-
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 -
CaaX-motif-adjacent residues influence G protein gamma (Gγ) prenylation under suboptimal conditions.The Journal of Biological Chemistry Nov 2023Prenylation is an irreversible post-translational modification that supports membrane interactions of proteins involved in various cellular processes, including...
Prenylation is an irreversible post-translational modification that supports membrane interactions of proteins involved in various cellular processes, including migration, proliferation, and survival. Dysregulation of prenylation contributes to multiple disorders, including cancers and vascular and neurodegenerative diseases. Prenyltransferases tether isoprenoid lipids to proteins via a thioether linkage during prenylation. Pharmacological inhibition of the lipid synthesis pathway by statins is a therapeutic approach to control hyperlipidemia. Building on our previous finding that statins inhibit membrane association of G protein γ (Gγ) in a subtype-dependent manner, we investigated the molecular reasoning for this differential inhibition. We examined the prenylation of carboxy-terminus (Ct) mutated Gγ in cells exposed to Fluvastatin and prenyl transferase inhibitors and monitored the subcellular localization of fluorescently tagged Gγ subunits and their mutants using live-cell confocal imaging. Reversible optogenetic unmasking-masking of Ct residues was used to probe their contribution to prenylation and membrane interactions of the prenylated proteins. Our findings suggest that specific Ct residues regulate membrane interactions of the Gγ polypeptide, statin sensitivity, and extent of prenylation. Our results also show a few hydrophobic and charged residues at the Ct are crucial determinants of a protein's prenylation ability, especially under suboptimal conditions. Given the cell and tissue-specific expression of different Gγ subtypes, our findings indicate a plausible mechanism allowing for statins to differentially perturb heterotrimeric G protein signaling in cells depending on their Gγ-subtype composition. Our results may also provide molecular reasoning for repurposing statins as Ras oncogene inhibitors and the failure of using prenyltransferase inhibitors in cancer treatment.
Topics: Humans; Amino Acid Motifs; Drug Resistance; HeLa Cells; Heterotrimeric GTP-Binding Proteins; Hydroxymethylglutaryl-CoA Reductase Inhibitors; Models, Molecular; Mutation; Protein Prenylation; Protein Structure, Tertiary; Protein Transport; Signal Transduction
PubMed: 37739036
DOI: 10.1016/j.jbc.2023.105269 -
Hepatology (Baltimore, Md.) Apr 2018
Topics: Hepatitis Delta Virus; Piperidines; Prenylation; Pyridines
PubMed: 29365356
DOI: 10.1002/hep.29807 -
The Journal of Pathology Mar 2022Investigations of major mevalonate pathway enzymes have demonstrated the importance of local isoprenoid synthesis in cardiac homeostasis. Farnesyl diphosphate synthase...
Investigations of major mevalonate pathway enzymes have demonstrated the importance of local isoprenoid synthesis in cardiac homeostasis. Farnesyl diphosphate synthase (FPPS) synthesizes isoprenoid precursors needed for cholesterol biosynthesis and protein prenylation. Wang, Zhang, Chen et al, in a recently published article in The Journal of Pathology, elegantly elucidated the pathological outcomes of FPPS deficiency in cardiomyocytes, which paradoxically resulted in increased prenylation of the small GTPases Ras and Rheb. Cardiomyocyte FPPS depletion caused severe dilated cardiomyopathy that was associated with enhanced GTP-loading and abundance of Ras and Rheb in lipidated protein-enriched cardiac fractions and robust activation of downstream hypertrophic ERK1/2 and mTOR signaling pathways. Cardiomyopathy and activation of ERK1/2 and mTOR caused by loss of FPPS were ameliorated by inhibition of farnesyltransferase, suggesting that impairment of FPPS activity results in promiscuous activation of Ras and Rheb through non-canonical actions of farnesyltransferase. Here, we discuss the findings and adaptive signaling mechanisms in response to disruption of local cardiomyocyte mevalonate pathway activity, highlighting how alteration in a key branch point in the mevalonate pathway affects cardiac biology and function and perturbs protein prenylation, which might unveil novel strategies and intricacies of targeting the mevalonate pathway to treat cardiovascular diseases. © 2021 The Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.
Topics: Heart Failure; Humans; Mevalonic Acid; Monomeric GTP-Binding Proteins; Myocytes, Cardiac; Prenylation; Protein Prenylation
PubMed: 34783037
DOI: 10.1002/path.5837 -
Molecular and Biochemical Parasitology Jul 1997Prenyl modification of proteins by farnesyl and geranylgeranyl isoprenoids occurs in a variety of eukaryotic cells. Culturing of Trypanosoma brucei in the presence of...
Prenyl modification of proteins by farnesyl and geranylgeranyl isoprenoids occurs in a variety of eukaryotic cells. Culturing of Trypanosoma brucei in the presence of [3H]mevalonolactone (which is hydrolyzed in cells to give mevalonic acid, the precursor of protein prenyl groups) and an inhibitor of mevalonic acid biosynthesis leads to the radiolabeling of a specific set of proteins when analyzed by gel electrophoresis. T. brucei proteins were also labeled when cells were cultured in the presence of [3H]farnesol or [3H]geranylgeraniol, and each prenol labels a distinct set of proteins. Unlike mammalian cells, only a few T. brucei proteins of molecular weights similar to those of the mammalian Ras superfamily of GTPase (20-30 kDa) were labeled with [3H]farnesol or [3H]geranylgeraniol. When the 0-55% ammonium sulfate fraction of T. brucei cytosol was fractionated on anion exchange chromatography, protein farnesyltransferase (PFT) and protein geranylgeranyltransferase-I (PGGT-I) activities were detected and elute as two distinct peaks. Partially purified T. brucei PFT and PGGT-I display partly different specificities toward prenyl acceptor substrates from those of mammalian protein prenyltransferases. As shown previously, rat PFT utilizes proteins ending in CVLS and CVIM as efficient prenyl acceptors and rat PGGT-I utilizes proteins ending in CVLL and CVIM in vitro. On the contrary, T. brucei PFT farnesylates a protein ending in CVIM but not CVLS or CVLL, and T. brucei PGGT-I preferentially geranylgeranylates a protein ending in CVLL.
Topics: Alkyl and Aryl Transferases; Animals; Cells, Cultured; Farnesyltranstransferase; Gene Expression Regulation; Protein Prenylation; Protein Processing, Post-Translational; Protozoan Proteins; Substrate Specificity; Transferases; Trypanosoma brucei brucei
PubMed: 9233673
DOI: 10.1016/s0166-6851(97)00043-1 -
Journal of Natural Products Aug 2017Isoprene units derived from dimethylallyl diphosphate (DMAPP) are an important motif in many natural products including terpenoids, carotenoids, steroids, and natural...
Isoprene units derived from dimethylallyl diphosphate (DMAPP) are an important motif in many natural products including terpenoids, carotenoids, steroids, and natural rubber. Understanding the chemical characteristics of DMAPP is an important topic in natural products chemistry, organic chemistry, and biochemistry. We have developed a direct bioinspired indole prenylation reaction using DMAPP or its equivalents as the electrophile in homogeneous aqueous acidic media in the absence of enzyme to provide prenylated indole products. After establishing the bioinspired indole prenylation reaction, this was then used to achieve the synthesis of a series of natural products, namely, N-prenylcyclo-l-tryptophyl-l-proline, tryprostatins, rhinocladins, and terezine D.
Topics: Biological Products; Butadienes; Dipeptides; Hemiterpenes; Indoles; Molecular Structure; Organophosphorus Compounds; Pentanes; Prenylation; Proline; Terpenes; Water
PubMed: 28803474
DOI: 10.1021/acs.jnatprod.7b00464 -
ACS Synthetic Biology Jan 2023Reversible membrane targeting of proteins is one of the key regulators of cellular interaction networks, for example, for signaling and polarization. So-called "membrane...
Reversible membrane targeting of proteins is one of the key regulators of cellular interaction networks, for example, for signaling and polarization. So-called "membrane switches" are thus highly attractive targets for the design of minimal cells but have so far been tricky to reconstitute in vitro. Here, we introduce cell-free prenylated protein synthesis (CFpPS), which enables the synthesis and membrane targeting of proteins in a single reaction mix including the prenylation machinery. CFpPS can confer membrane affinity to any protein via addition of a 4-peptide motif to its C-terminus and offers robust production of prenylated proteins not only in their soluble forms but also in the direct vicinity of biomimetic membranes. Thus, CFpPS enabled us to reconstitute the prenylated polarity hub Cdc42 and its regulatory protein in vitro, implementing a key membrane switch. We propose CFpPS to be a versatile and effective platform for engineering complex features, such as polarity induction, in synthetic cells.
Topics: Protein Prenylation; Peptides; Transcription Factors
PubMed: 36445320
DOI: 10.1021/acssynbio.2c00406 -
Natural Product Reports Apr 2000
Review
Topics: Enzymes; Kinetics; Mutagenesis, Site-Directed; Protein Binding; Protein Prenylation; Protein Processing, Post-Translational; Substrate Specificity
PubMed: 10821108
DOI: 10.1039/a904110i -
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 -
Development (Cambridge, England) Jun 2022A complete picture of how signaling pathways lead to multicellularity is largely unknown. Previously, we generated mutations in a protein prenylation enzyme, GGB, and...
A complete picture of how signaling pathways lead to multicellularity is largely unknown. Previously, we generated mutations in a protein prenylation enzyme, GGB, and showed that it is essential for maintaining multicellularity in the moss Physcomitrium patens. Here, we show that ROP GTPases act as downstream factors that are prenylated by GGB and themselves play an important role in the multicellularity of P. patens. We also show that the loss of multicellularity caused by the suppression of GGB or ROP GTPases is due to uncoordinated cell expansion, defects in cell wall integrity and the disturbance of the directional control of cell plate orientation. Expressing prenylatable ROP in the ggb mutant not only rescues multicellularity in protonemata but also results in development of gametophores. Although the prenylation of ROP is important for multicellularity, a higher threshold of active ROP is required for gametophore development. Thus, our results suggest that ROP activation via prenylation by GGB is a key process at both cell and tissue levels, facilitating the developmental transition from one dimension to two dimensions and to three dimensions in P. patens.
Topics: Bryopsida; Cell Wall; GTP Phosphohydrolases; Prenylation; Signal Transduction
PubMed: 35660859
DOI: 10.1242/dev.200279