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Accounts of Chemical Research Jul 2017Ribosomally synthesized and Post-translationally modified Peptides (RiPPs) take advantage of the ribosomal translation machinery to generate linear peptides that are...
Ribosomally synthesized and Post-translationally modified Peptides (RiPPs) take advantage of the ribosomal translation machinery to generate linear peptides that are subsequently modified with heterocycles and/or macrocycles to impose three-dimensional structure and thwart degradation by proteases. Although RiPP precursors are limited to proteinogenic amino acids, post-translational modifications (PTMs) can alter the structure of individual amino acids and thereby improve the stability and biological activity of the molecule. These "tailoring modifications" often occur on amino acid side chains-for example, hydroxylation, methylation, halogenation, prenylation, and acylation-but can also take place within the backbone, as in epimerization, or can result in capping of the N- or C-terminus. At one extreme, these modifications can be essential to the activity of the RiPP, either as a compulsory step in reaching the final molecule or by imparting chemical functionality required for biological activity. At the other extreme, tailoring PTMs may have little effect on the activity in an in vitro setting-possibly because of test conditions that do not match the biological context in which the PTMs evolved. Establishing the molecular basis for the function of tailoring PTMs often requires a three-dimensional structure of the RiPP bound to its biological target. These structures have revealed roles for tailoring PTMs that include providing additional hydrogen bonds to targets, rigidifying the RiPP structure to reduce the entropic cost of binding, or altering the secondary structure of the peptide backbone. Bacterial RiPPs are particularly suited to structural characterization, as they are relatively easy to isolate from laboratory cultures or to produce in a heterologous host. The identification of new tailoring PTMs within bacteria is also facilitated by clustering of the genes encoding tailoring enzymes with those of the RiPP precursor and primary modification enzymes. In this Account, we describe the effects of tailoring PTMs on RiPP structure, their interactions with biological targets, and their influence on RiPP stability, with a focus on bacterial RiPP classes. We also discuss the enzymes that generate tailoring PTMs and highlight examples of and prospects for engineering of RiPPs.
Topics: Acylation; Biological Products; Halogens; Hydroxylation; Methylation; Peptides; Prenylation; Protein Conformation; Protein Stability; Ribosomes
PubMed: 28682627
DOI: 10.1021/acs.accounts.7b00175 -
Plant Physiology Feb 2011Prenylation primarily by geranylgeranylation is required for membrane attachment and function of type I Rho of Plants (ROPs) and Gγ proteins, while type II ROPs are...
Prenylation primarily by geranylgeranylation is required for membrane attachment and function of type I Rho of Plants (ROPs) and Gγ proteins, while type II ROPs are attached to the plasma membrane by S-acylation. Yet, it is not known how prenylation affects ROP membrane interaction dynamics and what are the functional redundancy and specificity of type I and type II ROPs. Here, we have used the expression of ROPs in mammalian cells together with geranylgeranylation and CaaX prenylation-deficient mutants to answer these questions. Our results show that the mechanism of type II ROP S-acylation and membrane attachment is unique to plants and likely responsible for the viability of plants in the absence of CaaX prenylation activity. The prenylation of ROPs determines their steady-state distribution between the plasma membrane and the cytosol but has little effect on membrane interaction dynamics. In addition, the prenyl group type has only minor effects on ROP function. Phenotypic analysis of the CaaX prenylation-deficient pluripetala mutant epidermal cells revealed that type I ROPs affect cell structure primarily on the adaxial side, while type II ROPs are functional and induce a novel cell division phenotype in this genetic background. Taken together, our studies show how prenyl and S-acyl lipid modifications affect ROP subcellular distribution, membrane interaction dynamics, and function.
Topics: Acylation; Animals; Arabidopsis; Arabidopsis Proteins; Cell Line; Cell Membrane; Cloning, Molecular; Escherichia coli; Gas Chromatography-Mass Spectrometry; Insecta; Membrane Proteins; Mice; Monomeric GTP-Binding Proteins; Mutation; NIH 3T3 Cells; Phenotype; Plant Epidermis; Protein Prenylation
PubMed: 21139084
DOI: 10.1104/pp.110.166850 -
Translational Vision Science &... Jul 2021Choroideremia results from the deficiency of Rab Escort Protein 1 (REP1), encoded by CHM, involved in the prenylation of Rab GTPases. Here, we investigate whether the...
PURPOSE
Choroideremia results from the deficiency of Rab Escort Protein 1 (REP1), encoded by CHM, involved in the prenylation of Rab GTPases. Here, we investigate whether the transcription and expression of other genes involved in the prenylation of Rab proteins correlates with disease progression in a cohort of patients with choroideremia.
METHODS
Rates of retinal pigment epithelial area loss in 41 patients with choroideremia were measured using fundus autofluorescence imaging for up to 4 years. From lysates of cultured skin fibroblasts donated by patients (n = 15) and controls (n = 14), CHM, CHML, RABGGTB and RAB27A mRNA expression, and REP1 and REP2 protein expression were compared.
RESULTS
The central autofluorescent island area loss in patients with choroideremia occurred with a mean half-life of 5.89 years (95% confidence interval [CI] = 5.09-6.70), with some patients demonstrating relatively fast or slow rates of progression (range = 3.3-14.1 years). Expression of CHM mRNA and REP1 protein were significantly decreased in all patients. No difference in expression of CHML, RABGGTB, RAB27A, or REP2 was seen between patients and controls. No correlation was seen between expression of the genes analyzed and rates of retinal degeneration. Non-sense induced transcriptional compensation of CHML, a CHM-like retrogene, was not observed in patients with CHM variants predicted to undergo non-sense mediated decay.
CONCLUSIONS
Patients with choroideremia, who are deficient for REP1, show normal levels of expression of other genes involved in Rab prenylation, which do not appear to play any modifying role in the rate of disease progression.
TRANSLATIONAL RELEVANCE
There remains little evidence for selection of patients for choroideremia gene therapy based on genotype.
Topics: Adaptor Proteins, Signal Transducing; Choroideremia; Disease Progression; Humans; Prenylation; Retinal Degeneration
PubMed: 34254989
DOI: 10.1167/tvst.10.8.12 -
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 -
Cell Death & Disease Nov 2022Cutaneous melanoma is one of the most aggressive and lethal forms of skin cancer. Some specific driver mutations have been described in multiple oncogenes including BRAF...
Cutaneous melanoma is one of the most aggressive and lethal forms of skin cancer. Some specific driver mutations have been described in multiple oncogenes including BRAF and NRAS that are mutated in 60-70% and 15-20% of melanoma, respectively. The aim of this study was to evaluate the role of Small Heat Shock Protein B8 (HSPB8) on cell growth and migration of both BLM (BRAF/NRAS) and A375 (BRAF/NRAS) human melanoma cell lines. HSPB8 is a member of the HSPB family of chaperones involved in protein quality control (PQC) system and contributes to chaperone assisted selective autophagy (CASA) as well as in the regulation of mitotic spindle. In cancer, HSPB8 has anti- or pro-tumoral action depending on tumor type. In melanoma cell lines characterized by low HSPB8 levels, we demonstrated that the restoration of HSPB8 expression causes cell growth arrest, reversion of EMT (Epithelial-Mesenchymal Transition)-like phenotype switching and antimigratory effect, independently from the cell mutational status. We demonstrated that HSPB8 regulates the levels of the active prenylated form of NRAS in NRAS-mutant and NRAS-wild-type melanoma cell lines. Consequently, the inhibition of NRAS impairs the activation of Akt/mTOR pathway inducing autophagy activation. Autophagy can play a dual role in regulating cell death and survival. We have therefore demonstrated that HSPB8-induced autophagy is a crucial event that counteracts cell growth in melanoma. Collectively, our results suggest that HSPB8 has an antitumoral action in melanoma cells characterized by BRAF and NRAS mutations.
Topics: Humans; Melanoma; Skin Neoplasms; Proto-Oncogene Proteins B-raf; GTP Phosphohydrolases; Heat-Shock Proteins; Membrane Proteins; Prenylation; Autophagy; Molecular Chaperones
PubMed: 36400750
DOI: 10.1038/s41419-022-05365-9 -
Characteristic metabolites of Hypericum plants: their chemical structures and biological activities.Journal of Natural Medicines Jun 2021Plants belonging to the genus Hypericum (Hypericaceae) are recognized as an abundant source of natural products with interesting chemical structures and intriguing... (Review)
Review
Plants belonging to the genus Hypericum (Hypericaceae) are recognized as an abundant source of natural products with interesting chemical structures and intriguing biological activities. In the course of our continuing study on constituents of Hypericum plants, aiming at searching natural product-based lead compounds for therapeutic agents, we have isolated more than 100 new characteristic metabolites classified as prenylated acylphloroglucinols, meroterpenes, ketides, dibenzo-1,4-dioxane derivatives, and xanthones including prenylated xanthones, phenylxanthones, and xanthonolignoids from 11 Hypericum plants and one Triadenum plant collected in Japan, China, and Uzbekistan or cultivated in Japan. This review summarizes their chemical structures and biological activities.
Topics: Biological Products; China; Hypericum; Japan; Molecular Structure; Phytochemicals; Prenylation; Uzbekistan
PubMed: 33555487
DOI: 10.1007/s11418-021-01489-y -
Phytochemistry 2009Prenylation plays a major role in the diversification of aromatic natural products, such as phenylpropanoids, flavonoids, and coumarins. This biosynthetic reaction... (Review)
Review
Prenylation plays a major role in the diversification of aromatic natural products, such as phenylpropanoids, flavonoids, and coumarins. This biosynthetic reaction represents the crucial coupling process of the shikimate or polyketide pathway providing an aromatic moiety and the isoprenoid pathway derived from the mevalonate or methyl erythritol phosphate (MEP) pathway, which provides the prenyl (isoprenoid) chain. In particular, prenylation contributes strongly to the diversification of flavonoids, due to differences in the prenylation position on the aromatic rings, various lengths of prenyl chain, and further modifications of the prenyl moiety, e.g., cyclization and hydroxylation, resulting in the occurrence of ca. 1000 prenylated flavonoids in plants. Many prenylated flavonoids have been identified as active components in medicinal plants with biological activities, such as anti-cancer, anti-androgen, anti-leishmania, and anti-nitric oxide production. Due to their beneficial effects on human health, prenylated flavonoids are of particular interest as lead compounds for producing drugs and functional foods. However, the gene coding for prenyltransferases that catalyze the key step of flavonoid prenylation have remained unidentified for more than three decades, because of the membrane-bound nature of these enzymes. Recently, we have succeeded in identifying the first prenyltransferase gene SfN8DT-1 from Sophora flavescens, which is responsible for the prenylation of the flavonoid naringenin at the 8-position, and is specific for flavanones and dimethylallyl diphosphate (DMAPP) as substrates. Phylogenetic analysis showed that SfN8DT-1 has the same evolutionary origin as prenyltransferases for vitamin E and plastoquinone. A prenyltransferase GmG4DT from soybean, which is involved in the formation of glyceollin, was also identified recently. This enzyme was specific for pterocarpan as its aromatic substrate, and (-)-glycinol was the native substrate yielding the direct precursor of glyceollin I. These enzymes are localized to plastids and the prenyl chain is derived from the MEP pathway. Further relevant genes involved in the prenylation of other types of polyphenol are expected to be cloned by utilizing the sequence information provided by the above studies.
Topics: Biological Products; Dimethylallyltranstransferase; Molecular Structure; Plants; Prenylation
PubMed: 19819506
DOI: 10.1016/j.phytochem.2009.08.023 -
Cell Communication and Signaling : CCS Aug 2022The CAAX-prenyltransferases farnesyltransferase (FTase) and geranylgeranyltransferase I (GGTase I) are heterodimers with a common α- (FTα) and unique β-subunits....
BACKGROUND
The CAAX-prenyltransferases farnesyltransferase (FTase) and geranylgeranyltransferase I (GGTase I) are heterodimers with a common α- (FTα) and unique β-subunits. Recently, α-subunits of species (e.g., human) that harbour an N-terminal proline-rich region (PRR) showed different dimerization behaviours than α-subunits without PRR (e.g., yeast). However, the specific function of the PRR has not been elucidated so far.
METHODS
To determine whether the PRR is a conserved motif throughout eukaryotes, we performed phylogenetics. Elucidating the impact of the PRR on enzyme properties, we cloned human as well as rat PRR deficient FTα, expressed them heterologously and compared protein-protein interaction by pull-down as well as crosslinking experiments. Substrate binding, enzyme activity and sensitivity towards common FTase inhibitors of full length and PRR-deletion α-subunits and their physiological partners was determined by continuous fluorescence assays.
RESULTS
The PRR is highly conserved in mammals, with an exception for marsupials harbouring a poly-alanine region instead. The PRR shows similarities to canonical SH3-binding domains and to profilin-binding domains. Independent of the PRR, the α-subunits were able to dimerize with the different physiological β-subunits in in vitro as well as in yeast two-hybrid experiments. FTase and GGTase I with truncated FTα were active. The K values for both substrates are in the single-digit µM range and show no significant differences between enzymes with full length and PRR deficient α-subunits within the species.
CONCLUSIONS
Our data demonstrate that an N-terminal PRR of FTα is highly conserved in mammals. We could show that the activity and inhibitability is not influenced by the truncation of the N-terminal region. Nevertheless, this region shows common binding motifs for other proteins involved in cell-signalling, trafficking and phosphorylation, suggesting that this PRR might have other or additional functions in mammals. Our results provide new starting points due to the relevant but only partly understood role of FTα in eukaryotic FTase and GGTase I. Video Abstract.
Topics: Animals; Dimethylallyltranstransferase; Humans; Mammals; Proline; Protein Prenylation; Rats; Saccharomyces cerevisiae; Substrate Specificity
PubMed: 35941619
DOI: 10.1186/s12964-022-00929-w -
Current Opinion in Chemical Biology Dec 2012Protein post-translational modifications increase the functional diversity of the proteome by covalently adding chemical moieties onto proteins thereby changing their... (Review)
Review
Protein post-translational modifications increase the functional diversity of the proteome by covalently adding chemical moieties onto proteins thereby changing their activation state, cellular localization, interacting partners, and life cycle. Lipidation is one such modification that enables membrane association of naturally cytosolic proteins. Protein prenyltransferases irreversibly install isoprenoid units of varying length via a thioether linkage onto proteins that exert their cellular activity at membranes. Substrates of prenyltransferases are involved in countless signaling pathways and processes within the cell. Identification of new prenylation substrates, prenylation pathway regulators, and dynamic trafficking of prenylated proteins are all avenues of intense, ongoing research that are challenging, exciting, and have the potential to significantly advance the field in the near future.
Topics: Animals; Bacterial Infections; Bacterial Physiological Phenomena; Dimethylallyltranstransferase; Host-Pathogen Interactions; Humans; Neoplasms; Protein Prenylation; Substrate Specificity
PubMed: 23141597
DOI: 10.1016/j.cbpa.2012.10.015 -
Biochemistry Sep 2022The regiospecific prenylation of an aromatic amino acid catalyzed by a dimethylallyl-l-tryptophan synthase (DMATS) is a key step in the biosynthesis of many fungal and...
The regiospecific prenylation of an aromatic amino acid catalyzed by a dimethylallyl-l-tryptophan synthase (DMATS) is a key step in the biosynthesis of many fungal and bacterial natural products. DMATS enzymes share a common "ABBA" fold with divergent active site contours that direct alternative C-C, C-N, and C-O bond-forming trajectories. DMATS1 from catalyzes the reverse N-prenylation of l-Trp by generating an allylic carbocation from dimethylallyl diphosphate (DMAPP) that then alkylates the indole nitrogen of l-Trp. DMATS1 stands out among the greater DMATS family because it exhibits unusually broad substrate specificity: it can utilize geranyl diphosphate (GPP) or l-Tyr as an alternative prenyl donor or acceptor, respectively; it can catalyze both forward and reverse prenylation, i.e., at C1 or C3 of DMAPP; and it can catalyze C-N and C-O bond-forming reactions. Here, we report the crystal structures of DMATS1 and its complexes with l-Trp or l-Tyr and unreactive thiolodiphosphate analogues of the prenyl donors DMAPP and GPP. Structures of ternary complexes mimic Michaelis complexes with actual substrates and illuminate active site features that govern prenylation regiochemistry. Comparison with CymD, a bacterial enzyme that catalyzes the reverse N-prenylation of l-Trp with DMAPP, indicates that bacterial and fungal DMATS enzymes share a conserved reaction mechanism. However, the narrower active site contour of CymD enforces narrower substrate specificity. Structure-function relationships established for DMATS enzymes will ultimately inform protein engineering experiments that will broaden the utility of these enzymes as useful tools for synthetic biology.
Topics: Biological Products; Catalysis; Dimethylallyltranstransferase; Fusarium; Hemiterpenes; Indoles; Neoprene; Nitrogen; Organophosphorus Compounds; Prenylation; Substrate Specificity; Tryptophan; Tryptophan Synthase
PubMed: 36084241
DOI: 10.1021/acs.biochem.2c00350