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Developmental Cell Jun 2020Compartmentalized signaling is critical for cellular organization and specificity of functional outcomes in neurons. Here, we report that post-translational lipidation...
Compartmentalized signaling is critical for cellular organization and specificity of functional outcomes in neurons. Here, we report that post-translational lipidation of newly synthesized proteins in axonal compartments allows for short-term and autonomous responses to extrinsic cues. Using conditional mutant mice, we found that protein prenylation is essential for sympathetic axon innervation of target organs. We identify a localized requirement for prenylation in sympathetic axons to promote axonal growth in response to the neurotrophin, nerve growth factor (NGF). NGF triggers prenylation of proteins including the Rac1 GTPase in axons, counter to the canonical view of prenylation as constitutive, and strikingly, in a manner dependent on axonal protein synthesis. Newly prenylated proteins localize to TrkA-harboring endosomes in axons and promote receptor trafficking necessary for axonal growth. Thus, coupling of prenylation to local protein synthesis presents a mechanism for spatially segregated cellular functions during neuronal development.
Topics: Animals; Axon Guidance; Axons; Cells, Cultured; Endosomes; HEK293 Cells; Humans; Mice; Mice, Inbred C57BL; Nerve Growth Factor; Neuropeptides; PC12 Cells; Protein Prenylation; Rats; Rats, Sprague-Dawley; Receptor, trkA; rac1 GTP-Binding Protein
PubMed: 32533921
DOI: 10.1016/j.devcel.2020.05.020 -
Molecular Neurobiology Dec 2019Early activation of cytosolic NADPH oxidase-2 (Nox2) in diabetes increases retinal ROS production, damaging their mitochondria. The assembly of Nox2 holoenzyme requires...
PURPOSE
Early activation of cytosolic NADPH oxidase-2 (Nox2) in diabetes increases retinal ROS production, damaging their mitochondria. The assembly of Nox2 holoenzyme requires activation of a small molecular weight G protein Rac1. Rac1 activation is regulated by guanine exchange factors and guanine nucleotide-dissociation inhibitors, and post-translational modifications assist in its association with exchange factors and dissociation inhibitors. The goal of this study is to investigate the mechanisms of Rac1 activation in the development of diabetic retinopathy.
METHODS
The levels of the dissociation inhibitor, prenylating enzyme (farnesyltransferase, FNTA), and exchange factor Vav2 were quantified in human retinal endothelial cells, incubated in normal or high glucose for 96 h. The roles of prenylation and Vav2 in Rac1-Nox2-ROS mitochondrial damage were confirmed in FNTA-siRNA-transfected cells and using the Vav2 inhibitor EHop, respectively. Retinal histopathology and functional changes associated with diabetic retinopathy were analyzed in diabetic mice receiving EHop for 6 months. Key parameters of Rac1 activation were confirmed in the retinal microvasculature from human donors with diabetic retinopathy.
RESULTS
In HRECs, glucose increased FNTA and Vav2 and decreased the dissociation inhibitor. FNTA-siRNA and EHop inhibited glucose-induced activation of Rac1-Nox2-ROS signaling. In diabetic mice, EHop ameliorated the development of retinopathy and functional/structural abnormalities and attenuated Rac1-Nox2-mitochondrial damage. Similar alterations in Rac1 regulators were observed in retinal microvasculature from human donors with diabetic retinopathy. In diabetes, Rac1 prenylation and its interactions with Vav2 contribute to Nox2-ROS-mitochondrial damage, and the pharmacological inhibitors to attenuate Rac1 interactions with its regulators could have the potential to halt/inhibit the development of diabetic retinopathy. Graphical Abstract Activation of prenylating enzyme farnesyltransferase (FNTA) in diabetes, prenylates Rac1. The binding of Rac1 with guanine nucleotide-dissociation inhibitor (GDI) is decreased, but its association with the guanine exchange factor, Vav2, is increased, resulting in Rac1 activation. Active Rac1 helps in the assembly of Nox2 holoenzyme, and Nox2 activation increases cytosolic ROS production, damaging the mitochondria. Damaged mitochondria accelerate capillary cell apoptosis, and ultimately, results in the development of diabetic retinopathy.
Topics: Aged; Animals; Diabetic Retinopathy; Endothelial Cells; Glucose; Humans; Mice, Inbred C57BL; Microvessels; Middle Aged; NADPH Oxidase 2; Oxidative Stress; Prenylation; Proto-Oncogene Proteins c-vav; Tissue Donors; rac1 GTP-Binding Protein
PubMed: 31300985
DOI: 10.1007/s12035-019-01696-5 -
Biological & Pharmaceutical Bulletin 2024CsPT4 is an aromatic prenyltransferase that synthesizes cannabigerolic acid (CBGA), the key intermediate of cannabinoid biosynthesis in Cannabis sativa, from olivetolic...
CsPT4 is an aromatic prenyltransferase that synthesizes cannabigerolic acid (CBGA), the key intermediate of cannabinoid biosynthesis in Cannabis sativa, from olivetolic acid (OA) and geranyl diphosphate (GPP). CsPT4 has a catalytic potential to produce a variety of CBGA analogs via regioselective C-prenylation of aromatic substrates having resorcylic acid skeletons including bibenzyl 2,4-dihydroxy-6-phenylethylbenzoic acid (DPA). In this study, we further investigated the substrate specificity of CsPT4 using phlorocaprophenone (PCP) and 2',4',6'-trihydroxydihydrochalcone (THDC), the isomers of OA and DPA, respectively, and demonstrated that CsPT4 catalyzed both C-prenylation and O-prenylation reactions on PCP and THDC that share acylphloroglucinol substructures. Interestingly, the kinetic parameters of CsPT4 for these substrates differed depending on whether they underwent C-prenylation or O-prenylation, suggesting that this enzyme utilized different substrate-binding modes suitable for the respective reactions. Aromatic prenyltransferases that catalyze O-prenylation are rare in the plant kingdom, and CsPT4 was notable for altering the reaction specificity between C- and O-prenylations depending on the skeletons of aromatic substrates. We also demonstrated that enzymatically synthesized geranylated acylphloroglucinols had potent antiausterity activity against PANC-1 human pancreatic cancer cells, with 4'-O-geranyl THDC being the most effective. We suggest that CsPT4 is a valuable catalyst to generate biologically active C- and O-prenylated molecules that could be anticancer lead compounds.
Topics: Humans; Cannabis; Dimethylallyltranstransferase; Prenylation; Catalysis; Substrate Specificity
PubMed: 38369346
DOI: 10.1248/bpb.b23-00868 -
Heliyon Feb 2023Tryptophan and its derived metabolites have been assumed to play important roles in the development and survival of organisms. However, the links of tryptophan and its...
Tryptophan and its derived metabolites have been assumed to play important roles in the development and survival of organisms. However, the links of tryptophan and its derived metabolites to temperature change remained largely cryptic. Here we presented that a class of prenyl indole alkaloids biosynthesized from tryptophan dramatically accumulated in thermophilic fungus under cold stress, in which lipid droplets were also highly accumulated and whose conidiophores were highly build-up. Concurrently, disruption of the key gene involved in the biosynthesis of prenyl indole alkaloids, resulted in decreased lipid and shrunken mitochondria but enlarged vacuoles. Moreover, the Fe and superoxide levels in were significantly increased but the reactive oxygen species lipid peroxidation and autophagy levels decreased. Metabolomics study revealed that most enriched metabolites in were mainly composed of tryptophan degraded metabolites including well known ROS scavenger kynurenamines, and lipid-inhibitors, anthranilic acid and indoleacetic acid, and free radical reaction suppressor free fatty acids. Transcriptomic analysis suggested that the key gene involved in tryptophan metabolism, coinciding with the lipid metabolic processes and ion transports were most up-regulated in under stress. Our results confirmed a lipid-mediated fungal response to cold stress and unveiled a link of tryptophan-based metabolic reprogramming to the fungal cold adaption.
PubMed: 36747564
DOI: 10.1016/j.heliyon.2023.e13066 -
BMC Cancer Jul 2020Ovarian cancer remains the most fatal gynecological malignancy. Current therapeutic options are limited due to late diagnosis in the majority of the cases, metastatic...
BACKGROUND
Ovarian cancer remains the most fatal gynecological malignancy. Current therapeutic options are limited due to late diagnosis in the majority of the cases, metastatic spread to the peritoneal cavity and the onset of chemo-resistance. Thus, novel therapeutic approaches are required. Statins and amino-bisphosphonates are inhibitors of the mevalonate pathway, which is a fundamental pathway of cellular metabolism, essential for cholesterol production and posttranslational protein farnesylation and geranylgeranylation. While this pathway has emerged as a promising treatment target in several human malignancies, its potential as a therapeutic approach in ovarian cancer is still not fully understood.
METHODS
Human ovarian cancer cell lines (IGROV-1, A2780, A2780cis) were treated with increasing concentrations (0.5-100 μM) of statins (simvastatin, atorvastatin, rosuvastatin) and zoledronic acid. Effects on cell vitality and apoptosis were assessed using Cell Titer Blue®, Caspase 3/7 Glo®, clonogenic assays as well as cleaved poly (ADP-ribose) polymerase (cPARP) detection. The inhibition of the mevalonate pathway was confirmed using Western Blot of unprenylated Ras and Rap1a proteins. Quantitative real-time PCR and ELISA were used to analyze modulations on several key regulators of ovarian cancer tumorigenesis.
RESULTS
The treatment of IGROV-1 and A2780 cells with statins and zoledronic acid reduced vitality (by up to 80%; p < 0.001) and induced apoptosis by up to 8-folds (p < 0.001) in a dose-dependent fashion. Rescue experiments using farnesyl pyrophosphate or geranylgeranyl pyrophosphate evidenced that blocked geranylgeranylation is the major underlying mechanism of the pro-apoptotic effects. Gene expression of the tumor-promoting cytokines and mediators, such as transforming growth factor (TGF)-β1, vascular endothelial growth factor (VEGF), interleukin (IL)-8, and IL-6 were significantly suppressed by statins and zoledronic acid by up to 90% (p < 0.001). For all readouts, simvastatin was most potent of all agents used. Cisplatin-resistant A2780cis cells showed a relative resistance to statins and zoledronic acid. However, similar to the effects in A2780 cells, simvastatin and zoledronic acid significantly induced caspase 3/7 activation (6-folds; p < 0.001).
CONCLUSION
Our in vitro findings point to promising anti-tumor effects of statins and zoledronic acid in ovarian cancer and warrant additional validation in preclinical and clinical settings.
Topics: Apoptosis; Atorvastatin; Cell Line, Tumor; Cell Survival; Drug Resistance, Neoplasm; Female; Gene Expression; Humans; Hydroxymethylglutaryl-CoA Reductase Inhibitors; Interleukin-6; Interleukin-8; Mevalonic Acid; Ovarian Neoplasms; Polyisoprenyl Phosphates; Prenylation; Rosuvastatin Calcium; Sesquiterpenes; Simvastatin; Transforming Growth Factor beta1; Vascular Endothelial Growth Factor A; Zoledronic Acid
PubMed: 32727400
DOI: 10.1186/s12885-020-07164-x -
Bioorganic Chemistry Feb 2016Protein prenylation is a posttranslational modification catalyzed by prenyltransferases involving the attachment of farnesyl or geranylgeranyl groups to residues near... (Review)
Review
Protein prenylation is a posttranslational modification catalyzed by prenyltransferases involving the attachment of farnesyl or geranylgeranyl groups to residues near the C-termini of proteins. This irreversible covalent modification is important for membrane localization and proper signal transduction. Here, the use of isoprenoid analogues for studying prenylated proteins is reviewed. First, experiments with analogues containing small fluorophores that are alternative substrates for prenyltransferases are described. Those analogues have been useful for quantifying binding affinity and for the production of fluorescently labeled proteins. Next, the use of analogues that incorporate biotin, bioorthogonal groups or antigenic moieties is described. Such probes have been particularly useful for identifying proteins that are naturally prenylated within mammalian cells. Overall, the use of isoprenoid analogues has contributed significantly to the understanding of protein prenlation.
Topics: Alkyl and Aryl Transferases; Fluorescent Dyes; Molecular Probes; Protein Prenylation; Proteins; Proteomics; Terpenes
PubMed: 26709869
DOI: 10.1016/j.bioorg.2015.12.003 -
Molecules (Basel, Switzerland) Dec 2022Prenylated diresorcinols exhibit various bioactivities, including cytotoxic, antibacterial, and antifungal activities. Therefore, establishing facile and efficient...
Prenylated diresorcinols exhibit various bioactivities, including cytotoxic, antibacterial, and antifungal activities. Therefore, establishing facile and efficient synthetic routes for prenylated diresorcinols facilitates their development as chemical probes or drugs with a novel mode of action. In this study, microwave-assisted copper catalysis was explored as a cost-effective and environmentally friendly method for the cross-coupling of sterically hindered -prenylated phenols and aryl halides to produce bioactive prenylated diresorcinols, diorcinol I and leotiomycene B. Notable advantages of microwave-assisted catalysis include not only operational simplicity and rapid heating but also shorter reaction times and higher chemical yields. In addition, highly regioselective prenylation of phenol was achieved for the preparation of -prenyl phenol via directed lithiation and subsequent alkylation. This study provides valuable insights for the preparation of other bioactive prenylated diresorcinols. Furthermore, considering that prenylated benzenoids are biosynthetic precursors of various polycyclic natural products, this synthetic route could be expanded to more complex bioactive compounds possessing diaryl ethers.
Topics: Phenol; Microwaves; Phenols; Ethers; Catalysis
PubMed: 36615257
DOI: 10.3390/molecules28010062 -
Cardiovascular Research Mar 2017RhoB plays a key role in the pathogenesis of hypoxia-induced pulmonary hypertension. Farnesylated RhoB promotes growth responses in cancer cells and we investigated...
AIMS
RhoB plays a key role in the pathogenesis of hypoxia-induced pulmonary hypertension. Farnesylated RhoB promotes growth responses in cancer cells and we investigated whether inhibition of protein farnesylation will have a protective effect.
METHODS AND RESULTS
The analysis of lung tissues from rodent models and pulmonary hypertensive patients showed increased levels of protein farnesylation. Oral farnesyltransferase inhibitor tipifarnib prevented development of hypoxia-induced pulmonary hypertension in mice. Tipifarnib reduced hypoxia-induced vascular cell proliferation, increased endothelium-dependent vasodilatation and reduced vasoconstriction of intrapulmonary arteries without affecting cell viability. Protective effects of tipifarnib were associated with inhibition of Ras and RhoB, actin depolymerization and increased eNOS expression in vitro and in vivo. Farnesylated-only RhoB (F-RhoB) increased proliferative responses in cultured pulmonary vascular cells, mimicking the effects of hypoxia, while both geranylgeranylated-only RhoB (GG-RhoB), and tipifarnib had an inhibitory effect. Label-free proteomics linked F-RhoB with cell survival, activation of cell cycle and mitochondrial biogenesis. Hypoxia increased and tipifarnib reduced the levels of F-RhoB-regulated proteins in the lung, reinforcing the importance of RhoB as a signalling mediator. Unlike simvastatin, tipifarnib did not increase the expression levels of Rho proteins.
CONCLUSIONS
Our study demonstrates the importance of protein farnesylation in pulmonary vascular remodelling and provides a rationale for selective targeting of this pathway in pulmonary hypertension.
Topics: Animals; Antihypertensive Agents; Cell Proliferation; Cells, Cultured; Disease Models, Animal; Endothelial Cells; Enzyme Inhibitors; Farnesyltranstransferase; Humans; Hypertension, Pulmonary; Hypoxia; Male; Mice, Inbred C57BL; Phenotype; Protein Prenylation; Proteomics; Pulmonary Artery; Quinolones; Time Factors; Transfection; Vasoconstriction; Vasodilation; rhoB GTP-Binding Protein
PubMed: 28395021
DOI: 10.1093/cvr/cvw258 -
Natural Product Reports Jun 2018Covering: up to February 2017 Various fungi of the genera Aspergillus, Penicillium, and Malbranchea produce prenylated indole alkaloids possessing a... (Review)
Review
Covering: up to February 2017 Various fungi of the genera Aspergillus, Penicillium, and Malbranchea produce prenylated indole alkaloids possessing a bicyclo[2.2.2]diazaoctane ring system. After the discovery of distinct enantiomers of the natural alkaloids stephacidin A and notoamide B, from A. protuberus MF297-2 and A. amoenus NRRL 35660, another fungi, A. taichungensis, was found to produce their diastereomers, 6-epi-stephacidin A and versicolamide B, as major metabolites. Distinct enantiomers of stephacidin A and 6-epi-stephacidin A may be derived from a common precursor, notoamide S, by enzymes that form a bicyclo[2.2.2]diazaoctane core via a putative intramolecular hetero-Diels-Alder cycloaddition. This review provides our current understanding of the structural and stereochemical homologies and disparities of these alkaloids. Through the deployment of biomimetic syntheses, whole-genome sequencing, and biochemical studies, a unified biogenesis of both the dioxopiperazine and the monooxopiperazine families of prenylated indole alkaloids constituted of bicyclo[2.2.2]diazaoctane ring systems is presented.
Topics: Aquatic Organisms; Aspergillus; Fungi; Indole Alkaloids; Molecular Structure; Prenylation; Stereoisomerism
PubMed: 29632911
DOI: 10.1039/c7np00042a -
ACS Chemical Biology Oct 2022Photoswitchable lipids have emerged as attractive tools for the optical control of lipid bioactivity, metabolism, and biophysical properties. Their design is typically...
Photoswitchable lipids have emerged as attractive tools for the optical control of lipid bioactivity, metabolism, and biophysical properties. Their design is typically based on the incorporation of an azobenzene photoswitch into the hydrophobic lipid tail, which can be switched between its - and -form using two different wavelengths of light. While glycero- and sphingolipids have been successfully designed to be photoswitchable, isoprenoid lipids have not yet been investigated. Herein, we describe the development of photoswitchable analogs of an isoprenoid lipid and systematically assess their potential for the optical control of various steps in the isoprenylation processing pathway of CaaX proteins in . One photoswitchable analog of farnesyl diphosphate () allowed effective optical control of substrate prenylation by farnesyltransferase. The subsequent steps of isoprenylation processing (proteolysis by either Ste24 or Rce1 and carboxyl methylation by Ste14) were less affected by photoisomerization of the group introduced into the lipid moiety of the substrate a-factor, a mating pheromone from yeast. We assessed both proteolysis and methylation of the a-factor analogs and the bioactivity of a fully processed a-factor analog containing the photoswitch, exogenously added to cognate yeast cells. Combined, these data describe the first successful conversion of an isoprenoid lipid into a photolipid and suggest the utility of this approach for the optical control of protein prenylation.
Topics: Saccharomyces cerevisiae; Terpenes; Farnesyltranstransferase; Peptides; Protein Prenylation; Pheromones; Lipids; Sphingolipids; Membrane Proteins; Metalloendopeptidases; Saccharomyces cerevisiae Proteins
PubMed: 36194691
DOI: 10.1021/acschembio.2c00645