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Biochemistry Jul 2019Indole prenyltransferases catalyze the prenylation of l-tryptophan (l-Trp) and other indoles to produce a diverse set of natural products in bacteria, fungi, and plants,...
Indole prenyltransferases catalyze the prenylation of l-tryptophan (l-Trp) and other indoles to produce a diverse set of natural products in bacteria, fungi, and plants, many of which possess useful biological properties. Among this family of enzymes, CymD from catalyzes the reverse N1 prenylation of l-Trp, an unusual reaction given the poor nucleophilicity of the indole nitrogen. CymD utilizes dimethylallyl diphosphate (DMAPP) as the prenyl donor, catalyzing the dissociation of the diphosphate leaving group followed by nucleophilic attack of the indole nitrogen at the tertiary carbon of the dimethylallyl cation. To better understand the structural basis of selective indole N-alkylation reactions in biology, we have determined the X-ray crystal structures of CymD, the CymD-l-Trp complex, and the CymD-l-Trp-DMSPP complex (DMSPP is dimethylallyl -thiolodiphosphate, an unreactive analogue of DMAPP). The orientation of l-Trp with respect to DMSPP reveals how the active site contour of CymD serves as a template to direct the reverse prenylation of the indole nitrogen. Comparison to PriB, a C6 bacterial indole prenyltransferase, offers further insight regarding the structural basis of regioselective indole prenylation. Isothermal titration calorimetry measurements indicate a synergistic relationship between l-Trp and DMSPP binding. Finally, activity assays demonstrate the selectivity of CymD for l-Trp and indole as prenyl acceptors. Collectively, these data establish a foundation for understanding and engineering the regioselectivity of indole prenylation by members of the prenyltransferase protein family.
Topics: Catalysis; Dimethylallyltranstransferase; Micromonosporaceae; Protein Prenylation; Protein Structure, Secondary; Protein Structure, Tertiary; Tryptophan; X-Ray Diffraction
PubMed: 31251043
DOI: 10.1021/acs.biochem.9b00399 -
Biochemical and Biophysical Research... Oct 2014Protein prenylation is a post-translational modification where farnesyl or geranylgeranyl groups are enzymatically attached to a C-terminal cysteine residue. This...
Protein prenylation is a post-translational modification where farnesyl or geranylgeranyl groups are enzymatically attached to a C-terminal cysteine residue. This modification is essential for the activity of small cellular GTPases, as it allows them to associate with intracellular membranes. Dissociated from membranes, prenylated proteins need to be transported through the aqueous cytoplasm by protein carriers that shield the hydrophobic anchor from the solvent. One such carrier is Rho GDP dissociation inhibitor (RhoGDI). Recently, it was shown that prenylated Rho proteins that are not associated with RhoGDI are subjected to proteolysis in the cell. We hypothesized that the role of RhoGDI might be not only to associate with prenylated proteins but also to regulate the prenylation process in the cell. This idea is supported by the fact that RhoGDI binds both unprenylated and prenylated Rho proteins with high affinity in vitro, and hence, these interactions may affect the kinetics of prenylation. We addressed this question experimentally and found that RhoGDI increased the catalytic efficiency of geranylgeranyl transferase-I in RhoA prenylation. Nevertheless, we did not observe formation of a ternary RhoGDI∗RhoA∗GGTase-I complex, indicating sequential operation of geranylgeranyltransferase-I and RhoGDI. Our results suggest that RhoGDI accelerates Rho prenylation by kinetically trapping the reaction product, thereby increasing the rate of product release.
Topics: Alkyl and Aryl Transferases; Animals; Catalysis; Cell Line; Cricetinae; Fibroblasts; Protein Prenylation; rho-Specific Guanine Nucleotide Dissociation Inhibitors; rhoA GTP-Binding Protein
PubMed: 25223799
DOI: 10.1016/j.bbrc.2014.09.024 -
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 -
Current Protocols in Protein Science May 2001This unit describes methods for analysis of prenylation and the carboxyl-methylation that often accompanies it. The two prenoid groups that have been found attached to...
This unit describes methods for analysis of prenylation and the carboxyl-methylation that often accompanies it. The two prenoid groups that have been found attached to proteins--farnesyl (C15) and geranylgeranyl (C20)--are both derived from intermediates in the isoprenoid biosynthetic pathway that utilizes mevalonic acid. In the protocols described here, radiolabeled mevalonate is used to label these intermediates in either intact cells or in vitro; the labeled intermediates then become incorporated into proteins. Alternatively, the preformed radioactive prenyl diphosphates can be used for in vitro translations, as described here. Carboxyl-methylation of C-terminal prenylated cysteine residues often occurs subsequent to prenylation. Methods are given for radiolabeling of the methyl group with [(3)H-methyl]methionine, that is converted intracellularly into S-adenosylmethionine, and for radiolabeling with preformed S-adenosyl[(3)H]methionine.
Topics: Animals; Biochemistry; Cells, Cultured; Methylation; Protein Biosynthesis; Protein Prenylation; Proteins; Rabbits
PubMed: 18429123
DOI: 10.1002/0471140864.ps1403s05 -
Biochimica Et Biophysica Acta Aug 2010The mevalonate pathway is a crucial metabolic pathway for most eukaryotic cells. Cholesterol is a highly recognized product of this pathway but growing interest is being... (Review)
Review
The mevalonate pathway is a crucial metabolic pathway for most eukaryotic cells. Cholesterol is a highly recognized product of this pathway but growing interest is being given to the synthesis and functions of isoprenoids. Isoprenoids are a complex class of biologically active lipids including for example, dolichol, ubiquinone, farnesylpyrophosphate (FPP) and geranylgeranyl pyrophosphate (GGPP). Early work had shown that the long-chain isoprenoid dolichol is decreased but that dolichyl phosphate and ubiquinone are elevated in brains of Alzheimer's disease (AD) patients. Until recently, levels of their biological active precursors FPP and GGPP were unknown. These short-chain isoprenoids are critical in the post-translational modification of certain proteins which function as molecular switches in numerous signaling pathways. The major protein families belong to the superfamily of small GTPases, consisting of roughly 150 members. Recent experimental evidence indicated that members of the small GTPases are involved in AD pathogenesis and stimulated interest in the role of FPP and GGPP in protein prenylation and cell function. A straightforward prediction derived from those studies was that FPP and GGPP levels would be elevated in AD brains as compared with normal neurological controls. For the first time, recent evidence shows significantly elevated levels of FPP and GGPP in human AD brain tissue. Cholesterol levels did not differ between AD and control samples. One obvious conclusion is that homeostasis of FPP and GGPP but not of cholesterol is specifically targeted in AD. Since prenylation of small GTPases by FPP or GGPP is indispensable for their proper function we are proposing that these two isoprenoids are up-regulated in AD resulting in an over abundance of certain prenylated proteins which contributes to neuronal dysfunction.
Topics: Aging; Alzheimer Disease; Animals; Cholesterol; Humans; Metabolic Networks and Pathways; Mevalonic Acid; Models, Biological; Monomeric GTP-Binding Proteins; Nerve Degeneration; Protein Prenylation; Terpenes
PubMed: 20382260
DOI: 10.1016/j.bbalip.2010.03.014 -
ACS Applied Bio Materials May 2022Despite broad interest in understanding the biological implications of protein farnesylation in regulating different facets of cell biology, the use of this... (Review)
Review
Despite broad interest in understanding the biological implications of protein farnesylation in regulating different facets of cell biology, the use of this post-translational modification to develop protein-based materials and therapies remains underexplored. The progress has been slow due to the lack of accessible methodologies to generate farnesylated proteins with broad physicochemical diversities rapidly. This limitation, in turn, has hindered the empirical elucidation of farnesylated proteins' sequence-structure-function rules. To address this gap, we genetically engineered prokaryotes to develop operationally simple, high-yield biosynthetic routes to produce farnesylated proteins and revealed determinants of their emergent material properties (nano-aggregation and phase-behavior) using scattering, calorimetry, and microscopy. These outcomes foster the development of farnesylated proteins as recombinant therapeutics or biomaterials with molecularly programmable assembly.
Topics: Biocompatible Materials; Genetic Engineering; Protein Prenylation; Proteins; Temperature
PubMed: 35044146
DOI: 10.1021/acsabm.1c01162 -
Advances in Immunology 1998
Review
Topics: Animals; GTP Phosphohydrolases; Humans; Immunity; Protein Prenylation; ras Proteins
PubMed: 9505089
DOI: 10.1016/s0065-2776(08)60559-3 -
Current Topics in Medicinal Chemistry 2003R115777 (R)-6-amino[(4-chlorophenyl)(1-methyl-1H-imidazol-5-yl)methyl]-4-(3-chlorophenyl)-1-methyl-2(1H)-quinolinone is a potent and selective inhibitor of farnesyl... (Review)
Review
R115777 (R)-6-amino[(4-chlorophenyl)(1-methyl-1H-imidazol-5-yl)methyl]-4-(3-chlorophenyl)-1-methyl-2(1H)-quinolinone is a potent and selective inhibitor of farnesyl protein transferase with significant antitumor effects in vivo subsequent to oral administration in mice. Taking its roots into Janssen's ketoconazole and retinoic acid catabolism programs, our interest into Ras prenylation process led us stepwise to identify the key structural features of R115777. Methodology, structure activity relationships, and pharmacology will be presented. R115777 is currently in phase III clinical evaluation.
Topics: Alkyl and Aryl Transferases; Animals; Antineoplastic Agents; Clinical Trials as Topic; Enzyme Inhibitors; Humans; Mice; Molecular Structure; Neoplasms; Protein Prenylation; Quinolones; Structure-Activity Relationship
PubMed: 12769710
DOI: 10.2174/1568026033452050 -
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 -
Chemical Biology & Drug Design Dec 2010Protein prenyltransferases catalyze the attachment of C15 (farnesyl) and C20 (geranylgeranyl) groups to proteins at specific sequences localized at or near the C-termini...
Protein prenyltransferases catalyze the attachment of C15 (farnesyl) and C20 (geranylgeranyl) groups to proteins at specific sequences localized at or near the C-termini of specific proteins. Determination of the specific protein prenyltransferase substrates affected by the inhibition of these enzymes is critical for enhancing knowledge of the mechanism of such potential drugs. Here, we investigate the utility of alkyne-containing isoprenoid analogs for chemical proteomics experiments by showing that these compounds readily penetrate mammalian cells in culture and become incorporated into proteins that are normally prenylated. Derivatization via Cu(I) catalyzed click reaction with a fluorescent azide reagent allows the proteins to be visualized and their relative levels to be analyzed. Simultaneous treatment of cells with these probes and inhibitors of prenylation reveals decreases in the levels of some but not all of the labeled proteins. Two-dimensional electrophoretic separation of these labeled proteins followed by mass spectrometric analysis allowed several labeled proteins to be unambiguously identified. Docking experiments and density functional theory calculations suggest that the substrate specificity of protein farnesyl transferase may vary depending on whether azide- or alkyne-based isoprenoid analogs is employed. These results demonstrate the utility of alkyne-containing analogs for chemical proteomic applications.
Topics: Alkynes; Animals; Azides; Biomarkers; Catalytic Domain; Cell Line; Electrophoresis, Gel, Two-Dimensional; HeLa Cells; Humans; Protein Prenylation; Proteomics; Quantum Theory; Substrate Specificity; Terpenes
PubMed: 21040496
DOI: 10.1111/j.1747-0285.2010.01037.x