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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 -
Molecular Neurobiology Mar 2021Protein prenylation is a post-translational lipid modification that governs a variety of important cellular signaling pathways, including those regulating synaptic...
Protein prenylation is a post-translational lipid modification that governs a variety of important cellular signaling pathways, including those regulating synaptic functions and cognition in the nervous system. Two enzymes, farnesyltransferase (FT) and geranylgeranyltransferase type I (GGT), are essential for the prenylation process. Genetic reduction of FT or GGT ameliorates neuropathology but only FT haplodeficiency rescues cognitive function in transgenic mice of Alzheimer's disease. A follow-up study showed that systemic or forebrain neuron-specific deficiency of GGT leads to synaptic and cognitive deficits under physiological conditions. Whether FT plays different roles in shaping neuronal functions and cognition remains elusive. This study shows that in contrast to the detrimental effects of GGT reduction, systemic haplodeficiency of FT has little to no impact on hippocampal synaptic plasticity and cognition. However, forebrain neuron-specific FT deletion also leads to reduced synaptic plasticity, memory retention, and hippocampal dendritic spine density. Furthermore, a novel prenylomic analysis identifies distinct pools of prenylated proteins that are affected in the brain of forebrain neuron-specific FT and GGT knockout mice, respectively. Taken together, this study uncovers that physiological levels of FT and GGT in neurons are essential for normal synaptic/cognitive functions and that the prenylation status of specific signaling molecules regulates neuronal functions.
Topics: Alkyl and Aryl Transferases; Animals; Cognition; Dendritic Spines; Hippocampus; Long-Term Potentiation; Maze Learning; Mice; Neuronal Plasticity; Neurons; Protein Prenylation; Spatial Learning; Spatial Memory; Synapses
PubMed: 33098528
DOI: 10.1007/s12035-020-02169-w -
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 -
Nature Communications Mar 2016Prenylation reactions play crucial roles in controlling the activities of biomolecules. Bacterial prenyltransferases, TleC from Streptomyces blastmyceticus and MpnD from...
Prenylation reactions play crucial roles in controlling the activities of biomolecules. Bacterial prenyltransferases, TleC from Streptomyces blastmyceticus and MpnD from Marinactinospora thermotolerans, catalyse the 'reverse' prenylation of (-)-indolactam V at the C-7 position of the indole ring with geranyl pyrophosphate or dimethylallyl pyrophosphate, to produce lyngbyatoxin or pendolmycin, respectively. Using in vitro analyses, here we show that both TleC and MpnD exhibit relaxed substrate specificities and accept various chain lengths (C5-C25) of the prenyl donors. Comparisons of the crystal structures and their ternary complexes with (-)-indolactam V and dimethylallyl S-thiophosphate revealed the intimate structural details of the enzyme-catalysed 'reverse' prenylation reactions and identified the active-site residues governing the selection of the substrates. Furthermore, structure-based enzyme engineering successfully altered the preference for the prenyl chain length of the substrates, as well as the regio- and stereo-selectivities of the prenylation reactions, to produce a series of unnatural novel indolactams.
Topics: Actinomycetales; Bacterial Proteins; Dimethylallyltranstransferase; Indoles; Lactams; Molecular Structure; Prenylation; Protein Engineering; Streptomyces; Substrate Specificity
PubMed: 26952246
DOI: 10.1038/ncomms10849 -
Current Protocols in Chemical Biology Sep 2018Protein prenylation involves the attachment of a farnesyl or geranylgeranyl group onto a cysteine residue located near the C-terminus of a protein, recognized via a...
Protein prenylation involves the attachment of a farnesyl or geranylgeranyl group onto a cysteine residue located near the C-terminus of a protein, recognized via a specific prenylation motif, and results in the formation of a thioether bond. To identify putative prenylated proteins and investigate changes in their levels of expression, metabolic labeling and subsequent bioorthogonal labeling has become one of the methods of choice. In that strategy, synthetic analogues of biosynthetic precursors for post-translational modification bearing bioorthogonal functionality are added to the growth medium from which they enter cells and become incorporated into proteins. Subsequently, the cells are lysed and proteins bearing the analogues are then covalently modified using selective chemical reagents that react via bioorthogonal processes, allowing a variety of probes for visualization or enrichment to be attached for subsequent analysis. Here, we describe protocols for synthesizing several different isoprenoid analogues and describe how they are metabolically incorporated into mammalian cells, and the incorporation into prenylated proteins visualized via in-gel fluorescence analysis. © 2018 by John Wiley & Sons, Inc.
Topics: Alkynes; Molecular Structure; Protein Prenylation; Proteins; Terpenes
PubMed: 30058775
DOI: 10.1002/cpch.46 -
International Journal of Molecular... Mar 2021Geranylgeranyltransferase type-I (GGTase-I) represents an important drug target since it contributes to the function of many proteins that are involved in tumor...
Geranylgeranyltransferase type-I (GGTase-I) represents an important drug target since it contributes to the function of many proteins that are involved in tumor development and metastasis. This led to the development of GGTase-I inhibitors as anti-cancer drugs blocking the protein function and membrane association of e.g., Rap subfamilies that are involved in cell differentiation and cell growth. In the present study, we developed a new NanoBiT assay to monitor the interaction of human GGTase-I and its substrate Rap1B. Different Rap1B prenylation-deficient mutants (C181G, C181S, and ΔCQLL) were designed and investigated for their interaction with GGTase-I. While the Rap1B mutants C181G and C181S still exhibited interaction with human GGTase-I, mutant ΔCQLL, lacking the entire CAAX motif (defined by a cysteine residue, two aliphatic residues, and the C-terminal residue), showed reduced interaction. Moreover, a specific, peptidomimetic and competitive CAAX inhibitor was able to block the interaction of Rap1B with GGTase-I. Furthermore, activation of both Gα-coupled human adenosine receptors, A (AAR) and A (AAR), increased the interaction between GGTase-I and Rap1B, probably representing a way to modulate prenylation and function of Rap1B. Thus, AAR and AAR antagonists might be promising candidates for therapeutic intervention for different types of cancer that overexpress Rap1B. Finally, the NanoBiT assay provides a tool to investigate the pharmacology of GGTase-I inhibitors.
Topics: Adenosine A2 Receptor Antagonists; Alkyl and Aryl Transferases; Enzyme Inhibitors; Humans; Peptide Fragments; Protein Interaction Domains and Motifs; Protein Prenylation; Substrate Specificity; Xanthines; rap GTP-Binding Proteins
PubMed: 33801503
DOI: 10.3390/ijms22052501 -
ELife May 2020Endosomes and lysosomes harbor Rab5 and Rab7 on their surface as key proteins involved in their identity, biogenesis, and fusion. Rab activation requires a guanine...
Endosomes and lysosomes harbor Rab5 and Rab7 on their surface as key proteins involved in their identity, biogenesis, and fusion. Rab activation requires a guanine nucleotide exchange factor (GEF), which is Mon1-Ccz1 for Rab7. During endosome maturation, Rab5 is replaced by Rab7, though the underlying mechanism remains poorly understood. Here, we identify the molecular determinants for Rab conversion in vivo and in vitro, and reconstitute Rab7 activation with yeast and metazoan proteins. We show (i) that Mon1-Ccz1 is an effector of Rab5, (ii) that membrane-bound Rab5 is the key factor to directly promote Mon1-Ccz1 dependent Rab7 activation and Rab7-dependent membrane fusion, and (iii) that this process is regulated in yeast by the casein kinase Yck3, which phosphorylates Mon1 and blocks Rab5 binding. Our study thus uncovers the minimal feed-forward machinery of the endosomal Rab cascade and a novel regulatory mechanism controlling this pathway.
Topics: Animals; Casein Kinase I; Drosophila; Drosophila Proteins; Endosomes; Guanine Nucleotide Exchange Factors; Liposomes; Membrane Fusion; Phosphatidylinositol Phosphates; Phosphorylation; Protein Binding; Protein Prenylation; Saccharomyces cerevisiae Proteins; Sf9 Cells; Vacuoles; Vesicular Transport Proteins; rab GTP-Binding Proteins; rab5 GTP-Binding Proteins; rab7 GTP-Binding Proteins
PubMed: 32391792
DOI: 10.7554/eLife.56090 -
Bioorganic & Medicinal Chemistry Letters Oct 2019The enzyme geranylgeranyl diphosphate synthase (GGDPS) is a potential therapeutic target for multiple myeloma. Malignant plasma cells produce and secrete large amounts...
The enzyme geranylgeranyl diphosphate synthase (GGDPS) is a potential therapeutic target for multiple myeloma. Malignant plasma cells produce and secrete large amounts of monoclonal protein, and inhibition of GGDPS results in disruption of protein geranylgeranylation which in turn impairs intracellular protein trafficking. Our previous work has demonstrated that some isoprenoid triazole bisphosphonates are potent and selective inhibitors of GGDPS. To explore the possibility of selective delivery of such compounds to plasma cells, new analogues with an ω-hydroxy group have been synthesized and examined for their enzymatic and cellular activity. These studies demonstrate that incorporation of the ω-hydroxy group minimally impairs GGDPS inhibitory activity. Furthermore conjugation of one of the novel ω-hydroxy GGDPS inhibitors to hyaluronic acid resulted in enhanced cellular activity. These results will allow future studies to focus on the in vivo biodistribution of HA-conjugated GGDPS inhibitors.
Topics: Antineoplastic Agents; Apoptosis; Cell Proliferation; Diphosphonates; Enzyme Inhibitors; Farnesyltranstransferase; Humans; Models, Molecular; Molecular Structure; Multiple Myeloma; Protein Prenylation; Structure-Activity Relationship; Terpenes; Tumor Cells, Cultured
PubMed: 31474482
DOI: 10.1016/j.bmcl.2019.126633 -
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 -
Proceedings of the National Academy of... May 2017Cerebral cavernous malformations (CCMs) are common vascular anomalies that develop in the central nervous system and, more rarely, the retina. The lesions can cause...
Cerebral cavernous malformations (CCMs) are common vascular anomalies that develop in the central nervous system and, more rarely, the retina. The lesions can cause headache, seizures, focal neurological deficits, and hemorrhagic stroke. Symptomatic lesions are treated according to their presentation; however, targeted pharmacological therapies that improve the outcome of CCM disease are currently lacking. We performed a high-throughput screen to identify Food and Drug Administration-approved drugs or other bioactive compounds that could effectively suppress hyperproliferation of mouse brain primary astrocytes deficient for CCM3. We demonstrate that fluvastatin, an inhibitor of 3-hydroxy-3-methyl-glutaryl (HMG)-CoA reductase and the -bisphosphonate zoledronic acid monohydrate, an inhibitor of protein prenylation, act synergistically to reverse outcomes of CCM3 loss in cultured mouse primary astrocytes and in glial cells in vivo. Further, the two drugs effectively attenuate neural and vascular deficits in chronic and acute mouse models of CCM3 loss in vivo, significantly reducing lesion burden and extending longevity. Sustained inhibition of the mevalonate pathway represents a potential pharmacological treatment option and suggests advantages of combination therapy for CCM disease.
Topics: Animals; Astrocytes; Diphosphonates; Drosophila; Drug Evaluation, Preclinical; Drug Therapy, Combination; Endothelial Cells; Fatty Acids, Monounsaturated; Female; Fluvastatin; Hemangioma, Cavernous, Central Nervous System; High-Throughput Screening Assays; Hydroxymethylglutaryl-CoA Reductase Inhibitors; Imidazoles; Indoles; MAP Kinase Signaling System; Male; Mice; Pregnancy; Protein Prenylation; Zoledronic Acid
PubMed: 28500274
DOI: 10.1073/pnas.1702942114