-
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 -
Bioconjugate Chemistry Dec 2015Site-specific protein labeling is an important technique in protein chemistry and is used for diverse applications ranging from creating protein conjugates to protein...
Site-specific protein labeling is an important technique in protein chemistry and is used for diverse applications ranging from creating protein conjugates to protein immobilization. Enzymatic reactions, including protein prenylation, have been widely exploited as methods to accomplish site-specific labeling. Enzymatic prenylation is catalyzed by prenyltransferases, including protein farnesyltransferase (PFTase) and geranylgeranyltransferase type I (GGTase-I), both of which recognize C-terminal CaaX motifs with different specificities and transfer prenyl groups from isoprenoid diphosphates to their respective target proteins. A number of isoprenoid analogues containing bioorthogonal functional groups have been used to label proteins of interest via PFTase-catalyzed reaction. In this study, we sought to expand the scope of prenyltransferase-mediated protein labeling by exploring the utility of rat GGTase-I (rGGTase-I). First, the isoprenoid specificity of rGGTase-I was evaluated by screening eight different analogues and it was found that those with bulky moieties and longer backbone length were recognized by rGGTase-I more efficiently. Taking advantage of the different substrate specificities of rat PFTase (rPFTase) and rGGTase-I, we then developed a simultaneous dual labeling method to selectively label two different proteins by using isoprenoid analogue and CaaX substrate pairs that were specific to only one of the prenyltransferases. Using two model proteins, green fluorescent protein with a C-terminal CVLL sequence (GFP-CVLL) and red fluorescent protein with a C-terminal CVIA sequence (RFP-CVIA), we demonstrated that when incubated together with both prenyltransferases and the selected isoprenoid analogues, GFP-CVLL was specifically modified with a ketone-functionalized analogue by rGGTase-I and RFP-CVIA was selectively labeled with an alkyne-containing analogue by rPFTase. By switching the ketone-containing analogue to an azide-containing analogue, it was possible to create protein tail-to-tail dimers in a one-pot procedure through the copper(I)-catalyzed alkyne-azide cycloaddition (CuAAC) reaction. Overall, with the flexibility of using different isoprenoid analogues, this system greatly extends the utility of protein labeling using prenyltransferases.
Topics: Alkyl and Aryl Transferases; Animals; Green Fluorescent Proteins; Luminescent Proteins; Models, Molecular; Protein Prenylation; Rats; Staining and Labeling; Substrate Specificity; Terpenes; Red Fluorescent Protein
PubMed: 26561785
DOI: 10.1021/acs.bioconjchem.5b00553 -
The Journal of Biological Chemistry Dec 2006Hutchinson-Gilford progeria syndrome (HGPS) is caused by a LMNA mutation that leads to the synthesis of a mutant prelamin A that is farnesylated but cannot be further... (Review)
Review
Hutchinson-Gilford progeria syndrome (HGPS) is caused by a LMNA mutation that leads to the synthesis of a mutant prelamin A that is farnesylated but cannot be further processed to mature lamin A. A more severe progeroid disorder, restrictive dermopathy (RD), is caused by the loss of the prelamin A-processing enzyme, ZMPSTE24. The absence of ZMPSTE24 prevents the endoproteolytic processing of farnesyl-prelamin A to mature lamin A and leads to the accumulation of farnesyl-prelamin A. In both HGPS and RD, the farnesyl-prelamin A is targeted to the nuclear envelope, where it interferes with the integrity of the nuclear envelope and causes misshapen cell nuclei. Recent studies have shown that the frequency of misshapen nuclei can be reduced by treating cells with a farnesyltransferase inhibitor (FTI). Also, administering an FTI to mouse models of HGPS and RD ameliorates the phenotypes of progeria. These studies have prompted interest in testing the efficacy of FTIs in children with HGPS.
Topics: Animals; Humans; Lamin Type A; Nuclear Proteins; Progeria; Protein Precursors; Protein Prenylation; Syndrome
PubMed: 17090536
DOI: 10.1074/jbc.R600033200 -
Proceedings of the National Academy of... Aug 1999Protein prenylation in plants was studied by in vivo metabolic (3)H-mevalonate labeling in combination with a range of protein synthesis inhibitors. In spinach...
Protein prenylation in plants was studied by in vivo metabolic (3)H-mevalonate labeling in combination with a range of protein synthesis inhibitors. In spinach cotyledons, this posttranslational protein modification was found to be divided into two categories, one representing the conventional prenylation involving farnesyl and geranylgeranyl groups bound to cysteine residues via thioether linkages. This category revealed a similar pattern of prenylated proteins to that observed in mammalian cells and depends on nuclear gene expression. The other category was shown to represent a type of prenylation confined to chloroplasts. It depends on plastid gene expression and does not involve a thioether bond. The modifying isoprenoid could be released from the chloroplastic polypeptides by alkaline treatment and was identified as phytol upon GC-MS analysis. The phytol could readily be derived from all-trans-[(3)H]farnesol, which, like all-trans-[(3)H]geranylgeraniol, was taken up by the cotyledons, resulting in incorporation of radiolabel into proteins.
Topics: Animals; Cell Fractionation; Chloroplasts; Cotyledon; Diterpenes; Farnesol; Gas Chromatography-Mass Spectrometry; Mammals; Mevalonic Acid; Plant Proteins; Protein Prenylation; Protein Synthesis Inhibitors; Spinacia oleracea; Tritium
PubMed: 10468564
DOI: 10.1073/pnas.96.18.10074 -
Nature Reviews. Cancer Oct 2011Protein farnesylation and geranylgeranylation, together referred to as prenylation, are lipid post-translational modifications that are required for the transforming...
Protein farnesylation and geranylgeranylation, together referred to as prenylation, are lipid post-translational modifications that are required for the transforming activity of many oncogenic proteins, including some RAS family members. This observation prompted the development of inhibitors of farnesyltransferase (FT) and geranylgeranyl-transferase 1 (GGT1) as potential anticancer drugs. In this Review, we discuss the mechanisms by which FT and GGT1 inhibitors (FTIs and GGTIs, respectively) affect signal transduction pathways, cell cycle progression, proliferation and cell survival. In contrast to their preclinical efficacy, only a small subset of patients responds to FTIs. Identifying tumours that depend on farnesylation for survival remains a challenge, and strategies to overcome this are discussed. One GGTI has recently entered the clinic, and the safety and efficacy of GGTIs await results from clinical trials.
Topics: Alkyl and Aryl Transferases; Animals; Antineoplastic Agents; Cell Cycle; Cell Proliferation; Cell Survival; Enzyme Inhibitors; Farnesyltranstransferase; Humans; Molecular Targeted Therapy; Monomeric GTP-Binding Proteins; Neoplasms; Neuropeptides; Protein Prenylation; Randomized Controlled Trials as Topic; Ras Homolog Enriched in Brain Protein; Signal Transduction
PubMed: 22020205
DOI: 10.1038/nrc3151 -
Molecular and Cellular Biology Mar 2000We have recently reported that skeletal muscle of the ob/ob mouse, an animal model of genetic obesity with extreme insulin resistance, exhibits alterations in the...
We have recently reported that skeletal muscle of the ob/ob mouse, an animal model of genetic obesity with extreme insulin resistance, exhibits alterations in the expression of multiple genes. Analysis and cloning of a full-length cDNA of one of the overexpressed mRNAs revealed a 300-amino-acid protein that could be identified as the mouse geranylgeranyl diphosphate synthase (GGPP synthase) based on its homology to proteins cloned from yeast and fungus. GGPP synthase catalyzes the synthesis of all-trans-geranylgeranyl diphosphate (GGPP), an isoprenoid used for protein isoprenylation in animal cells, and is a branch point enzyme in the mevalonic acid pathway. Three mRNAs for GGPP synthase of 4.3, 3.2, and 1.7 kb were detected in Northern blot analysis. Western blot analysis of tissue homogenates using specific antipeptide antibodies revealed a single band of 34.8 kDa. Expression level of this protein in different tissues correlated with expression of the 4.3- and 3.2-kb mRNAs. GGPP synthase mRNA expression was increased 5- to 20-fold in skeletal muscle, liver, and fat of ob/ob mice by Northern blot analysis. Western blot analysis also showed a twofold overexpression of the protein in muscle and fat but not in liver, where the dominant isoform is encoded by the 1.7-kb mRNA. Differentiation of 3T3-L1 fibroblasts into adipocytes induced GGPP synthase expression more than 20-fold. Using the immunoprecipitated protein, we found that mammalian GGPP synthase synthesizes not only GGPP but also its metabolic precursor farnesyl diphosphate. Thus, the expression of GGPP synthase is regulated in multiple tissues in obesity and is induced during adipocyte differentiation. Altered regulation in the synthesis of isoprenoids for protein prenylation in obesity might be a factor determining the ability of the cells to respond to hormonal stimulation requiring both Ras-related small GTPases and trimeric G protein-coupled receptors.
Topics: Adipocytes; Alkyl and Aryl Transferases; Amino Acid Sequence; Animals; Cell Differentiation; Cloning, Molecular; Farnesyltranstransferase; Mice; Mice, Obese; Molecular Sequence Data; Obesity; Organ Specificity; Protein Isoforms; Protein Prenylation; RNA, Messenger; Sequence Alignment
PubMed: 10688662
DOI: 10.1128/MCB.20.6.2158-2166.2000 -
Acta Neuropathologica Communications Jul 2021The pathogenic mechanisms underlying the development of Alzheimer's disease (AD) remain elusive and to date there are no effective prevention or treatment for AD....
Protein farnesylation is upregulated in Alzheimer's human brains and neuron-specific suppression of farnesyltransferase mitigates pathogenic processes in Alzheimer's model mice.
The pathogenic mechanisms underlying the development of Alzheimer's disease (AD) remain elusive and to date there are no effective prevention or treatment for AD. Farnesyltransferase (FT) catalyzes a key posttranslational modification process called farnesylation, in which the isoprenoid farnesyl pyrophosphate is attached to target proteins, facilitating their membrane localization and their interactions with downstream effectors. Farnesylated proteins, including the Ras superfamily of small GTPases, are involved in regulating diverse physiological and pathological processes. Emerging evidence suggests that isoprenoids and farnesylated proteins may play an important role in the pathogenesis of AD. However, the dynamics of FT and protein farnesylation in human brains and the specific role of neuronal FT in the pathogenic progression of AD are not known. Here, using postmortem brain tissue from individuals with no cognitive impairment (NCI), mild cognitive impairment (MCI), or Alzheimer's dementia, we found that the levels of FT and membrane-associated H-Ras, an exclusively farnesylated protein, and its downstream effector ERK were markedly increased in AD and MCI compared with NCI. To elucidate the specific role of neuronal FT in AD pathogenesis, we generated the transgenic AD model APP/PS1 mice with forebrain neuron-specific FT knockout, followed by a battery of behavioral assessments, biochemical assays, and unbiased transcriptomic analysis. Our results showed that the neuronal FT deletion mitigates memory impairment and amyloid neuropathology in APP/PS1 mice through suppressing amyloid generation and reversing the pathogenic hyperactivation of mTORC1 signaling. These findings suggest that aberrant upregulation of protein farnesylation is an early driving force in the pathogenic cascade of AD and that targeting FT or its downstream signaling pathways presents a viable therapeutic strategy against AD.
Topics: Aged, 80 and over; Alzheimer Disease; Amyloid beta-Protein Precursor; Animals; Behavior, Animal; Brain; Cognitive Dysfunction; Disease Models, Animal; Extracellular Signal-Regulated MAP Kinases; Farnesyltranstransferase; Female; Humans; Male; Mechanistic Target of Rapamycin Complex 1; Mice; Mice, Knockout; Mice, Transgenic; Neurons; Plaque, Amyloid; Presenilin-1; Protein Prenylation; Proto-Oncogene Proteins p21(ras); Signal Transduction
PubMed: 34315531
DOI: 10.1186/s40478-021-01231-5 -
Memorias Do Instituto Oswaldo Cruz Aug 2011The development of new drugs is one strategy for malaria control. Biochemical pathways localised in the apicoplast of the parasite, such as the synthesis of isoprenic... (Review)
Review
The development of new drugs is one strategy for malaria control. Biochemical pathways localised in the apicoplast of the parasite, such as the synthesis of isoprenic precursors, are excellent targets because they are different or absent in the human host. Isoprenoids are a large and highly diverse group of natural products with many functions and their synthesis is essential for the parasite's survival. During the last few years, the genes, enzymes, intermediates and mechanisms of this biosynthetic route have been elucidated. In this review, we comment on some aspects of the methylerythritol phosphate pathway and discuss the presence of diverse isoprenic products such as dolichol, ubiquinone, carotenoids, menaquinone and isoprenylated proteins, which are biosynthesised during the intraerythrocytic stages of Plasmodium falciparum.
Topics: Carotenoids; Dolichols; Erythrocytes; Humans; Plasmodium falciparum; Protein Prenylation; Terpenes; Ubiquinone; Vitamin K 2
PubMed: 21881768
DOI: 10.1590/s0074-02762011000900018 -
Neuroscience Aug 2016Evidence suggests that 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors, or statins, may reduce the risk of Alzheimer's disease (AD). Statin action in patients...
Evidence suggests that 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors, or statins, may reduce the risk of Alzheimer's disease (AD). Statin action in patients with AD, as in those with heart disease, is likely to be at least partly independent of the effects of statins on cholesterol. Statins can alter cellular signaling and protein trafficking through inhibition of isoprenylation of Rho, Cdc42, and Rab family GTPases. The effects of statins on protein isoprenylation in vivo, particularly in the central nervous system, are poorly studied. We utilized two-dimensional gel electrophoresis approaches to directly monitor the levels of isoprenylated and non-isoprenylated forms of Rho and Rab family GTPases. We report that simvastatin significantly inhibits RhoA and Rab4, and Rab6 isoprenylation at doses as low as 50nM in vitro. We also provide the first in vivo evidence that statins inhibit the isoprenylation of RhoA in the brains of rats and RhoA, Cdc42, and H-Ras in the brains of mice treated with clinically relevant doses of simvastatin.
Topics: Animals; Blotting, Western; Brain; Cell Line, Tumor; Central Nervous System Agents; Drug Evaluation, Preclinical; Electrophoresis, Gel, Two-Dimensional; Hydroxymethylglutaryl-CoA Reductase Inhibitors; Isoelectric Focusing; Mice, Inbred C57BL; Protein Prenylation; Proto-Oncogene Proteins p21(ras); Rats, Inbred SHR; Simvastatin; cdc42 GTP-Binding Protein; rhoA GTP-Binding Protein
PubMed: 27180285
DOI: 10.1016/j.neuroscience.2016.04.053 -
Molecular Biology of the Cell Aug 2021G protein beta-gamma (Gβγ) subunits anchor to the plasma membrane (PM) through the carboxy-terminal (CT) prenyl group in Gγ. This interaction is crucial for the PM...
G protein beta-gamma (Gβγ) subunits anchor to the plasma membrane (PM) through the carboxy-terminal (CT) prenyl group in Gγ. This interaction is crucial for the PM localization and functioning of Gβγ, allowing GPCR-G protein signaling to proceed. The diverse Gγ family has 12 members, and we have recently shown that the signaling efficacies of major Gβγ effectors are Gγ-type dependent. This dependency is due to the distinct series of membrane-interacting abilities of Gγ. However, the molecular process allowing for Gβγ subunits to exhibit a discrete and diverse range of Gγ-type-dependent membrane affinities is unclear and cannot be explained using only the type of prenylation. The present work explores the unique designs of membrane-interacting CT residues in Gγ as a major source for this Gγ-type-dependent Gβγ signaling. Despite the type of prenylation, the results show signaling efficacy at the PM, and associated cell behaviors of Gβγ are governed by crucially located specific amino acids in the five to six residue preprenylation region of Gγ. The provided molecular picture of Gγ-membrane interactions may explain how cells gain Gγ-type-dependent G protein-GPCR signaling as well as how Gβγ elicits selective signaling at various subcellular compartments.
Topics: Cell Membrane; GTP-Binding Protein beta Subunits; GTP-Binding Protein gamma Subunits; HeLa Cells; Humans; Protein Prenylation; Signal Transduction
PubMed: 34106735
DOI: 10.1091/mbc.E20-11-0750