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PubMed1996

Heterotrimeric G proteins.

Over the past year, the thrust of work in the field of heterotrimeric G proteins has been primarily in the following areas: first, resolution of their three-dimensional structures by X-ray crystallography; second, elucidation of the effect of lipid modifications on the Galpha and Ggamma subunits; third, understanding the role of the Gbetagamma dimer in stimulation of a variety of effectors following receptor activation; and fourth, identification of the points of contact among the Galpha, Gbeta, and Ggamma subunits, and between these subunits and their cognate receptor or effector molecules.

Animals↗
PubMed1997

Reversible palmitoylation of signaling proteins.

Palmitoylation is unique among lipid modifications of proteins in that it is reversible and regulable. Recent advances in the study of palmitoylation include the following: the correlation of this modification with the localization of a signaling protein to specific membrane subdomains; the demonstration of a specific protein-protein interaction that is promoted by palmitoylation; and the identification, characterization, and purification of enzymes catalyzing this modification.

Cell Membrane↗
PubMed1999

Probing the hydrophobic pocket of farnesyltransferase: aromatic substitution of CAAX peptidomimetics leads to highly potent inhibitors.

Cysteine farnesylation at the carboxylate terminal tetrapeptide CAAX of Ras protein is catalyzed by farnesyltransferase. This lipid modification is necessary for regulatory function of both normal and oncogenic Ras. The high frequency of Ras mutation in human cancers has prompted an intensive study on finding ways of controlling oncogenic Ras function. Inhibition of farnesyltransferase is among the most sought after targets for cancer chemotherapy. We report here the design, synthesis and biological characterization of a series of peptidomimetics as farnesyltransferase inhibitors. These compounds are extremely potent towards farnesyltransferase with IC50 values ranging from subnanomolar to low nanomolar concentrations. They have a high selectivity for farnesyltransferase over the closely related geranylgeranyltransferase-I. Structure-activity relationship studies demonstrated that a properly positioned hydrophobic group significantly enhanced inhibition potency, reflecting an improved complementarity to the large hydrophobic pocket in the CAAX binding site.

Alkyl and Aryl Transferases↗
PubMed1998

Coronary artery surgery: the end of the beginning.

In each of the first three decades of myocardial revascularization, conventional treatment has been revised completely. This lecture comments on three areas of discovery that have shaped the evolution of myocardial revascularization: science, technology development, and revascularization. The discoveries in all three areas are inexorably interrelated. The single greatest lesson learned so far is that conduit performance carries more prognostic weight than any other factor. We have observed that vein graft atherosclerosis is predictable, and that the first-generation lipid lowering drugs have a favorable effect in patients who achieve marked LDL reduction. Biologically better revascularization begins with use of the internal thoracic artery for grafting to the anterior descending coronary artery. As the results of internal thoracic artery grafting are widely reported, arterial bypass revascularization has expanded, notably by radial and gastroepiploic arteries. The results of bilateral internal thoracic artery grafting are discussed, including large-scale registry results of internal thoracic artery usage in the United States. The internal thoracic artery is significantly underutilized. Diabetes affects both endoluminal and surgical revascularization. The new pharmacology in cardiology interventions shows promise in diminishing restenosis and thrombosis even in diabetic patients. Conversely, extended internal thoracic artery grafting may also benefit diabetic patients. Now we are entering a new age of minimally invasive coronary surgery. We have passed through the early stages of mini-thoracotomy, and we are moving on to access through 1-cm ports, intrathoracic cannulation, antegrade and retrograde myocardial protection, and computer guided three-dimensional vision and instrumentation. The potential for robotic control adds greater precision, ease of use, and safety. This new technology will be integrated with diagnostic information, intraoperative monitoring, anesthesia and perfusion data, cost accounting, and surgical note transcription. The operating room of the future will package intraoperative information and is adaptable to all surgical specialties. The future of coronary artery surgery will depend on minimally invasive techniques, all-arterial grafting, and selective lipid modification to reduce progressive atherosclerosis. The conclusion of this decade marks the end of the beginning. The new generation of cardiothoracic surgeons will share in an array of technology and research unmatched in previous decades.

Coronary Artery Disease↗
PubMed1999

Protein farnesylation in plants: a greasy tale.

Although farnesylation is required for a number of abscisic acid mediated responses in plants, knowledge of how this lipid modification of proteins regulates specific developmental and physiological processes remains unclear. Recent information from the Arabidopsis genome-sequencing project in combination with mutants deficient in farnesylation should unravel the role(s) of this process in plant signaling.

Abscisic Acid↗
PubMed1999

Plant desaturases: harvesting the fat of the land.

The past few years have witnessed a major upsurge in research towards the goal of modifying the lipid composition of plants. Genes encoding a range of different fatty acid desaturase activities have been cloned, and the evolutionary relationships between and within different classes of enzymes have tentatively been established. The effects of expressing some of these desaturases in heterologous hosts have also been studied, often producing unexpected results which contribute further to our understanding of plant lipid modification. It is to be hoped that, in the near future, the goal of producing unusual and valuable fatty acids in transgenic oilseeds will be achieved on a commercial scale.

Arabidopsis↗
PubMed2001

The treatment potential in preventive cardiology.

The Joint European Societies--European Society of Cardiology, European Atherosclerosis Society and European Society of Hypertension--1998 recommendations on prevention of coronary heart disease (CHD) in clinical practice set priorities and goals. The top priority is patients with established CHD, or other atherosclerotic disease, because they are already under the care of cardiologists and are at high risk of further morbidity and mortality. The lifestyle goals are to stop smoking, make healthy food choices and be active physically. The risk factor goals are a BP < 140/90 mmHg, total cholesterol < 5.0 mmol/l (190 mg/dl) and LDL-cholesterol < 3.0 mmol/l (115 mg/dl). The appropriate use of prophylactic drug therapies--aspirin, beta-blockers, ACE inhibitors, lipid modification therapies and anticoagulants--is also a recommended goal. The final goal is to screen relatives of patients with premature CHD (men < 55 years and women < 65 years). Surveys of clinical practice such as EUROASPIRE (European Action on Secondary Prevention) have shown risk can be further reduced in patients with established CHD because many are not achieving these lifestyle and risk factor goals. So there is considerable potential to raise the standard of preventive care for coronary patients through more effective lifestyle intervention and the use of drug therapies with proven efficacy. For the patient, this will mean a longer life with better quality.

Blood Pressure↗
PubMed2000

Conversion of Tyr361 beta to Leu in mammalian protein farnesyltransferase impairs product release but not substrate recognition.

Protein farnesyltransferase catalyzes the lipid modification of protein substrates containing Met, Ser, Gln, or Ala at their C-terminus. A closely related enzyme, protein geranylgeranyltransferase type I, carries out a similar modification of protein substrates containing a C-terminal Leu residue. Analysis of a mutant of protein farnesyltransferase containing a Tyr-to-Leu substitution at position 361 in the beta subunit led to the conclusion that the side chain of this Tyr residue played a major role in recognition of the protein substrates. However, no interactions have been observed between this Tyr residue and peptide substrates in the crystal structures of protein farnesyltransferase. In an attempt to reconcile these apparently conflicting data, a thorough kinetic characterization of the Y361L variant of mammalian protein farnesyltransferase was performed. Direct binding measurements for the Y361L variant yielded peptide substrate binding that was actually some 40-fold tighter than that with the wild-type enzyme. In contrast, binding of the peptide substrate for protein geranylgeranyltransferase type I was very weak. The basis for the discrepancy was uncovered in a pre-steady-state kinetic analysis, which revealed that the Y361L variant catalyzed farnesylation of a normal peptide substrate at a rate similar to that of the wild-type enzyme in a single turnover, but that subsequent turnover was prevented. These and additional studies revealed that the Y361L variant does not "switch" protein substrate specificity as concluded from steady-state parameters; rather, this variant exhibits severely impaired product dissociation with its normal substrate, a situation resulting in a greatly compromised steady-state activity.

Alkyl and Aryl Transferases↗
PubMed2002

Structural homology discloses a bifunctional structural motif at the N-termini of G alpha proteins.

In a family of proteins, often the three-dimensional structure has been experimentally determined only for one member or a few members of the family. Homology modeling can be used to model the structures of all other members of the family and thus allow comparison of these structures. This approach was applied to heterotrimeric G proteins that require anchorage to the plasma membrane to properly interact with membrane-bound receptors and downstream effectors. Lipid modification by palmitoylation is a fundamental contributor to this localization, but the signals leading to this modification are still unknown. In this work, homology models of all the different human G(alpha) paralogs were generated using automated homology modeling, and the electrostatic potential of these proteins was calculated and visualized. This approach identifies a basic, positively charged, structural motif in the N-termini of heterotrimeric G proteins, which is not readily discernible from sequence alone. The basic motif is much reduced in those G(alpha) subunits that also undergo myristoylation, suggesting that the basic patches and myristoylation play overlapping roles. These motifs can affect both membrane affinity and orientation and determine the palmitoylation of G(alpha) subunits in cooperation with the G(betagamma) subunits, as has been corroborated by previous experimental studies. Furthermore, other palmitoylated proteins such as GAP-43 and RGS proteins share this alpha-helical basic motif in their N-terminus. It therefore appears that this structural motif is more widely applicable as a membrane-targeting and palmitoylation-determining signal. The work presented here highlights the possibilities available for experimentalists to discover structural motifs that are not readily observed by analysis of the linear sequence.

Amino Acid Motifs↗
PubMed1997

On the mechanism of the inhibition of transducin function by farnesylcysteine analogs.

The gamma subunits of heterotrimeric G proteins are isoprenylated/methylated on their carboxy termini. The photoreceptor G protein, transducin, is farnesylated/methylated at this position. Since the isoprenyl group is required for G protein function, it is of great interest to determine the mechanism by which the farnesyl group of Tgamma interacts with the other transducin subunits and/or the activated photoreceptor, rhodopsin. Farnesylcysteine derivatives (N-acetyl-S-farnesyl-L-cysteine and farnesylated peptides) have been previously shown to have effects on transducin activity at high concentrations. Here, an extensive survey is done of farnesylcysteine analogs and other lipid molecules, which are tested for their ability to inhibit GTP/GDP exchange in transducin catalyzed by photolyzed rhodopsin. These studies are carried out to determine the nature of the inhibition process. While it does not appear that these molecules exhibit the specificity which would characterize a ligand-receptor type mechanism, the results suggest that these compounds are not acting in a nonspecific detergent-like manner either. The most likely mode of action of famesylcysteine analogs is that they interfere with the lipid-lipid based association of Talpha and Tbetagamma through the lipid modifications present on each subunit.

Acetylcysteine↗
PubMed1997

G-protein-mediated signaling in cholesterol-enriched arterial smooth muscle cells. 1. Reduced membrane-associated G-protein content due to diminished isoprenylation of G-gamma subunits and p21ras.

Mechanisms contributing to altered heterotrimeric G-protein expression and subsequent signaling events during cholesterol accretion have been unexplored. The influence of cholesterol enrichment on G-protein expression was examined in cultured smooth muscle cells that resemble human atherosclerotic cells by exposure to cationized LDL (cLDL). cLDL, which increases cellular free and esterified cholesterol 2-fold and 10-fold, respectively, reduced the cell membrane content of Galphai-1, Galphai-2, Galphai-3, Gq/11, and Galphas. The following evidence supports the premise that the mechanism by which this occurs is due to reduced isoprenylation of the Ggamma-subunit. First, the inhibitory effect of cholesterol enrichment on the membrane content of Galphai subunits was found to be post-transcriptional, since the mRNA steady-state levels of Galphai(1-3) were unchanged following cholesterol enrichment. Second, the membrane expression of alpha and beta subunits was mimicked by cholesterol and 17-ketocholesterol, both of which inhibit HMG-CoA reductase. Third, inhibition of Galphai and Gbeta expression in cholesterol-enriched cells was overcome by mevalonate, the immediate product of HMG-CoA reductase. Fourth, pulse-chase experiments revealed that cholesterol enrichment did not reduce the degradation rate of membrane-associated Galphai subunits. Fifth, cholesterol enrichment also reduced membrane expression of Ggamma-5, Ggamma-7upper; these gamma subunits are responsible for trafficking of the heterotrimeric G-protein complex to the cell membrane as a result of HMG-CoA reductase-dependent post-translational lipid modification (geranylgeranylation) and subsequent membrane association. Cholesterol enrichment did not alter expression of G-gamma-5 mRNA, as assessed by reverse transcriptase polymerase chain reaction, supporting a post-transcriptional defect in Ggamma subunit expression. Fifth, cholesterol enrichment also reduced the membrane content of p21ras (a low molecular weight G-protein requiring farnesylation for membrane targeting) but did not alter the membrane content of the two proteins that do not require isoprenylation for membrane association&sbd;PDGF-receptor or p60-src. Reduced G-protein content in cholesterol-laden cells was reflected by reduced G-protein-mediated signaling events, including ATP-induced GTPase activity, thrombin-induced inhibition of cyclic AMP accumulation, and MAP kinase activity. Collectively, these results demonstrate that cholesterol enrichment reduces G-protein expression and signaling by inhibiting isoprenylation and subsequent membrane targeting. These results provide a molecular basis for altered G-protein-mediated cell signaling processes in cholesterol-enriched cells.

Animals↗
PubMed1998

Galpha12 requires acylation for its transforming activity.

The alpha subunit of the heterotrimeric G protein G12, harboring a mutation in the GTP binding domain (Q229L), behaves as a potent oncogene in NIH 3T3 cells. This alpha subunit, like most other G protein alpha subunits, undergoes palmitoylation, the reversible posttranslational addition of palmitate to cysteine residues. We investigated the role of palmitoylation of alpha12 in membrane localization and transformation efficiency and whether another lipid modification, myristoylation, could substitute for palmitoylation. NIH 3T3 cells were stably transfected with plasmids that expressed the wild-type alpha12, the constitutively active Q229L (QL) mutant, and mutants in which C11 was changed to S (C11S) and S2 and R6 were changed to G and S, respectively (S2G). Incorporation of [3H]palmitate was found in the endogenous and expressed alpha12 but not in the C11S mutants. Incorporation of [3H]myristate was found only in the S2G mutants. The wild type, QL mutant, and all the acylation mutants were found in the particulate fraction. Cells expressing the nonpalmitoylated C11S,QL mutant did not undergo transformation. The S2G mutation in the nonpalmitoylated C11S,QL mutant restored the transformation efficiency to a greater level than that of the palmitoylated QL mutant as measured by foci formation, growth in soft agar, and growth rate. Palmitoylation was critical for the transformation efficiency of alpha12 but not specifically required because myristoylation could substitute for these functions.

3T3 Cells↗
PubMed2002

Photoaffinity analogues of farnesyl pyrophosphate transferable by protein farnesyl transferase.

Farnesylation is a posttranslational lipid modification in which a 15-carbon farnesyl isoprenoid is linked via a thioether bond to specific cysteine residues of proteins in a reaction catalyzed by protein farnesyltransferase (FTase). We synthesized analogues (3-6) of farnesyl pyrophosphate (FPP) to probe the range of modifications possible to the FPP skeleton which allow for efficient transfer by FTase. Photoaffinity analogues of FPP (5, 6) were prepared by substituting perfluorophenyl azide functional groups for the omega-terminal isoprene of FPP. Substituted anilines replace the omega-terminal isoprene in analogues 3 and 4. Compounds 3-5 were prepared by reductive amination of the appropriate anilines with 8-oxo-geranyl acetate, followed by ester hydrolysis, chlorination, and pyrophosphorylation. Additional substitution of three methylenes for the beta-isoprene of FPP gave photoprobe 6 in nine steps. Preparation of the analogues required TiCl(4)-mediated imine formation prior to NaBH(OAc)(3) reduction for anilines with a pK(a) < 1. The azide moiety was not affected by Ph(3)PCl(2) conversion of allylic alcohols 13-16 into corresponding chlorides 17-20. Analogues 3-6 are efficiently transferred to target N-dansyl-GCVLS peptide substrate by mammalian FTase. Comparison of analogue structures and kinetics of transfer to those of FPP reveals that ring fluorination and para substituents have little effect on the affinity of the analogue pyrophosphate for FTase and its transfer efficiency. These results are also supported with models of the analogue binding modes in the active site of FTase. The transferable azide photoprobe 5 photoinactivates FTase. Transferable analogues 5 and 6 allow the formation of appropriately posttranslationally modified photoreactive peptide probes of isoprene function.

Alkyl and Aryl Transferases↗
PubMed2002

Sensitive and rapid analysis of protein palmitoylation with a synthetic cell-permeable mimic of SRC oncoproteins.

The localization of oncogenic Src and Ras proteins to cellular plasma membranes is critical for the proliferation of specific cancers. In addition to other lipid modifications, these proteins require posttranslational palmitoylation of specific cysteine residues by the enzyme palmitoyl acyltransferase (PAT) in order to be stably anchored at plasma membranes. Hence, the identification of inhibitors of protein palmitoylation has significant potential to define a new class of antitumor agents. However, studies of protein palmitoylation have been hindered by the dynamic and reversible nature of cysteine acylation and the lack of sensitive and convenient assays of PAT activity. To facilitate the rapid identification of compounds that affect protein palmitoylation, we report the solid-phase synthesis of a fluorescent cell-permeable palmitoyl acyltransferase substrate that mimics the N-terminus of Src family proteins. Metabolic radiolabeling and epifluorescence microscopy of Jurkat lymphocytes treated with this Src-mimetic lipopeptide revealed that this compound is palmitoylated intracellularly, which confers localization at cellular plasma membranes. Addition of the palmitoylation inhibitor 2-bromopalmitic acid to substrate-treated cells blocked palmitoylation and diminished substrate-mediated plasma membrane fluorescence. Analysis of inhibition of palmitoylation by flow cytometry revealed that this fluorescent lipopeptide substrate represents a highly sensitive molecular probe of palmitoyl acyltransferase activity that enables unprecedented high-throughput assays of protein palmitoylation.

Acyltransferases↗
PubMed2001

Synthesis of farnesyl diphosphate analogues containing ether-linked photoactive benzophenones and their application in studies of protein prenyltransferases.

Protein prenylation is a posttranslational lipid modification in which C(15) and C(20) isoprenoid units are linked to specific protein-derived cysteine residues through a thioether linkage. This process is catalyzed by a class of enzymes called prenyltransferases that are being intensively studied due to the finding that Ras protein is farnesylated coupled with the observation that mutant forms of Ras are implicated in a variety of human cancers. Inhibition of this posttranslational modification may serve as a possible cancer chemotherapy. Here, the syntheses of two new farnesyl diphosphate (FPP) analogues containing photoactive benzophenone groups are described. Each of these compounds was prepared in six steps from dimethylallyl alcohol. Substrate studies, inhibition kinetics, photoinactivation studies, and photolabeling experiments are also included; these experiments were performed with a number of protein prenyltransferases from different sources. A X-ray crystal structure of one of these analogues bound to rat farnesyltransferase illustrates that they are good substrate mimics. Of particular importance, these new analogues can be enzymatically incorporated into Ras-based peptide substrates allowing the preparation of molecules with photoactive isoprenoids that may serve as valuable probes for the study of prenylation function. Photoaffinity labeling of human protein geranylgeranyltransferase with (32)P-labeled forms of these analogues suggests that the C-10 locus of bound geranylgeranyl diphosphate (GGPP) is in close proximity to residues from the beta-subunit of this enzyme. These results clearly demonstrate the utility of these compounds as photoaffinity labeling analogues for the study of a variety of protein prenyltransferases and other enzymes that employ FPP or GGPP as their substrates.

Animals↗
PubMed1998

Pre-clinical development of farnesyltransferase inhibitors.

ras is the oncogene most frequently found in human cancers, being detected in 30% of most human cancers and at significantly higher rates in certain cancers including pancreatic (90%) and colon (50%) [1]. Almost 10 years ago it was shown that a C-terminal lipid modification of Ras, catalyzed by a specific farnesyl-protein transferase (FPTase), was required for the function of both normal and oncogenic Ras proteins. This finding spurred the development of FPTase inhibitors (FTIs) as a potential cancer therapy directed at the ras oncogene. FTIs have exhibited potent antiproliferative activity in cell culture and animal tumor models with a surprising lack of toxicity to normal tissues. However, while FTIs were originally conceptualized as Ras-specific agents, their mechanism of action is significantly more complicated than originally envisioned.

Alkyl and Aryl Transferases↗
PubMed2003

Tamoxifen and the farnesyl transferase inhibitor FTI-277 synergize to inhibit growth in estrogen receptor-positive breast tumor cell lines.

Farnesyl transferase inhibitors (FTIs) serve to specifically inhibit farnesyl isoprenoid lipid modification of proteins. Although originally developed as anti-Ras oncoprotein drugs, it now appears that these compounds function independently of Ras. FTIs have been shown to inhibit transformation by a variety of mechanisms, including apoptosis involving cytochrome c release from mitochondria. Tamoxifen exhibits both anti-estrogenic and estrogenic properties and is widely used as an estrogen antagonist for the treatment of estrogen receptor (ER) positive human breast tumors. Tamoxifen can induce ER-dependent apoptosis in human breast tumor cells by a mechanism involving the Bcl2/mitochondrial arm of the apoptotic machinery. Since tamoxifen and FTIs may stimulate distinct components of the mitochondrial-based apoptotic machinery, we reasoned that their effects might be synergistic. Here we show that anti-estrogens and an FTI (FTI-277) synergize to inhibit cell growth and enhance cell death in ER positive, human breast tumor cell lines. However, the drugs exhibited only additive effects on an ER negative cell line. Analysis of treated ER positive T-47D cells demonstrated that a synergistic increase in apoptosis was induced, as measured by increased caspase 3 activity. Thus, tamoxifen and FTIs may synergize to promote apoptotic cell death in ER positive human breast tumor cells.

Antineoplastic Agents↗
PubMed1998

Regulation of cytoskeletal association by a basic amino acid motif in polyoma virus middle T antigen.

The subcellular localization of many oncogenic proteins is thought to be important for their function. In the case of the middle T antigen of the DNA tumour virus, polyoma, localization to membranes in a specific manner is essential for its cellular transforming activity. To investigate factors that influence this localization, heterologous membrane targetting sequences were substituted for the middle T antigen transmembrane domain and the properties of the resulting proteins studied. Whereas C-terminal lipid modification derived from the H-ras CaaX box restored oncogenic activity to non-transforming truncated middle T antigen species, N-terminal myristylation from pp60c-src did not. Furthermore, a region, rich in basic amino acids and adjacent to the middle T transmembrane domain, was found to mediate association with detergent-insoluble cytoskeleton. Co-operation between the basic motif and neighbouring membrane binding domains resulted in specific localization of proteins to particular membrane sites, characterized by the association with subcellular structures, likely to be cytoskeletal in nature. These results demonstrate that the cellular localization of MT is regulated by at least two determinants, a transmembrane sequence which confers membrane binding and a basic motif which specifies a particular site within the membrane.

Amino Acid Sequence↗