Remodeling the isoprenoid pathway in tobacco by expressing the cytoplasmic mevalonate pathway in chloroplasts

Metabolic engineering to enhance production of isoprenoid metabolites for industrial and medical purposes is an important goal. The substrate for isoprenoid synthesis in plants is produced by the mevalonate pathway (MEV) in the cytosol and by the 2-C-methyl-d-erythritol 4-phosphate (MEP) pathway in plastids. A multi-gene approach was employed to insert the entire cytosolic MEV pathway into the tobacco chloroplast genome. Molecular analysis confirmed the site-specific insertion of seven transgenes and homoplasmy. Functionality was demonstrated by unimpeded growth on fosmidomycin, which specifically inhibits the MEP pathway. Transplastomic plants containing the MEV pathway genes accumulated higher levels of mevalonate, carotenoids, squalene, sterols, and triacyglycerols than control plants. This is the first time an entire eukaryotic pathway with six enzymes has been transplastomically expressed in plants. Thus, we have developed an important tool to redirect metabolic fluxes in the isoprenoid biosynthesis pathway and a viable multigene strategy for engineering metabolism in plants.     

Targeting the RAS pathway in melanoma

Metastatic melanoma is a highly lethal type of skin cancer and is often refractory to all traditional chemotherapeutic agents. Key insights into the genetic makeup of melanoma tumors have led to the development of promising targeted agents. An activated RAS pathway, anchored by oncogenic BRAF, appears to be the central motor driving melanoma proliferation. Although recent clinical trials have brought enormous hope to patients with melanoma, adverse effects and novel escape mechanisms of these inhibitors have already emerged. Definition of the limits of the first successful targeted therapies will provide the basis for further advances in management of disseminated melanoma. In this review, the current state of targeted therapy for melanoma is discussed, including the potent BRAF(V600E) inhibitor vemurafenib.     

Enzymes of the mevalonate pathway of isoprenoid biosynthesis

The mevalonate pathway accounts for conversion of acetyl-CoA to isopentenyl 5-diphosphate, the versatile precursor of polyisoprenoid metabolites and natural products. The pathway functions in most eukaryotes, archaea, and some eubacteria. Only recently has much of the functional and structural basis for this metabolism been reported. The biosynthetic acetoacetyl-CoA thiolase and HMG-CoA synthase reactions rely on key amino acids that are different but are situated in active sites that are similar throughout the family of initial condensation enzymes. Both bacterial and animal HMG-CoA reductases have been extensively studied and the contrasts between these proteins and their interactions with statin inhibitors defined. The conversion of mevalonic acid to isopentenyl 5-diphosphate involves three ATP-dependent phosphorylation reactions. While bacterial enzymes responsible for these three reactions share a common protein fold, animal enzymes differ in this respect as the recently reported structure of human phosphomevalonate kinase demonstrates. There are significant contrasts between observations on metabolite inhibition of mevalonate phosphorylation in bacteria and animals. The structural basis for these contrasts has also recently been reported. Alternatives to the phosphomevalonate kinase and mevalonate diphosphate decarboxylase reactions may exist in archaea. Thus, new details regarding isopentenyl diphosphate synthesis from acetyl-CoA continue to emerge.     

Biosynthesis of abscisic acid by the non-mevalonate pathway in plants, and by the mevalonate pathway in fungi

The biosynthetic pathways to abscisic acid (ABA) were investigated by feeding [1-(13)C]-D-glucose to cuttings from young tulip tree shoots and to two ABA-producing phytopathogenic fungi. 13C-NMR spectra of the ABA samples isolated showed that the carbons at 1, 5, 6, 4', 7' and 9' of ABA from the tulip tree were labeled with 13C, while the carbons at 2, 4, 6, 1', 3', 5', 7', 8' and 9' of ABA from the fungi were labeled with 13C. The former corresponds to C-1 and -5 of isopentenyl pyrophosphate, and the latter to C-2, -4 and -5 of isopentenyl pyrophosphate. This finding reveals that ABA was biosynthesized by the non-mevalonate pathway in the plant, and by the mevalonate pathway in the fungi. 13C-Labeled beta-carotene from the tulip tree showed that the positions of the labeled carbons were the same as those of ABA, being consistent with the biosynthesis of ABA via carotenoids. Lipiferolide of the tulip tree was also biosynthesized by the non-mevalonate pathway.     

Effects of estrogen and testosterone on the metabolism of mevalonate by the shunt pathway

Mevalonate is metabolized by a sterol-forming and a non-sterol-forming, also called the "shunt", pathway. Effects of estrogen and testosterone administration on the shunt activity were examined in male and female Wistar and Sprague-Dawley rats. Shunt activity was determined in vivo from the yield of expired 14CO2 following [5-14C]mevalonate injection. Total mevalonate utilized was determined from the yield of expired 14CO2 following [1-14C]mevalonate injection. In the female, about 45% of mevalonate appears to be metabolized via the shunt, and in the male about 20%. This difference between male and female rats is dependent on both testosterone and estrogen, and apparently on testosterone to a greater extent. Thus estrogen treatment produced a 20-35% increase in shunt activity over castrated controls, while castration of males without hormonal treatment resulted in about a 50% increase in shunt activity, and testosterone administration returned castrated male and female shunt activity to that of intact males, or nearly so. Light/dark cycle had no effect in vivo on shunt activity, but may be critical in demonstrating sex differences in shunt activity in kidney slices.     

Acetoacetyl-CoA thiolase regulates the mevalonate pathway during abiotic stress adaptation

Acetoacetyl-CoA thiolase (EC 2.3.1.9), also called thiolase II, condenses two molecules of acetyl-CoA to give acetoacetyl-CoA. This is the first enzymatic step in the biosynthesis of isoprenoids via mevalonate (MVA). In this work, thiolase II from alfalfa (MsAACT1) was identified and cloned. The enzymatic activity was experimentally demonstrated in planta and in heterologous systems. The condensation reaction by MsAACT1 was proved to be inhibited by CoA suggesting a negative feedback regulation of isoprenoid production. Real-time RT-PCR analysis indicated that MsAACT1 expression is highly increased in roots and leaves under cold and salinity stress. Treatment with mevastatin, a specific inhibitor of the MVA pathway, resulted in a decrease in squalene production, antioxidant activity, and the survival of stressed plants. As expected, the presence of mevastatin did not change chlorophyll and carotenoid levels, isoprenoids synthesized via the plastidial MVA-independent pathway. The addition of vitamin C suppressed the sensitive phenotype of plants challenged with mevastatin, suggesting a critical function of the MVA pathway in abiotic stress-inducible antioxidant defence. MsAACT1 over-expressing transgenic plants showed salinity tolerance comparable with empty vector transformed plants and enhanced production of squalene without altering the 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR) activity in salt-stress conditions. Thus, acetoacetyl-CoA thiolase is a regulatory enzyme in isoprenoid biosynthesis involved in abiotic stress adaptation.     

Regulation of activin beta A mRNA level by cAMP.

We demonstrated the presence of five species of the activin beta A mRNA in human placenta and one major RNA associated with two minor RNAs of the activin in the fetal membrane. We investigated the effect of 8-bromo-cAMP (8-Br-cAMP) on accumulation of activin beta A subunit mRNA in human fibrosarcoma HT1080 cells. Although low levels of the activin mRNA were detectable in the untreated cells, the one main RNA species was predominantly accumulated by 8-Br-cAMP. We propose that generation of multiple activin mRNAs in the fetal membrane and cAMP-treated HT1080 cells is presumably due to a cell-specific alternative polyadenylation.     

Interrelationships between mevalonate metabolism and the mitogenic signaling pathway in T lymphocyte proliferation.

Upon stimulation with antigen or antibodies directed at the CD3.T cell receptor complex, T lymphocytes undergo a series of biochemical events that result in DNA synthesis and cellular proliferation. The purpose of the current study was to explore the role of mevalonic acid and its metabolites in this process. Stimulation of freshly isolated human T cells with immobilized anti-CD3 monoclonal antibody (mAb) results in the induction of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase message, with maximum induction occurring at 24 h of culture, approximately 12 h before the onset of DNA synthesis. Protein kinase C (PKC) probably mediates this induction, as H7, which inhibits PKC and cyclic nucleotide-dependent protein kinases, but not HA1004, which inhibits all of these protein kinases except PKC, completely abrogates the appearance of HMG-CoA reductase message. The importance of HMG-CoA reductase induction and mevalonate production in cell cycle progression was demonstrated by the observation that either 25-hydroxycholesterol, which inhibits this induction, or lovastatin, a competitive inhibitor of HMG-CoA reductase, inhibited anti-CD3-induced T cell mitogenesis in a dose-dependent manner. The presence of lovastatin during the first 24-36 h of culture results in a progressive delay of cell cycle progression, whereas this agent, when present only for the first 12 h of culture, had no effect on T cell proliferation. These results suggest that mevalonate is required for cell cycle progression from mid-G1 into late G1. Exogenous mevalonate overcomes the antiproliferative effect of lovastatin but not of 25-hydroxycholesterol. Since 25-hydroxycholesterol suppresses the metabolism of mevalonic acid at multiple points, this result suggests that one or more metabolites of mevalonate, rather than mevalonate itself, plays an essential role in cell cycle progression. One metabolite of mevalonate, farnesol pyrophosphate, may play such a role, since free farnesol suppresses anti-CD3 mAb-induced T cell proliferation in a concentration-dependent manner. In mAb is associated with PKC-dependent induction of HMG-CoA reductase which, in turn, leads to the generation of mevalonic acid and its metabolites, one or more of which play a requisite role in cell cycle progression.     

A gene cluster for the mevalonate pathway from Streptomyces sp. Strain CL190

A biosynthetic 3-hydroxy-3-methylglutaryl coenzyme A reductase (EC 1. 1.1.34), the rate-limiting enzyme of the mevalonate pathway for isopentenyl diphosphate biosynthesis, had previously been purified from Streptomyces sp. strain CL190 and its corresponding gene (hmgr) had been cloned (S. Takahashi, T. Kuzuyama, and H. Seto, J. Bacteriol. 181:1256-1263, 1999). Sequence analysis of the flanking regions of the hmgr gene revealed five new open reading frames, orfA to -E, which showed similarity to those encoding eucaryotic and archaebacterial enzymes for the mevalonate pathway. Feeding experiments with [1-(13)C]acetate demonstrated that Escherichia coli JM109 harboring the hmgr gene and these open reading frames used the mevalonate pathway under induction with isopropyl beta-D-thiogalactopyranoside. This transformant could grow in the presence of fosmidomycin, a potent and specific inhibitor of the nonmevalonate pathway, indicating that the mevalonate pathway, intrinsically absent in E. coli, is operating in the E. coli transformant. The hmgr gene and orfABCDE are thus unambiguously shown to be responsible for the mevalonate pathway and to form a gene cluster in the genome of Streptomyces sp. strain CL190.     

Negative regulation of cell proliferation by mevalonate or one of the mevalonate phosphates

The role of mevalonate and its products in the regulation of cellular proliferation was examined using 6-fluoromevalonate (Fmev), a compound that blocks the conversion of mevalonate pyrophosphate to isopentenyl pyrophosphate. Fmev suppressed DNA synthesis by a variety of transformed and malignant T cell, B cell, and myeloid cell lines. In contrast to results previously reported with mitogen-stimulated human peripheral blood T cell DNA synthesis, low concentrations of low density lipoprotein (LDL) alone could not restore proliferation to these cell lines. The same concentrations of LDL were able to provide sufficient cholesterol and support the growth of all cell lines when mevalonate synthesis was blocked with a specific inhibitor of 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase, lovastatin. Fmev-mediated inhibition was totally prevented in some but not all cell lines when the concentration of exogenous LDL was increased 5-10-fold above that required to permit proliferation of lovastatin-blocked cells. Residual HMG-CoA reductase activity of cells cultured with LDL inversely correlated with the restoration of growth to Fmev-blocked cultures. Confirmation of the critical role of HMG-CoA reductase activity and mevalonate synthesis in the inhibition of cellular proliferation by Fmev was obtained by demonstrating that the specific inhibitor of this enzyme, lovastatin, restored proliferation of Fmev-blocked cells. Furthermore, supplementation of cultures with mevalonate, the product of HMG-CoA reductase activity, markedly inhibited proliferation of Fmev-blocked cells. These findings indicate that mevalonate or one of the mevalonate phosphates, which accumulates in Fmev-blocked cells, is a critical negative regulator of cellular proliferation.     

The shunt pathway of mevalonate metabolism in the isolated perfused rat liver

The shunt pathway of mevalonate metabolism (Edmond, J., and Popják, G. (1974) J. Biol. Chem. 249, 66-71) has been studied in isolated livers from fed rats perfused with physiological concentrations of variously labeled [14C]mevalonates. The measured rates of 14CO2 production were converted to rates of mitochondrial acetyl-CoA production from mevalonate by methods which take into account underestimations of metabolic rates derived from 14CO2 production. Our data confirm that the shunt pathway leads to mitochondrial acetyl-CoA. The apparent negligible rate of mevalonate shunting in liver, previously reported by others, stems from the very low contribution (congruent to 0.1%) of plasma mevalonate to total mevalonate metabolism in the liver. This contribution was assessed from the relative incorporations of 3H2O and [5-14C]mevalonate into sterols. In livers from fed rats, the shunt diverts about 5% of the production of mevalonate. The total rate of mevalonate shunting in the liver is about 200 times greater than in two kidneys. The liver is therefore the main site of mevalonate shunting in the rat.     

The shunt pathway of mevalonate metabolism in the isolated perfused rat kidney

The shunt pathway of mevalonate metabolism (Edmond, J., and Popják, G. (1974) J. Biol. Chem. 249, 66-71) has been studied in isolated kidneys from rats perfused with physiological concentrations of variously labeled [14C]- and [3H]mevalonates. The rate of operation of the shunt pathway was quantified by the production of either 14CO2 or 3H2O from the tracers. The measured rates of 14CO2 production from [14C] mevalonate were converted to rates of mitochondrial acetyl-CoA production by methods which take into account underestimations of metabolic rates derived from 14CO2 production. We have shown that the sex difference in renal shunting of mevalonate (Wiley, M. H., Howton, M. M., and Siperstein, M. D. (1979) J. Biol. Chem. 254, 837-842) occurs at physiological levels of substrate. The shunt pathway diverts up to 17% of the flux of mevalonate entering the cholesterol synthesis pathway in the kidney. It may, therefore, play a role in the long term regulation of cholesterol synthesis in this organ, as had been hypothesized by Edmond and Popják.     

Transport of mevalonate by Pseudomonas sp. strain M.

Pseudomonas sp. M, isolated from soil by elective culture on R,S-mevalonate as the sole source of carbon, possessed an inducible transport system for mevalonate. This high-affinity system had a pH optimum of 7.0, a temperature optimum of 30 degrees C, a Km for R,S-mevalonate of 88 microM, and a V max of 26 nmol of mevalonate transported per min/mg of cells (dry weight). Transport was energy dependent since azide, cyanide, or m-chlorophenylhydrazone caused complete cessation of transport activity. Transport of mevalonate was highly substrate specific. Of the 16 structural analogs of mevalonate tested, only acetoacetate, mevinolin, and mevaldehyde significantly inhibited transport. Growth of cells on mevalonate induced transport activity by 40- to 65-fold over that observed in cells grown on alternate carbon sources. A biphasic pattern for cell growth, as well as for induction of mevalonate transport activity, was observed when mevalonate was added to a culture actively growing on glucose. The induction of transport activity under these conditions began within 30 min after the addition of mevalonate and reached 60% of maximal activity during phase I. A further increase in mevalonate transport activity occurred during phase II of growth. Glucose was the preferred carbon source for growth during phase I, whereas mevalonate was preferred during phase II. Only one isomer of the R,S-mevalonate mixture appeared to be utilized, since growth ceased after 45 to 50% of the total mevalonate was depleted from the medium. However, nearly 30% of the preferred mevalonate isomer was depleted from the medium during phase I without significant metabolism to CO2. These results suggest that mevalonate or a mevalonate catabolite may accumulate in cells of Pseudomonas sp. M during phase I and that glucose metabolism may inhibit or repress the expression of enzymes further along the mevalonate catabolic pathway.