PFT) attach geranylgeranyl and farnesyl groups, respectively, to the C termini of...
Differential prenyl pyrophosphate binding to mammalian protein geranylgeranyltransferase-I and protein farnesyltransferase and its consequence on the specificity of protein prenylation.
Protein geranylgeranyltransferase-I (PGGT-I) and protein farnesyltransferase (PFT) attach geranylgeranyl and farnesyl groups, respectively, to the C termini of eukaryotic cell proteins. In vitro, PGGT-I and PFT can transfer both geranylgeranyl and farnesyl groups from geranylgeranyl pyrophosphate (GGPP) and farnesyl pyrophosphate (FPP) to their protein or peptide prenyl acceptor substrates. In the present study it is shown that PGGT-I binds GGPP 330-fold tighter than FPP and that PFT binds FPP 15-fold tighter than GGPP. Therefore, in vivo, where both GGPP and FPP compete for the binding to prenyltransferases, PGGT-I and PFT will likely be bound predominantly to GGPP and FPP, respectively. Previous studies have shown that K-Ras4B and the Ras-related GTPase TC21 are substrates for both PGGT-I and PFT in vitro. It is shown that TC21 can compete with the C-terminal peptide of the gamma subunit of heterotrimeric G proteins and with the C-terminal peptide of lamin B for geranylgeranylation by PGGT-I and for farnesylation by PFT, respectively. K-Ras4B competes in both cases but is almost exclusively farnesylated by PFT in the presence of the lamin B peptide competitor. Rapid and single turnover kinetic studies indicate that the rate constant for the PGGT-I-catalyzed geranylgeranyl transfer step of the reaction cycle is 14-fold larger than the steady-state turnover number, which indicates that the rate of the overall reaction is limited by a step subsequent to prenyl transfer such as release of products from the enzyme. PGGT-I-catalyzed farnesylation is 37-fold slower than geranylgeranylation and is limited by the farnesyl transfer step. These results together with earlier studies provide a paradigm for the substrate specificity of PGGT-I and PFT and provide information that is critical for the design of prenyltransferase inhibitors as anti-cancer agents.
Topics: Alkyl and Aryl Transferases; Animals; Cell Line; Organophosphorus Compounds; Protein Binding; Protein Prenylation; Rats; Spodoptera; Substrate Specificity; Transferases
PubMed: 9020098
DOI: 10.1074/jbc.272.7.3944 -
BMC Research Notes Oct 2012Heterotrimeric G-proteins, consisting of three subunits Gα, Gβ and Gγ are present in most eukaryotes and mediate signaling in numerous biological processes. In...
BACKGROUND
Heterotrimeric G-proteins, consisting of three subunits Gα, Gβ and Gγ are present in most eukaryotes and mediate signaling in numerous biological processes. In plants, Gγ subunits were shown to provide functional selectivity to G-proteins. Three unconventional Gγ subunits were recently reported in Arabidopsis, rice and soybean but no structural analysis has been reported so far. Their relationship with conventional Gγ subunits and taxonomical distribution has not been yet demonstrated.
RESULTS
After an extensive similarity search through plant genomes, transcriptomes and proteomes we assembled over 200 non-redundant proteins related to the known Gγ subunits. Structural analysis of these sequences revealed that most of them lack the obligatory C-terminal prenylation motif (CaaX). According to their C-terminal structures we classified the plant Gγ subunits into three distinct types. Type A consists of Gγ subunits with a putative prenylation motif. Type B subunits lack a prenylation motif and do not have any cysteine residues in the C-terminal region, while type C subunits contain an extended C-terminal domain highly enriched with cysteines. Comparative analysis of C-terminal domains of the proteins, intron-exon arrangement of the corresponding genes and phylogenetic studies suggested a common origin of all plant Gγ subunits.
CONCLUSION
Phylogenetic analyses suggest that types C and B most probably originated independently from type A ancestors. We speculate on a potential mechanism used by those Gγ subunits lacking isoprenylation motifs to anchor the Gβγ dimer to the plasma membrane and propose a new flexible nomenclature for plant Gγ subunits. Finally, in the light of our new classification, we give a word of caution about the interpretation of Gγ research in Arabidopsis and its generalization to other plant species.
Topics: Amino Acid Sequence; Heterotrimeric GTP-Binding Proteins; Molecular Sequence Data; Phylogeny; Plants; Protein Prenylation; Sequence Homology, Amino Acid
PubMed: 23113884
DOI: 10.1186/1756-0500-5-608 -
Current Biology : CB Jul 2004The passage of an individual's genome to future generations is essential for the maintenance of species and is mediated by highly specialized cells, the germ cells.... (Review)
Review
The passage of an individual's genome to future generations is essential for the maintenance of species and is mediated by highly specialized cells, the germ cells. Genetic studies in a number of model organisms have provided insight into the molecular mechanisms that control specification, migration and survival of early germ cells. Focusing on Drosophila, we will discuss the mechanisms by which germ cells initially form and remain transcriptionally silent while somatic cells are transcriptionally active. We will further discuss three separate attractive and repellent guidance pathways, mediated by a G-protein coupled receptor, two lipid phosphate phosphohydrolases, and isoprenylation. We will compare and contrast these findings with those obtained in other organisms, in particular zebrafish and mice. While aspects of germ cell specification are strikingly different between these species, germ cell specific gene functions have been conserved. In particular, mechanisms that sense directional cues during germ cell migration seem to be shared between invertebrates and vertebrates.
Topics: Animals; Cell Differentiation; Cell Movement; Drosophila; Drosophila Proteins; Gene Expression Regulation, Developmental; Genes; Germ Cells; Gonads; Hydroxymethylglutaryl CoA Reductases; Membrane Proteins; Mice; Models, Biological; Phosphatidate Phosphatase; Phosphoric Monoester Hydrolases; Protein Prenylation; Receptors, G-Protein-Coupled; Zebrafish
PubMed: 15268881
DOI: 10.1016/j.cub.2004.07.018 -
Analytical Chemistry Aug 2022Protein prenylation is an essential post-translational modification that plays a key role in facilitating protein localization. Aberrations in protein prenylation have...
Protein prenylation is an essential post-translational modification that plays a key role in facilitating protein localization. Aberrations in protein prenylation have been indicated in multiple disease pathologies including progeria, some forms of cancer, and Alzheimer's disease. While there are single-cell methods to study prenylation, these methods cannot simultaneously assess prenylation and other cellular changes in the complex cell environment. Here, we report a novel method to monitor, at the single-cell level, prenylation and expression of autophagy markers. An isoprenoid analogue containing a terminal alkyne, substrate of prenylation enzymes, was metabolically incorporated into cells in culture. Treatment with a terbium reporter containing an azide functional group, followed by copper-catalyzed azide-alkyne cycloaddition, covalently attached terbium ions to prenylated proteins within cells. In addition, simultaneous treatment with a holmium-containing analogue of the reporter, without an azide functional group, was used to correct for non-specific retention at the single-cell level. This procedure was compatible with other mass cytometric sample preparation steps that use metal-tagged antibodies. We demonstrate that this method reports changes in levels of prenylation in competitive and inhibitor assays, while tracking autophagy molecular markers with metal-tagged antibodies. The method reported here makes it possible to track prenylation along with other molecular pathways in single cells of complex systems, which is essential to elucidate the role of this post-translational modification in disease, cell response to pharmacological treatments, and aging.
Topics: Alkynes; Antibodies; Azides; Biomarkers; Protein Prenylation; Terbium; Terpenes
PubMed: 35952372
DOI: 10.1021/acs.analchem.2c01509 -
Biochemistry Jul 2018Protein prenylation involves the attachment of a hydrophobic isoprenoid moiety to the C-terminus of proteins. Several small GTPases, including members of the Ras and Rho...
Protein prenylation involves the attachment of a hydrophobic isoprenoid moiety to the C-terminus of proteins. Several small GTPases, including members of the Ras and Rho subfamilies, require prenylation for their normal and pathological functions. Recent work has suggested that SmgGDS proteins regulate the prenylation of small GTPases in vivo. Using RhoA as a representative small GTPase, we directly test this hypothesis using biochemical assays and present a mechanism describing the mode of prenylation regulation. SmgGDS-607 completely inhibits RhoA prenylation catalyzed by protein geranylgeranyltransferase I (GGTase-I) in an in vitro radiolabel incorporation assay. SmgGDS-607 inhibits prenylation by binding to and blocking access to the C-terminal tail of the small GTPase (substrate sequestration mechanism) rather than via inhibition of the prenyltransferase activity. The reactivity of GGTase-I with RhoA is unaffected by addition of nucleotides. In contrast, the affinity of SmgGDS-607 for RhoA varies with the nucleotide bound to RhoA; SmgGDS-607 has a higher affinity for RhoA-GDP compared to RhoA-GTP. Consequently, the prenylation blocking function of SmgGDS-607 is regulated by the bound nucleotide. This work provides mechanistic insight into a novel pathway for the regulation of small GTPase protein prenylation by SmgGDS-607 and demonstrates that peptides are a good mimic for full-length proteins when measuring GGTase-I activity.
Topics: Alkyl and Aryl Transferases; Guanine Nucleotide Exchange Factors; Guanosine Diphosphate; Guanosine Triphosphate; Humans; Protein Binding; Protein Prenylation; rhoA GTP-Binding Protein
PubMed: 29940100
DOI: 10.1021/acs.biochem.8b00567 -
Molecules (Basel, Switzerland) May 2017The enzyme geranylgeranyl diphosphate synthase (GGDPS) catalyzes the synthesis of the 20-carbon isoprenoid geranylgeranyl diphosphate (GGPP). GGPP is the isoprenoid... (Review)
Review
The enzyme geranylgeranyl diphosphate synthase (GGDPS) catalyzes the synthesis of the 20-carbon isoprenoid geranylgeranyl diphosphate (GGPP). GGPP is the isoprenoid donor for protein geranylgeranylation reactions catalyzed by the enzymes geranylgeranyl transferase (GGTase) I and II. Inhibitors of GGDPS result in diminution of protein geranylgeranylation through depletion of cellular GGPP levels, and there has been interest in GGDPS inhibitors as potential anti-cancer agents. Here we discuss recent advances in the development of GGDPS inhibitors, including insights gained by structure-function relationships, and review the preclinical data that support the continued development of this novel class of drugs.
Topics: Animals; Enzyme Inhibitors; Farnesyltranstransferase; Humans; Protein Prenylation; Structure-Activity Relationship; Terpenes
PubMed: 28555000
DOI: 10.3390/molecules22060886 -
The Plant Cell Apr 1993Many mammalian and yeast proteins, including small ras-like GTP binding proteins, heterotrimeric G protein gamma subunits, and nuclear lamins, have been shown to be...
Many mammalian and yeast proteins, including small ras-like GTP binding proteins, heterotrimeric G protein gamma subunits, and nuclear lamins, have been shown to be covalently linked to isoprenoid derivatives of mevalonic acid. Isoprenylation of these proteins is required for their assembly into membranes and, hence, for their biological activity. In this report, it is shown that cultured tobacco cells, when pretreated with an inhibitor of endogenous mevalonic acid synthesis (lovastatin), incorporate radioactivity from 14C-mevalonic acid into proteins. Most of these proteins are membrane associated, and many are similar in mass to mammalian ras-like GTP binding proteins and nuclear lamins. Furthermore, it is shown that tobacco cell extracts catalyze the transfer of radioactivity from 3H-farnesyl pyrophosphate and 3H-geranylgeranyl pyrophosphate to protein substrates in vitro. These studies indicate the presence of at least two distinct prenyl:protein transferases in tobacco extracts: one that utilizes farnesyl pyrophosphate and preferentially modifies a substrate protein with a CAIM carboxy terminus (farnesyl:protein transferase) and one that utilizes geranylgeranyl pyrophosphate and preferentially modifies a substrate protein with a CAIL carboxy terminus (geranylgeranyl:protein transferase type I). This work provides a basis for future work on the role of protein isoprenylation in plant cell growth, signal transduction, and membrane biogenesis.
Topics: Alkyl and Aryl Transferases; Cells, Cultured; Electrophoresis, Polyacrylamide Gel; Kinetics; Mevalonic Acid; Plant Proteins; Plants, Toxic; Protein Prenylation; Protein Processing, Post-Translational; Nicotiana; Transferases
PubMed: 8485402
DOI: 10.1105/tpc.5.4.433