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Journal of Cardiovascular Pharmacology Feb 2021Isoprenylation is an important post-transcriptional modification of small GTPases required for their activation and function. Isoprenoids, including farnesyl... (Review)
Review
Isoprenylation is an important post-transcriptional modification of small GTPases required for their activation and function. Isoprenoids, including farnesyl pyrophosphate (FPP) and geranylgeranyl pyrophosphate, are indispensable for isoprenylation by serving as donors of a prenyl moiety to small G proteins. In the human body, isoprenoids are mainly generated by the mevalonate pathway (also known as the cholesterol-synthesis pathway). The hydroxymethylglutaryl coenzyme A reductase catalyzes the first rate-limiting steps of the mevalonate pathway, and its inhibitor (statins) are widely used as lipid-lowering agents. In addition, the FPP synthase is also of critical importance for the regulation of the isoprenoids production, for which the inhibitor is mainly used in the treatment of osteoporosis. Synthetic FPP can be further used to generate geranylgeranyl pyrophosphate and cholesterol. Recent studies suggest a role for isoprenoids in the genesis and development of cardiovascular disorders, such as pathological cardiac hypertrophy, fibrosis, endothelial dysfunction, and fibrotic responses of smooth-muscle cells. Furthermore, statins and FPP synthase inhibitors have also been applied for the management of heart failure and other cardiovascular diseases rather than their clinical use for hyperlipidemia or bone diseases. In this review, we focus on the function of several critical enzymes, including hydroxymethylglutaryl coenzyme A reductase, FPP synthase, farnesyltransferase, and geranylgeranyltransferase in the mevalonate pathway which are involved in regulating the generation of isoprenoids and isoprenylation of small GTPases, and their pathophysiological role in the cardiovascular system. Moreover, we summarize recent research into applications of statins and the FPP synthase inhibitors to treat cardiovascular diseases, rather than for their traditional indications respectively.
Topics: Cardiovascular System; Cholesterol; Farnesyltranstransferase; Geranyltranstransferase; Humans; Hydroxymethylglutaryl CoA Reductases; Mevalonic Acid; Monomeric GTP-Binding Proteins; Polyisoprenyl Phosphates; Protein Prenylation; Sesquiterpenes
PubMed: 33538531
DOI: 10.1097/FJC.0000000000000952 -
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 -
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 -
Journal of Proteomics Aug 2021There are important challenges when investigating individual post-translational modifications (PTMs) or protein interaction network and delineating if PTMs or their... (Review)
Review
There are important challenges when investigating individual post-translational modifications (PTMs) or protein interaction network and delineating if PTMs or their changes and cross-talks are involved during infection, disease initiation or as a result of disease progression. Proteomics and in silico approaches now offer the possibility to complement each other to further understand the regulatory involvement of these modifications in parasites and infection biology. Accordingly, the current review highlights key expressed or altered proteins and PTMs are invisible switches that turn on and off the function of most of the proteins. PTMs include phosphorylation, glycosylation, ubiquitylation, palmitoylation, myristoylation, prenylation, acetylation, methylation, and epigenetic PTMs in P. falciparum which have been recently identified. But also other low-abundant or overlooked PTMs that might be important for the parasite's survival, infectivity, antigenicity, immunomodulation and pathogenesis. We here emphasize the PTMs as regulatory pathways playing major roles in the biology, pathogenicity, metabolic pathways, survival, host-parasite interactions and the life cycle of P. falciparum. Further validations and functional characterizations of such proteins might confirm the discovery of therapeutic targets and might most likely provide valuable data for the treatment of P. falciparum, the main cause of severe malaria in human.
Topics: Animals; Humans; Malaria, Falciparum; Plasmodium falciparum; Protein Processing, Post-Translational; Proteomics; Protozoan Proteins
PubMed: 34089893
DOI: 10.1016/j.jprot.2021.104279 -
Nature Communications Apr 2022The MARCH E3 ubiquitin (Ub) ligase MARCH1 regulates trafficking of major histocompatibility complex class II (MHC II) and CD86, molecules of critical importance to...
The MARCH E3 ubiquitin (Ub) ligase MARCH1 regulates trafficking of major histocompatibility complex class II (MHC II) and CD86, molecules of critical importance to immunity. Here we show, using a genome-wide CRISPR knockout screen, that ubiquitin-like protein 3 (UBL3) is a necessary component of ubiquitination-mediated trafficking of these molecules in mice and in humans. Ubl3-deficient mice have elevated MHC II and CD86 expression on the surface of professional and atypical antigen presenting cells. UBL3 also regulates MHC II and CD86 in human dendritic cells (DCs) and macrophages. UBL3 impacts ubiquitination of MARCH1 substrates, a mechanism that requires UBL3 plasma membrane anchoring via prenylation. Loss of UBL3 alters adaptive immunity with impaired development of thymic regulatory T cells, loss of conventional type 1 DCs, increased number of trogocytic marginal zone B cells, and defective in vivo MHC II and MHC I antigen presentation. In summary, we identify UBL3 as a conserved, critical factor in MARCH1-mediated ubiquitination with important roles in immune responses.
Topics: Animals; B7-2 Antigen; Dendritic Cells; Histocompatibility Antigens Class II; Major Histocompatibility Complex; Mice; Mice, Inbred C57BL; Ubiquitin-Protein Ligases; Ubiquitination; Ubiquitins
PubMed: 35411049
DOI: 10.1038/s41467-022-29524-w -
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 -
Chembiochem : a European Journal of... Sep 2022Regioselective carbon-carbon bond formation belongs to the challenging tasks in organic synthesis. In this context, C-C bond formation catalyzed by...
Regioselective carbon-carbon bond formation belongs to the challenging tasks in organic synthesis. In this context, C-C bond formation catalyzed by 4-dimethylallyltryptophan synthases (4-DMATSs) represents a possible tool to regioselectively synthesize C4-prenylated indole derivatives without site-specific preactivation and circumventing the need of protection groups as used in chemical synthetic approaches. In this study, a toolbox of 4-DMATSs to produce a set of 4-dimethylallyl tryptophan and indole derivatives was identified. Using three wild-type enzymes as well as variants, various C5-substituted tryptophan derivatives as well as N-methyl tryptophan were successfully prenylated with conversions up to 90 %. Even truncated tryptophan derivatives like tryptamine and 3-indole propanoic acid were regioselectively prenylated in position C4. The acceptance of C5-substituted tryptophan derivatives was improved up to 5-fold by generating variants (e. g. T108S). The feasibility of semi-preparative prenylation of selected tryptophan derivatives was successfully demonstrated on 100 mg scale at 15 mM substrate concentration, allowing to reduce the previously published multistep chemical synthetic sequence to just a single step.
Topics: Biocatalysis; Carbon; Dimethylallyltranstransferase; Indoles; Prenylation; Substrate Specificity; Tryptophan
PubMed: 35770709
DOI: 10.1002/cbic.202200311