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International Journal of Molecular... Apr 2023Protein prenylation is an important protein modification that is responsible for diverse physiological activities in eukaryotic cells. This modification is generally...
Protein prenylation is an important protein modification that is responsible for diverse physiological activities in eukaryotic cells. This modification is generally catalyzed by three types of prenyl transferases, which include farnesyl transferase (FT), geranylgeranyl transferase (GGT-1) and Rab geranylgeranyl transferase (GGT-2). Studies in malaria parasites showed that these parasites contain prenylated proteins, which are proposed to play multiple functions in parasites. However, the prenyl transferases have not been functionally characterized in parasites of subphylum Apicomplexa. Here, we functionally dissected functions of three of the prenyl transferases in the Apicomplexa model organism () using a plant auxin-inducible degron system. The homologous genes of the beta subunit of FT, GGT-1 and GGT-2 were endogenously tagged with AID at the C-terminus in the TIR1 parental line using a CRISPR-Cas9 approach. Upon depletion of these prenyl transferases, GGT-1 and GGT-2 had a strong defect on parasite replication. Fluorescent assay using diverse protein markers showed that the protein markers ROP5 and GRA7 were diffused in the parasites depleted with GGT-1 and GGT-2, while the mitochondrion was strongly affected in parasites depleted with GGT-1. Importantly, depletion of GGT-2 caused the stronger defect to the sorting of rhoptry protein and the parasite morphology. Furthermore, parasite motility was observed to be affected in parasites depleted with GGT-2. Taken together, this study functionally characterized the prenyl transferases, which contributed to an overall understanding of protein prenylation in and potentially in other related parasites.
Topics: Animals; Transferases; Parasites; Toxoplasma; Farnesyltranstransferase; Protein Prenylation; Protein Transport; Protozoan Proteins
PubMed: 37108334
DOI: 10.3390/ijms24087172 -
The EMBO Journal Apr 2020Protein prenylation is essential for many cellular processes including signal transduction, cytoskeletal reorganization, and membrane trafficking. Here, we identify a...
Protein prenylation is essential for many cellular processes including signal transduction, cytoskeletal reorganization, and membrane trafficking. Here, we identify a novel type of protein prenyltransferase, which we named geranylgeranyltransferase type-III (GGTase-III). GGTase-III consists of prenyltransferase alpha subunit repeat containing 1 (PTAR1) and the β subunit of RabGGTase. Using a biotinylated geranylgeranyl analogue, we identified the Golgi SNARE protein Ykt6 as a substrate of GGTase-III. GGTase-III transfers a geranylgeranyl group to mono-farnesylated Ykt6, generating doubly prenylated Ykt6. The crystal structure of GGTase-III in complex with Ykt6 provides structural basis for Ykt6 double prenylation. In GGTase-III-deficient cells, Ykt6 remained in a singly prenylated form, and the Golgi SNARE complex assembly was severely impaired. Consequently, the Golgi apparatus was structurally disorganized, and intra-Golgi protein trafficking was delayed. Our findings reveal a fourth type of protein prenyltransferase that generates geranylgeranyl-farnesyl Ykt6. Double prenylation of Ykt6 is essential for the structural and functional organization of the Golgi apparatus.
Topics: Alkyl and Aryl Transferases; Animals; Dimethylallyltranstransferase; Golgi Apparatus; Humans; Male; Membrane Fusion; Protein Binding; Protein Multimerization; Protein Prenylation; Protein Transport; R-SNARE Proteins; Rats; Rats, Wistar; SNARE Proteins
PubMed: 32128853
DOI: 10.15252/embj.2019104120 -
International Journal of Molecular... Oct 2023Choroideremia (CHM) is an X-linked chorioretinal dystrophy leading to progressive retinal degeneration that results in blindness by late adulthood. It is caused by...
Choroideremia (CHM) is an X-linked chorioretinal dystrophy leading to progressive retinal degeneration that results in blindness by late adulthood. It is caused by mutations in the gene encoding the Rab Escort Protein 1 (REP1), which plays a crucial role in the prenylation of Rab proteins ensuring correct intracellular trafficking. Gene augmentation is a promising therapeutic strategy, and there are several completed and ongoing clinical trials for treating CHM using adeno-associated virus (AAV) vectors. However, late-phase trials have failed to show significant functional improvements and have raised safety concerns about inflammatory events potentially caused by the use of viruses. Therefore, alternative non-viral therapies are desirable. Episomal scaffold/matrix attachment region (S/MAR)-based plasmid vectors were generated containing the human coding sequence, a GFP reporter gene, and ubiquitous promoters (pS/MAR-CHM). The vectors were assessed in two choroideremia disease model systems: (1) patient-derived fibroblasts and (2) zebrafish, using Western blotting to detect REP1 protein expression and in vitro prenylation assays to assess the rescue of prenylation function. Retinal immunohistochemistry was used to investigate vector expression and photoreceptor morphology in injected zebrafish retinas. The pS/MAR-CHM vectors generated persistent REP1 expression in patient fibroblasts and showed a significant rescue of prenylation function by 75%, indicating correction of the underlying biochemical defect associated with CHM. In addition, GFP and human REP1 expression were detected in zebrafish microinjected with the pS/MAR-CHM at the one-cell stage. Injected zebrafish showed increased survival, prenylation function, and improved retinal photoreceptor morphology. Non-viral S/MAR vectors show promise as a potential gene-augmentation strategy without the use of immunogenic viral components, which could be applicable to many inherited retinal disease genes.
Topics: Animals; Humans; Adult; Choroideremia; Zebrafish; Retina; Mutation; Retinal Dystrophies; Plasmids; Adaptor Proteins, Signal Transducing
PubMed: 37894906
DOI: 10.3390/ijms242015225 -
Journal of Visualized Experiments : JoVE Jan 2020Protein prenylation is a key modification that is responsible for targeting proteins to intracellular membranes. KRAS4b, which is mutated in 22% of human cancers, is...
Protein prenylation is a key modification that is responsible for targeting proteins to intracellular membranes. KRAS4b, which is mutated in 22% of human cancers, is processed by farnesylation and carboxymethylation due to the presence of a 'CAAX' box motif at the C-terminus. An engineered baculovirus system was used to express farnesylated and carboxymethylated KRAS4b in insect cells and has been described previously. Here, we describe the detailed, practical purification and biochemical characterization of the protein. Specifically, affinity and ion exchange chromatography were used to purify the protein to homogeneity. Intact and native mass spectrometry was used to validate the correct modification of KRAS4b and to verify nucleotide binding. Finally, membrane association of farnesylated and carboxymethylated KRAS4b to liposomes was measured using surface plasmon resonance spectroscopy.
Topics: Animals; Baculoviridae; Cell Line; Chromatography, Ion Exchange; Liposomes; Mass Spectrometry; Methylation; Protein Prenylation; Proto-Oncogene Proteins p21(ras); Recombinant Proteins; Reproducibility of Results; Surface Plasmon Resonance
PubMed: 32009649
DOI: 10.3791/60703 -
Angewandte Chemie (International Ed. in... Oct 2015Targeted therapy based on protein-drug conjugates has attracted significant attention owing to its high efficacy and low side effects. However, efficient and stable drug...
Targeted therapy based on protein-drug conjugates has attracted significant attention owing to its high efficacy and low side effects. However, efficient and stable drug conjugation to a protein binder remains a challenge. Herein, a chemoenzymatic method to generate highly stable and homogenous drug conjugates with high efficiency is presented. The approach comprises the insertion of the CaaX sequence at the C-terminal end of the protein binder, prenylation using farnesyltransferase, and drug conjugation through an oxime ligation reaction. MMAF and an EGFR-specific repebody are used as the antitumor agent and protein binder, respectively. The method enables the precisely controlled synthesis of repebody-drug conjugates with high yield and homogeneity. The utility of this approach is illustrated by the notable stability of the repebody-drug conjugates in human plasma, negligible off-target effects, and a remarkable antitumor activity in vivo. The present method can be widely used for generating highly homogeneous and stable PDCs for targeted therapy.
Topics: Animals; Antineoplastic Agents; Cell Line, Tumor; Drug Delivery Systems; ErbB Receptors; Farnesyltranstransferase; Humans; Mice, Nude; Models, Molecular; Neoplasms; Oligopeptides; Oximes; Protein Binding; Protein Prenylation; Proteins
PubMed: 26315561
DOI: 10.1002/anie.201505964 -
Nature May 2022Imbalances in lipid homeostasis can have deleterious effects on health. Yet how cells sense metabolic demand due to lipid depletion and respond by increasing nutrient...
Imbalances in lipid homeostasis can have deleterious effects on health. Yet how cells sense metabolic demand due to lipid depletion and respond by increasing nutrient absorption remains unclear. Here we describe a mechanism for intracellular lipid surveillance in Caenorhabditis elegans that involves transcriptional inactivation of the nuclear hormone receptor NHR-49 through its cytosolic sequestration to endocytic vesicles via geranylgeranyl conjugation to the small G protein RAB-11.1. Defective de novo isoprenoid synthesis caused by lipid depletion limits RAB-11.1 geranylgeranylation, which promotes nuclear translocation of NHR-49 and activation of rab-11.2 transcription to enhance transporter residency at the plasma membrane. Thus, we identify a critical lipid sensed by the cell, its conjugated G protein, and the nuclear receptor whose dynamic interactions enable cells to sense metabolic demand due to lipid depletion and respond by increasing nutrient absorption and lipid metabolism.
Topics: Animals; Caenorhabditis elegans; Caenorhabditis elegans Proteins; Lipids; Monomeric GTP-Binding Proteins; Protein Prenylation; Receptors, Cytoplasmic and Nuclear
PubMed: 35585236
DOI: 10.1038/s41586-022-04729-7 -
Nature Chemistry Jun 2019Post-translational farnesylation or geranylgeranylation at a C-terminal cysteine residue regulates the localization and function of over 100 proteins, including the Ras...
Post-translational farnesylation or geranylgeranylation at a C-terminal cysteine residue regulates the localization and function of over 100 proteins, including the Ras isoforms, and is a therapeutic target in diseases including cancer and infection. Here, we report global and selective profiling of prenylated proteins in living cells enabled by the development of isoprenoid analogues YnF and YnGG in combination with quantitative chemical proteomics. Eighty prenylated proteins were identified in a single human cell line, 64 for the first time at endogenous abundance without metabolic perturbation. We further demonstrate that YnF and YnGG enable direct identification of post-translationally processed prenylated peptides, proteome-wide quantitative analysis of prenylation dynamics and alternative prenylation in response to four different prenyltransferase inhibitors, and quantification of defective Rab prenylation in a model of the retinal degenerative disease choroideremia.
Topics: Adaptor Proteins, Signal Transducing; Alkynes; Animals; Cell Line; Gene Knockout Techniques; Humans; Mass Spectrometry; Mice, Knockout; Molecular Probes; Protein Prenylation; Proteins; Proteome; Proteomics
PubMed: 30936521
DOI: 10.1038/s41557-019-0237-6 -
Archives of Biochemistry and Biophysics Apr 2019In this study we examined whether the action of simvastatin affects re-differentiation of passaged chondrocytes and if so, whether this was mediated via changes in...
In this study we examined whether the action of simvastatin affects re-differentiation of passaged chondrocytes and if so, whether this was mediated via changes in cholesterol or cholesterol intermediates. Bovine articular chondrocytes, of varying passage number, human knee chondrocytes and rat chondrosarcoma chondrocytes were treated with simvastatin and examined for changes in mRNA and protein expression of markers of the chondrocyte phenotype as well as changes in cell shape, proliferation and proteoglycan production. In all three models, while still in monolayer culture, simvastatin treatment alone promoted changes in phenotype and morphology indicative of re-differentiation most prominent being an increase in SOX9 mRNA and protein expression. In passaged bovine chondrocytes, simvastatin stimulated the expression of SOX9, ACAN, BMP2 and inhibited the expression of COL1 and α-smooth muscle actin. Co-treatment of chondrocytes with simvastatin plus exogenous cholesterol-conditions that had previously reversed the inhibition on CD44 shedding, did not alter the effects of simvastatin on re-differentiation. However, the co-treatment of chondrocytes with simvastatin together with other pathway intermediates, mevalonate, geranylgeranylpyrophosphate and to a lesser extent, farnesylpyrophosphate, blocked the pro-differentiation effects of simvastatin. Treatment with simvastatin stimulated expression of SOX9 and COL2a and enhanced SOX9 protein in human OA chondrocytes. The co-treatment of OA chondrocytes with mevalonate or geranylgeranylpyrophosphate, but not cholesterol, blocked the simvastatin effects. These results lead us to conclude that the blocking of critical protein prenylation events is required for the positive effects of simvastatin on the re-differentiation of chondrocytes.
Topics: Animals; Cattle; Cells, Cultured; Chondrocytes; Humans; Rats; Simvastatin
PubMed: 30776329
DOI: 10.1016/j.abb.2019.01.038 -
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 -
PloS One 2015Protein prenylation is a widespread and highly conserved eukaryotic post-translational modification that endows proteins with the ability to reversibly attach to...
Protein prenylation is a widespread and highly conserved eukaryotic post-translational modification that endows proteins with the ability to reversibly attach to intracellular membranes. The dynamic interaction of prenylated proteins with intracellular membranes is essential for their signalling functions and is frequently deregulated in disease processes such as cancer. As a result, protein prenylation has been pharmacologically targeted by numerous drug discovery programs, albeit with limited success. To a large extent, this can be attributed to an insufficient understanding of the interplay of different protein prenyltransferases and the combinatorial diversity of the prenylatable sequence space. Here, we report a high-throughput, growth-based genetic selection assay in Saccharomyces cerevisiae based on the Ras Recruitment System which, for the first time, has allowed us to create a comprehensive map of prenylatable protein sequences in S. cerevisiae. We demonstrate that potential prenylatable space is sparsely (6.2%) occupied leaving room for creation of synthetic orthogonal prenylatable sequences. To experimentally demonstrate that, we used the developed platform to engineer mutant farnesyltransferases that efficiently prenylate substrate motives that are not recognised by endogenous protein prenyltransferases. These uncoupled mutants can now be used as starting points for the systematic engineering of the eukaryotic protein prenylation machinery.
Topics: Cloning, Molecular; Drug Discovery; Farnesyltranstransferase; High-Throughput Nucleotide Sequencing; Protein Engineering; Protein Prenylation; Saccharomyces cerevisiae
PubMed: 25768003
DOI: 10.1371/journal.pone.0120716