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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 -
PloS One 2014Dysregulation of Ras and Rho family small GTPases drives the invasion and metastasis of multiple cancers. For their biological functions, these GTPases require proper...
Dysregulation of Ras and Rho family small GTPases drives the invasion and metastasis of multiple cancers. For their biological functions, these GTPases require proper subcellular localization to cellular membranes, which is regulated by a series of post-translational modifications that result in either farnesylation or geranylgeranylation of the C-terminal CAAX motif. This concept provided the rationale for targeting farnesyltransferase (FTase) and geranylgeranyltransferases (GGTase) for cancer treatment. However, the resulting prenyl transferase inhibitors have not performed well in the clinic due to issues with alternative prenylation and toxicity. As an alternative, we have developed a unique class of potential anti-cancer therapeutics called Prenyl Function Inhibitors (PFIs), which are farnesol or geranyl-geraniol analogs that act as alternate substrates for FTase or GGTase. Here, we test the ability of our lead PFIs, anilinogeraniol (AGOH) and anilinofarnesol (AFOH), to block the invasion of breast cancer cells. We found that AGOH treatment effectively decreased invasion of MDA-MB-231 cells in a two-dimensional (2D) invasion assay at 100 µM while it blocked invasive growth in three-dimensional (3D) culture model at as little as 20 µM. Notably, the effect of AGOH on 3D invasive growth was phenocopied by electroporation of cells with C3 exotransferase. To determine if RhoA and RhoC were direct targets of AGOH, we performed Rho activity assays in MDA-MB-231 and MDA-MB-468 cells and found that AGOH blocked RhoA and RhoC activation in response to LPA and EGF stimulation. Notably, the geranylgeraniol analog AFOH was more potent than AGOH in inhibiting RhoA and RhoC activation and invasive growth. Interestingly, neither AGOH nor AFOH impacted 3D growth of MCF10A cells. Collectively, this study demonstrates that AGOH and AFOH dramatically inhibit breast cancer invasion, at least in part by blocking Rho function, thus, suggesting that targeting prenylation by using PFIs may offer a promising mechanism for treatment of invasive breast cancer.
Topics: Acyclic Monoterpenes; Antineoplastic Agents; Breast Neoplasms; Cell Culture Techniques; Farnesol; Female; Fluorescent Antibody Technique; Humans; Immunoblotting; Molecular Structure; Neoplasm Invasiveness; Protein Prenylation; Terpenes; rho GTP-Binding Proteins
PubMed: 24587105
DOI: 10.1371/journal.pone.0089892 -
Genome Biology 2005We refined the motifs for carboxy-terminal protein prenylation by analysis of known substrates for farnesyltransferase (FT), geranylgeranyltransferase I (GGT1) and...
We refined the motifs for carboxy-terminal protein prenylation by analysis of known substrates for farnesyltransferase (FT), geranylgeranyltransferase I (GGT1) and geranylgeranyltransferase II (GGT2). In addition to the CaaX box for the first two enzymes, we identify a preceding linker region that appears constrained in physicochemical properties, requiring small or flexible, preferably hydrophilic, amino acids. Predictors were constructed on the basis of sequence and physical property profiles, including interpositional correlations, and are available as the Prenylation Prediction Suite (PrePS, http://mendel.imp.univie.ac.at/sat/PrePS) which also allows evaluation of evolutionary motif conservation. PrePS can predict partially overlapping substrate specificities, which is of medical importance in the case of understanding cellular action of FT inhibitors as anticancer and anti-parasite agents.
Topics: Alkyl and Aryl Transferases; Amino Acid Motifs; Amino Acid Sequence; Animals; Computational Biology; Conserved Sequence; Evolution, Molecular; Humans; Internet; Models, Molecular; Molecular Sequence Data; Protein Prenylation; Reproducibility of Results; Sequence Alignment; Software; Substrate Specificity
PubMed: 15960807
DOI: 10.1186/gb-2005-6-6-r55 -
Disease Models & Mechanisms May 2024Prenylated proteins are prevalent in eukaryotic biology (∼1-2% of proteins) and are associated with human disease, including cancer, premature aging and infections....
Prenylated proteins are prevalent in eukaryotic biology (∼1-2% of proteins) and are associated with human disease, including cancer, premature aging and infections. Prenylated proteins with a C-terminal CaaX sequence are targeted by CaaX-type prenyltransferases and proteases. To aid investigations of these enzymes and their targets, we developed Saccharomyces cerevisiae strains that express these human enzymes instead of their yeast counterparts. These strains were developed in part to explore human prenyltransferase specificity because of findings that yeast FTase has expanded specificity for sequences deviating from the CaaX consensus (i.e. atypical sequence and length). The humanized yeast strains displayed robust prenyltransferase activity against CaaX sequences derived from human and pathogen proteins containing typical and atypical CaaX sequences. The system also recapitulated prenylation of heterologously expressed human proteins (i.e. HRas and DNAJA2). These results reveal that substrate specificity is conserved for yeast and human farnesyltransferases but is less conserved for type I geranylgeranyltransferases. These yeast systems can be easily adapted for investigating the prenylomes of other organisms and are valuable new tools for helping define the human prenylome, which includes physiologically important proteins for which the CaaX modification status is unknown.
Topics: Humans; Saccharomyces cerevisiae; Protein Prenylation; Substrate Specificity; Amino Acid Sequence; Dimethylallyltranstransferase; Viral Proteins; Alkyl and Aryl Transferases
PubMed: 38818856
DOI: 10.1242/dmm.050516 -
Journal of Lipid Research Jan 2006Some proteins undergo posttranslational modification by the addition of an isoprenyl lipid (farnesyl- or geranylgeranyl-isoprenoid) to a cysteine residue proximal to the... (Review)
Review
Some proteins undergo posttranslational modification by the addition of an isoprenyl lipid (farnesyl- or geranylgeranyl-isoprenoid) to a cysteine residue proximal to the C terminus. Protein isoprenylation promotes membrane association and contributes to protein-protein interactions. Farnesylated proteins include small GTPases, tyrosine phosphatases, nuclear lamina, cochaperones, and centromere-associated proteins. Prenylation is required for the transforming activity of Ras. Because of the high frequency of Ras mutations in cancer, farnesyl transferase inhibitors (FTIs) were investigated as a means to antagonize Ras function. Evaluation of FTIs led to the finding that both K- and N-Ras are alternatively modified by geranylgeranyl prenyltransferase-1 in FTI-treated cells. Geranylgeranylated forms of Ras retain the ability to associate with the plasma membrane and activate substrates. Despite this, FTIs are effective at inhibiting the growth of human tumor cells in vitro, suggesting that activity is dependent on blocking the farnesylation of other proteins. FTIs also inhibit the in vivo growth of human tumor xenografts and sensitize these models to chemotherapeutics, most notably taxanes. Several FTIs have entered clinical trials for various cancer indications. In some clinical settings, primarily hematologic malignancies, FTIs have displayed evidence of single-agent activity. Clinical studies in progress are exploring the antitumor activity of FTIs as single agents and in combination. This review will summarize the basic biology of FTIs, their antitumor activity in preclinical models, and the current status of clinical studies with these agents.
Topics: Animals; Antineoplastic Agents; Cell Line, Tumor; Enzyme Inhibitors; Farnesyltranstransferase; Humans; Lipid Metabolism; Neoplasms; Protein Prenylation; Protein Processing, Post-Translational; ras Proteins
PubMed: 16278491
DOI: 10.1194/jlr.R500012-JLR200 -
European Journal of Biochemistry Jul 2002In this review, we summarize recent progress in studying three main classes of prenyltransferases: (a) isoprenyl pyrophosphate synthases (IPPSs), which catalyze chain... (Comparative Study)
Comparative Study Review
In this review, we summarize recent progress in studying three main classes of prenyltransferases: (a) isoprenyl pyrophosphate synthases (IPPSs), which catalyze chain elongation of allylic pyrophosphate substrates via consecutive condensation reactions with isopentenyl pyrophosphate (IPP) to generate linear polymers with defined chain lengths; (b) protein prenyltransferases, which catalyze the transfer of an isoprenyl pyrophosphate (e.g. farnesyl pyrophosphate) to a protein or a peptide; (c) prenyltransferases, which catalyze the cyclization of isoprenyl pyrophosphates. The prenyltransferase products are widely distributed in nature and serve a variety of important biological functions. The catalytic mechanism deduced from the 3D structure and other biochemical studies of these prenyltransferases as well as how the protein functions are related to their reaction mechanism and structure are discussed. In the IPPS reaction, we focus on the mechanism that controls product chain length and the reaction kinetics of IPP condensation in the cis-type and trans-type enzymes. For protein prenyltransferases, the structures of Ras farnesyltransferase and Rab geranylgeranyltransferase are used to elucidate the reaction mechanism of this group of enzymes. For the enzymes involved in cyclic terpene biosynthesis, the structures and mechanisms of squalene cyclase, 5-epi-aristolochene synthase, pentalenene synthase, and trichodiene synthase are summarized.
Topics: Amino Acid Sequence; Animals; Binding Sites; Dimethylallyltranstransferase; Humans; Kinetics; Models, Molecular; Molecular Sequence Data; Oxidoreductases; Polyisoprenyl Phosphates; Protein Conformation; Protein Prenylation; Protein Processing, Post-Translational; Sequence Alignment; Sequence Homology, Amino Acid; Structure-Activity Relationship
PubMed: 12135472
DOI: 10.1046/j.1432-1033.2002.03014.x -
Small GTPases Mar 2018Rab proteins are the major regulators of vesicular trafficking in eukaryotic cells. Their activity can be tightly controlled within cells: Regulated by guanine... (Review)
Review
Rab proteins are the major regulators of vesicular trafficking in eukaryotic cells. Their activity can be tightly controlled within cells: Regulated by guanine nucleotide exchange factors (GEFs) and GTPase activating proteins (GAPs), they switch between an active GTP-bound state and an inactive GDP-bound state, interacting with downstream effector proteins only in the active state. Additionally, they can bind to membranes via C-terminal prenylated cysteine residues and they can be solubilized and shuttled between membranes by chaperone-like molecules called GDP dissociation inhibitors (GDIs). In this review we give an overview of Rab proteins with a focus on the current understanding of their regulation by GEFs, GAPs and GDI.
Topics: Amino Acid Sequence; Animals; Cell Membrane; GTPase-Activating Proteins; Guanine Nucleotide Dissociation Inhibitors; Guanine Nucleotide Exchange Factors; Humans; Protein Prenylation; rab GTP-Binding Proteins
PubMed: 28055292
DOI: 10.1080/21541248.2016.1276999 -
ACS Synthetic Biology Jan 2023Reversible membrane targeting of proteins is one of the key regulators of cellular interaction networks, for example, for signaling and polarization. So-called "membrane...
Reversible membrane targeting of proteins is one of the key regulators of cellular interaction networks, for example, for signaling and polarization. So-called "membrane switches" are thus highly attractive targets for the design of minimal cells but have so far been tricky to reconstitute in vitro. Here, we introduce cell-free prenylated protein synthesis (CFpPS), which enables the synthesis and membrane targeting of proteins in a single reaction mix including the prenylation machinery. CFpPS can confer membrane affinity to any protein via addition of a 4-peptide motif to its C-terminus and offers robust production of prenylated proteins not only in their soluble forms but also in the direct vicinity of biomimetic membranes. Thus, CFpPS enabled us to reconstitute the prenylated polarity hub Cdc42 and its regulatory protein in vitro, implementing a key membrane switch. We propose CFpPS to be a versatile and effective platform for engineering complex features, such as polarity induction, in synthetic cells.
Topics: Protein Prenylation; Peptides; Transcription Factors
PubMed: 36445320
DOI: 10.1021/acssynbio.2c00406 -
Frontiers in Immunology 2019The rare autoinflammatory disease mevalonate kinase deficiency (MKD, which includes HIDS and mevalonic aciduria) is caused by recessive, pathogenic variants in the gene...
The rare autoinflammatory disease mevalonate kinase deficiency (MKD, which includes HIDS and mevalonic aciduria) is caused by recessive, pathogenic variants in the gene encoding mevalonate kinase. Deficiency of this enzyme decreases the synthesis of isoprenoid lipids and thus prevents the normal post-translational prenylation of small GTPase proteins, which then accumulate in their unprenylated form. We recently optimized a sensitive assay capable of detecting unprenylated Rab GTPase proteins in peripheral blood mononuclear cells (PBMCs) and showed that this assay distinguished MKD from other autoinflammatory diseases. We have now analyzed PBMCs from an additional six patients with genetically-confirmed MKD (with different compound heterozygous genotypes), and compared these with PBMCs from three healthy volunteers and four unaffected control individuals heterozygous for the commonest pathogenic variant, . We detected a clear accumulation of unprenylated Rab proteins, as well as unprenylated Rap1A by western blotting, in all six genetically-confirmed MKD patients compared to heterozygous controls and healthy volunteers. Furthermore, in the three subjects for whom measurements of residual mevalonate kinase activity was available, enzymatic activity inversely correlated with the extent of the defect in protein prenylation. Finally, a heterozygous patient presenting with autoinflammatory symptoms did not have defective prenylation, indicating a different cause of disease. These findings support the notion that the extent of loss of enzyme function caused by biallelic variants determines the severity of defective protein prenylation, and the accumulation of unprenylated proteins in PBMCs may be a sensitive and consistent biomarker that could be used to aid, or help rule out, diagnosis of MKD.
Topics: Adult; Biomarkers; Cells, Cultured; Child; Female; Genotype; Hereditary Autoinflammatory Diseases; Humans; Leukocytes, Mononuclear; Male; Mevalonate Kinase Deficiency; Mutation; Phosphorylation; Phosphotransferases (Alcohol Group Acceptor); Protein Prenylation; rap1 GTP-Binding Proteins
PubMed: 31474985
DOI: 10.3389/fimmu.2019.01900 -
Biochemistry Mar 2020Protein prenylation is a posttranslational modification involving the attachment of a C15 or C20 isoprenoid group to a cysteine residue near the C-terminus of the target...
Protein prenylation is a posttranslational modification involving the attachment of a C15 or C20 isoprenoid group to a cysteine residue near the C-terminus of the target substrate by protein farnesyltransferase (FTase) or protein geranylgeranyltransferase type I (GGTase-I), respectively. Both of these protein prenyltransferases recognize a C-terminal "CaaX" sequence in their protein substrates, but recent studies in yeast- and mammalian-based systems have demonstrated FTase can also accept sequences that diverge in length from the canonical four-amino acid motif, such as the recently reported five-amino acid C(x)X motif. In this work, we further expand the substrate scope of FTase by demonstrating sequence-dependent farnesylation of shorter three-amino acid "Cxx" C-terminal sequences using both genetic and biochemical assays. Strikingly, biochemical assays utilizing purified mammalian FTase and Cxx substrates reveal prenyl donor promiscuity leading to both farnesylation and geranylgeranylation of these sequences. These findings expand the substrate pool of sequences that can be potentially prenylated, further refine our understanding of substrate recognition by FTase and GGTase-I, and suggest the possibility of a new class of prenylated proteins within proteomes.
Topics: Amino Acid Motifs; Farnesyltranstransferase; Kinetics; Prenylation; Protein Prenylation; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins; Substrate Specificity
PubMed: 32125828
DOI: 10.1021/acs.biochem.0c00081