-
Plants (Basel, Switzerland) Apr 2024In plants, the plastidial mevalonate (MVA)-independent pathway is required for the modification with geranylgeranyl groups of CaaL-motif proteins, which are substrates...
In plants, the plastidial mevalonate (MVA)-independent pathway is required for the modification with geranylgeranyl groups of CaaL-motif proteins, which are substrates of protein geranylgeranyltransferase type-I (PGGT-I). As a consequence, fosmidomycin, a specific inhibitor of 1-deoxy-d-xylulose (DX)-5 phosphate reductoisomerase/DXR, the second enzyme in this so-called methylerythritol phosphate (MEP) pathway, also acts as an effective inhibitor of protein prenylation. This can be visualized in plant cells by confocal microscopy by expressing GFP-CaM-CVIL, a prenylation sensor protein. After treatment with fosmidomycin, the plasma membrane localization of this GFP-based sensor is altered, and a nuclear distribution of fluorescence is observed instead. In tobacco cells, a visual screen of conditions allowing membrane localization in the presence of fosmidomycin identified jasmonic acid methyl esther (MeJA) as a chemical capable of gradually overcoming inhibition. Using Arabidopsis protein prenyltransferase loss-of-function mutant lines expressing GFP-CaM-CVIL proteins, we demonstrated that in the presence of MeJA, protein farnesyltransferase (PFT) can modify the GFP-CaM-CVIL sensor, a substrate the enzyme does not recognize under standard conditions. Similar to MeJA, farnesol and MVA also alter the protein substrate specificity of PFT, whereas DX and geranylgeraniol have limited or no effect. Our data suggest that MeJA adjusts the protein substrate specificity of PFT by promoting a metabolic cross-talk directing the origin of the prenyl group used to modify the protein. MVA, or an MVA-derived metabolite, appears to be a key metabolic intermediate for this change in substrate specificity.
PubMed: 38674519
DOI: 10.3390/plants13081110 -
Journal of Natural Products May 2024Collembola are closely related to insects, but our knowledge of their often unique chemistry is limited. Here we report the identification of the epicuticular lipid...
Collembola are closely related to insects, but our knowledge of their often unique chemistry is limited. Here we report the identification of the epicuticular lipid nitidane, representing a novel class of epicuticular lipids. Nitidane () is an irregular terpene consisting of seven isoprene units, made up of a diterpene core that is modified by a geranyl moiety that is itself prenylated. The observed [4+(2+1)]-terpene structure has not been reported before.
Topics: Animals; Diterpenes; Molecular Structure; Prenylation; Lipids
PubMed: 38668715
DOI: 10.1021/acs.jnatprod.4c00258 -
BioRxiv : the Preprint Server For... Apr 2024E3-ubiquitin ligases (E3s) are main components of the ubiquitin-proteasome system (UPS), as they determine substrate specificity in response to internal and external...
E3-ubiquitin ligases (E3s) are main components of the ubiquitin-proteasome system (UPS), as they determine substrate specificity in response to internal and external cues to regulate protein homeostasis. However, the regulation of membrane protein ubiquitination by E3s within distinct cell membrane compartments or organelles is not well understood. We show that FBXO10, the interchangeable component of the SKP1/CUL1/F-box ubiquitin ligase complex (SCF-E3), undergoes lipid-modification with geranylgeranyl isoprenoid at Cysteine953 (C953), facilitating its dynamic trafficking to the outer mitochondrial membrane (OMM). FBXO10 polypeptide does not contain a canonical mitochondrial targeting sequence (MTS); instead, its geranylgeranylation at C953 and the interaction with two cytosolic factors, PDE6δ (a prenyl group-binding protein), and HSP90 (a mitochondrial chaperone) orchestrate specific OMM targeting of prenyl-FBXO10 across diverse membrane compartments. The geranylgeranylation-deficient FBXO10(C953S) mutant redistributes away from the OMM, leading to impaired mitochondrial ATP production, decreased mitochondrial membrane potential, and increased mitochondrial fragmentation. Phosphoglycerate mutase 5 (PGAM5) was identified as a potential substrate of FBXO10 at the OMM using comparative quantitative mass spectrometry analyses of enriched mitochondria (LFQ-MS/MS), leveraging the redistribution of FBXO10(C953S). FBXO10, but not FBXO10(C953S), promoted polyubiquitylation and degradation of PGAM5. Examination of the role of this pathway in a physiological context revealed that the loss of FBXO10 or expression of prenylation-deficient-FBXO10(C953S) inhibited PGAM5 degradation, disrupted mitochondrial homeostasis, and impaired myogenic differentiation of human iPSCs and murine myoblasts. Our studies identify a mechanism for selective E3-ligase mediated regulation of mitochondrial membrane proteostasis and metabolic health, potentially amenable to therapeutic intervention.
PubMed: 38659932
DOI: 10.1101/2024.04.16.589745 -
Bioconjugate Chemistry Apr 2024Bioorthogonal chemistry has gained widespread use in the study of many biological systems of interest, including protein prenylation. Prenylation is a post-translational...
Bioorthogonal chemistry has gained widespread use in the study of many biological systems of interest, including protein prenylation. Prenylation is a post-translational modification, in which one or two 15- or 20-carbon isoprenoid chains are transferred onto cysteine residues near the C-terminus of a target protein. The three main enzymes─protein farnesyltransferase (FTase), geranylgeranyl transferase I (GGTase I), and geranylgeranyl transferase II (GGTase II)─that catalyze this process have been shown to tolerate numerous structural modifications in the isoprenoid substrate. This feature has previously been exploited to transfer an array of farnesyl diphosphate analogues with a range of functionalities, including an alkyne-containing analogue for copper-catalyzed bioconjugation reactions. Reported here is the synthesis of an analogue of the isoprenoid substrate embedded with norbornene functionality (C10NorOPP) that can be used for an array of applications, ranging from metabolic labeling to selective protein modification. The probe was synthesized in seven steps with an overall yield of 7% and underwent an inverse electron demand Diels-Alder (IEDDA) reaction with tetrazine-containing tags, allowing for copper-free labeling of proteins. The use of C10NorOPP for the study of prenylation was explored in the metabolic labeling of prenylated proteins in HeLa, COS-7, and astrocyte cells. Furthermore, in HeLa cells, these modified prenylated proteins were identified and quantified using label-free quantification (LFQ) proteomics with 25 enriched prenylated proteins. Additionally, the unique chemistry of C10NorOPP was utilized for the construction of a multiprotein-polymer conjugate for the targeted labeling of cancer cells. That construct was prepared using a combination of norbornene-tetrazine conjugation and azide-alkyne cycloaddition, highlighting the utility of the additional degree of orthogonality for the facile assembly of new protein conjugates with novel structures and functions.
PubMed: 38654427
DOI: 10.1021/acs.bioconjchem.4c00072 -
Angewandte Chemie (International Ed. in... Jul 2024cis-Prenyltransferases (cis-PTs) catalyze the sequential head-to-tail condensation of isopentenyl diphosphate (IPP) to allylic diphosphates, producing mixed E-Z prenyl...
cis-Prenyltransferases (cis-PTs) catalyze the sequential head-to-tail condensation of isopentenyl diphosphate (IPP) to allylic diphosphates, producing mixed E-Z prenyl diphosphates of varying lengths; however, the specific enzymes synthesizing cis-C prenyl diphosphates have not been identified. Herein, we present the discovery and characterization of a cis-geranylfarnesyl diphosphate synthase (ScGFPPS) from Streptomyces clavuligerus. This enzyme demonstrates high catalytic proficiency in generating six distinct cis-polyisoprenoids, including three C and three C variants. We determined the crystal structure of ScGFPPS. Additionally, we unveil the crystal structure of nerylneryl diphosphate synthase (NNPS), known for synthesizing an all-cis-C polyisoprenoid. Comparative structural analysis of ScGFPPS and NNPS has identified key differences that influence product specificity. Through site-directed mutagenesis, we have identified eight single mutations that significantly refine the selectivity of ScGFPPS for cis-polyisoprenoids. Our findings not only expand the functional spectrum of cis-PTs but also provide a structural comparison strategy in cis-PTs engineering.
Topics: Streptomyces; Protein Engineering; Crystallography, X-Ray; Alkyl and Aryl Transferases; Models, Molecular
PubMed: 38651244
DOI: 10.1002/anie.202401669 -
Journal of Ethnopharmacology Aug 2024Licorice is a frequently used herbal medicine worldwide, and is used to treat cough, hepatitis, cancer and influenza in clinical practice of traditional Chinese...
ETHNOPHARMACOLOGICAL RELEVANCE
Licorice is a frequently used herbal medicine worldwide, and is used to treat cough, hepatitis, cancer and influenza in clinical practice of traditional Chinese medicine. Modern pharmacological studies indicate that prenylated flavonoids play an important role in the anti-tumor activity of licorice, especially the tumors in stomach, lung, colon and liver. Wighteone is one of the main prenylated flavonoids in licorice, and its possible effect and target against colorectal cancer have not been investigated.
AIM OF THE STUDY
This study aimed to investigate the anti-colorectal cancer effect and underlying mechanism of wighteone.
MATERIALS AND METHODS
SW480 human colorectal cancer cells were used to evaluate the in vitro anti-colorectal cancer activity and Akt regulation effect of wighteone by flow cytometry, phosphoproteomic and Western blot analysis. Surface plasmon resonance (SPR) assay, molecular docking and dynamics simulation, and kinase activity assay were used to investigate the direct interaction between wighteone and Akt. A nude mouse xenograft model with SW480 cells was used to verify the in vivo anti-colorectal cancer activity of wighteone.
RESULTS
Wighteone inhibited phosphorylation of Akt and its downstream kinases in SW480 cells, which led to a reduction in cell viability. Wighteone had direct interaction with both PH and kinase domains of Akt, which locked Akt in a "closed" conformation with allosteric inhibition, and Gln79, Tyr272, Arg273 and Lys297 played the most critical role due to their hydrogen bond and hydrophobic interactions with wighteone. Based on Akt overexpression or activation in SW480 cells, further mechanistic studies suggested that wighteone-induced Akt inhibition led to cycle arrest, apoptosis and autophagic death of SW480 cells. Moreover, wighteone exerted in vivo anti-colorectal cancer effect and Akt inhibition activity in the nude mouse xenograft model.
CONCLUSION
Wighteone could inhibit growth of SW480 cells through allosteric inhibition of Akt, which led to cell cycle arrest, apoptosis and autophagic death. The results contributed to understanding of the anti-tumor mechanism of licorice, and also provided a rationale to design novel Akt allosteric inhibitors for the treatment of colorectal cancer.
Topics: Animals; Humans; Male; Mice; Allosteric Regulation; Antineoplastic Agents, Phytogenic; Apoptosis; Cell Line, Tumor; Cell Proliferation; Colorectal Neoplasms; Flavonoids; Glycyrrhiza; Mice, Inbred BALB C; Mice, Nude; Molecular Docking Simulation; Proto-Oncogene Proteins c-akt; Xenograft Model Antitumor Assays
PubMed: 38641080
DOI: 10.1016/j.jep.2024.118195 -
Frontiers in Chemistry 2024In natural products (NPs) research, methods for the efficient prioritization of natural extracts (NEs) are key for discovering novel bioactive NPs. In this study a...
Integration of Wnt-inhibitory activity and structural novelty scoring results to uncover novel bioactive natural products: new Bicyclo[3.3.1]non-3-ene-2,9-diones from the leaves of .
In natural products (NPs) research, methods for the efficient prioritization of natural extracts (NEs) are key for discovering novel bioactive NPs. In this study a biodiverse collection of 1,600 NEs, previously analyzed by UHPLC-HRMS metabolite profiling was screened for Wnt pathway regulation. The results of the biological screening drove the selection of a subset of 30 non-toxic NEs with an inhibitory IC ≤ 5 g/mL. To increase the chance of finding structurally novel bioactive NPs, , a computational tool for automated scoring of NEs based on structural novelty was used to mine the HRMS analysis and dereplication results. After this, four out of the 30 bioactive NEs were shortlisted by this approach. The most promising sample was the ethyl acetate extract of the leaves of (Phyllanthaceae). Further phytochemical investigations of this species resulted in the isolation of three known prenylated flavones (, , ) and ten novel bicyclo[3.3.1]non-3-ene-2,9-diones (, , , , -), named . Assessment of the Wnt inhibitory activity of these compounds revealed that two prenylated flavones and three novel bicyclic compounds showed interesting activity without apparent cytotoxicity. This study highlights the potential of combining 's structural novelty scores with biological screening results to effectively discover novel bioactive NPs in large NE collections.
PubMed: 38638877
DOI: 10.3389/fchem.2024.1371982 -
Biochimica Et Biophysica Acta.... Jun 2024Mevalonate kinase deficiency (MKD) is an autosomal recessive metabolic disorder associated with recurrent autoinflammatory episodes. The disorder is caused by bi-allelic...
Mevalonate kinase deficiency (MKD) is an autosomal recessive metabolic disorder associated with recurrent autoinflammatory episodes. The disorder is caused by bi-allelic loss-of-function variants in the MVK gene, which encodes mevalonate kinase (MK), an early enzyme in the isoprenoid biosynthesis pathway. To identify molecular and cellular consequences of MKD, we studied primary fibroblasts from severely affected patients with mevalonic aciduria (MKD-MA) and more mildly affected patients with hyper IgD and periodic fever syndrome (MKD-HIDS). As previous findings indicated that the deficient MK activity in MKD impacts protein prenylation in a temperature-sensitive manner, we compared the subcellular localization and activation of the small Rho GTPases RhoA, Rac1 and Cdc42 in control, MKD-HIDS and MKD-MA fibroblasts cultured at physiological and elevated temperatures. This revealed a temperature-induced altered subcellular localization and activation in the MKD cells. To study if and how the temperature-induced ectopic activation of these signalling proteins affects cellular processes, we performed comparative transcriptome analysis of control and MKD-MA fibroblasts cultured at 37 °C or 40 °C. This identified cell cycle and actin cytoskeleton organization as respectively most down- and upregulated gene clusters. Further studies confirmed that these processes were affected in fibroblasts from both patients with MKD-MA and MKD-HIDS. Finally, we found that, similar to immune cells, the MK deficiency causes metabolic reprogramming in MKD fibroblasts resulting in increased expression of genes involved in glycolysis and the PI3K/Akt/mTOR pathway. We postulate that the ectopic activation of small GTPases causes inappropriate signalling contributing to the molecular and cellular aberrations observed in MKD.
Topics: Mevalonate Kinase Deficiency; Humans; Fibroblasts; Phosphotransferases (Alcohol Group Acceptor); Cells, Cultured; Signal Transduction
PubMed: 38636615
DOI: 10.1016/j.bbadis.2024.167177 -
Bioorganic Chemistry Jun 2024Protein prenylation is one example of a broad class of post-translational modifications where proteins are covalently linked to various hydrophobic moieties. To globally...
Protein prenylation is one example of a broad class of post-translational modifications where proteins are covalently linked to various hydrophobic moieties. To globally identify and monitor levels of all prenylated proteins in a cell simultaneously, our laboratory and others have developed chemical proteomic approaches that rely on the metabolic incorporation of isoprenoid analogues bearing bio-orthogonal functionality followed by enrichment and subsequent quantitative proteomic analysis. Here, several improvements in the synthesis of the alkyne-containing isoprenoid analogue C15AlkOPP are reported to improve synthetic efficiency. Next, metabolic labeling with C15AlkOPP was optimized to obtain useful levels of metabolic incorporation of the probe in several types of primary cells. Those conditions were then used to study the prenylomes of motor neurons (ES-MNs), astrocytes (ES-As), and their embryonic stem cell progenitors (ESCs), which allowed for the identification of 54 prenylated proteins from ESCs, 50 from ES-MNs, and 84 from ES-As, representing all types of prenylation. Bioinformatic analysis revealed specific enriched pathways, including nervous system development, chemokine signaling, Rho GTPase signaling, and adhesion. Hierarchical clustering showed that most enriched pathways in all three cell types are related to GTPase activity and vesicular transport. In contrast, STRING analysis showed significant interactions in two populations that appear to be cell type dependent. The data provided herein demonstrates that robust incorporation of C15AlkOPP can be obtained in ES-MNs and related primary cells purified via magnetic-activated cell sorting allowing the identification and quantification of numerous prenylated proteins. These results suggest that metabolic labeling with C15AlkOPP should be an effective approach for investigating the role of prenylated proteins in primary cells in both normal cells and disease pathologies, including ALS.
Topics: Astrocytes; Animals; Protein Prenylation; Alkynes; Motor Neurons; Terpenes; Mice; Molecular Structure; Cells, Cultured
PubMed: 38636436
DOI: 10.1016/j.bioorg.2024.107365 -
Chemistry & Biodiversity Jun 2024Four undescribed prenylated flavonoids, sophoratones A-D (1-4), and 17 known flavonoids, were obtained from the aerial parts of Sophora tonkinensis. Their structures...
Four undescribed prenylated flavonoids, sophoratones A-D (1-4), and 17 known flavonoids, were obtained from the aerial parts of Sophora tonkinensis. Their structures with absolute configurations were elucidated by detailed interpretation of NMR spectroscopy, mass spectrometry, and ECD calculations. Meanwhile, the ability of these compounds to inhibit the release of nitric oxide (NO) by a lipopolysaccharide induced mouse in RAW 264.7 cells was assayed. The results indicated that some compounds exhibited clear inhibitory effects, with IC ranging from 19.91±1.08 to 35.72±2.92 μM. These results suggest that prenylated flavonoids from the aerial parts of S. tonkinensis could potentially be used as a latent source of anti-inflammatory agents.
Topics: Sophora; Animals; Mice; Flavonoids; RAW 264.7 Cells; Plant Components, Aerial; Nitric Oxide; Lipopolysaccharides; Anti-Inflammatory Agents; Structure-Activity Relationship; Anti-Inflammatory Agents, Non-Steroidal; Molecular Structure; Dose-Response Relationship, Drug; Cell Survival
PubMed: 38634752
DOI: 10.1002/cbdv.202400399