Mevalonate Pathway Intermediates Research Articles

Sensitive quantification of mevalonate pathway intermediates and prediction of relative novel analogs by chemical derivatization-based LC-MS/MS

The mevalonate (MVA) pathway plays a crucial role in the occurrence and progression of various diseases, such as osteoporosis, breast cancer, and lung cancer, etc. However, determining all the MVA pathway intermediates is still challenging due to their high polarity, low concentration, chelation effect with metal compartments, and poor mass spectrometric response. In this study, we established a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method coupled with N2, N2, N4, N4-tetramethyl-6-(4-(piperazin-1-ylsulfonyl) phenyl)-1,3,5-triazine-2,4-diamine (Tmt-PP) labeling for the simultaneous analysis of all MVA intermediates in biospecimens. Chemical derivatization significantly improved the chromatographic retention, peak shape, and detection sensitivity of the analytes. Moreover, we employed a method named mass spectrum calculation to achieve the absolute quantification of the isomers, i.e., isopentenyl pyrophosphate (IPP) and dimethylallyl pyrophosphate (DMAPP). The established method was fully qualified and applied to explore the difference of these metabolites in cisplatin-resistant non-small cell lung cancer (NSCLC) cells. Additionally, several MVA intermediate analogs, including isopentenyl monophosphate or dimethylallyl monophosphate (IMP/DMAMP), geranyl monophosphate (GMP), 5-triphosphomevalonate (MTP), and isopentenyl triphosphate or dimethylallyl triphosphate (ITP/DMATP), were identified for the first time using a knowledge-driven prediction strategy. We further explored the tissue distribution of these novel metabolites. Overall, this work developed a sensitive quantification method for all MVA intermediates, which will enhance our understanding of the role of this pathway in various health and disease conditions. The novel metabolites we discovered warrant further investigations into their biosynthesis and biological functions.

Machine-learning guided elucidation of contribution of individual steps in the mevalonate pathway and construction of a yeast platform strain for terpenoid production

The production of terpenoids from engineered microbes contributes markedly to the bioeconomy by providing essential medicines, sustainable materials, and renewable fuels. The mevalonate pathway leading to the synthesis of terpenoid precursors has been extensively targeted for engineering. Nevertheless, the importance of individual pathway enzymes to the overall pathway flux and final terpenoid yield is less known, especially enzymes that are thought to be non-rate-limiting. To investigate the individual contribution of the five non-rate-limiting enzymes in the mevalonate pathway, we created a combinatorial library of 243 Saccharomyces cerevisiae strains, each having an extra copy of the mevalonate pathway integrated into the genome and expressing the non-rate-limiting enzymes from a unique combination of promoters. High-throughput screening combined with machine learning algorithms revealed that the mevalonate kinase, Erg12p, stands out as the critical enzyme that influences product titer. ERG12 is ideally expressed from a medium-strength promoter which is the ‘sweet spot’ resulting in high product yield. Additionally, a platform strain was created by targeting the mevalonate pathway to both the cytosol and peroxisomes. The dual localization synergistically increased terpenoid production and implied that some mevalonate pathway intermediates, such as mevalonate, isopentyl pyrophosphate (IPP), and dimethylallyl pyrophosphate (DMAPP), are diffusible across peroxisome membranes. The platform strain resulted in 94-fold, 60-fold, and 35-fold improved titer of monoterpene geraniol, sesquiterpene α-humulene, and triterpene squalene, respectively. The terpenoid platform strain will serve as a chassis for producing any terpenoids and terpene derivatives.

Untargeted analysis of the serum metabolome in cats with exocrine pancreatic insufficiency.

Exocrine pancreatic insufficiency (EPI) causes chronic digestive dysfunction in cats, but its pathogenesis and pathophysiology are poorly understood. Untargeted metabolomics is a promising analytic methodology that can reveal novel metabolic features and biomarkers of clinical disease syndromes. The purpose of this preliminary study was to use untargeted analysis of the serum metabolome to discover novel aspects of the pathobiology of EPI in cats. Serum samples were collected from 5 cats with EPI and 8 healthy controls. The diagnosis of EPI was confirmed by measurement of subnormal serum feline trypsin-like immunoreactivity (fTLI). Untargeted quantification of serum metabolite utilized ultra-high-performance liquid chromatography-tandem mass spectroscopy. Cats with EPI had significantly increased serum quantities of long-chain fatty acids, polyunsaturated fatty acids, mevalonate pathway intermediates, and endocannabinoids compared with healthy controls. Diacylglycerols, phosphatidylethanolamines, amino acid derivatives, and microbial metabolites were significantly decreased in cats with EPI compared to healthy controls. Diacyclglycerols and amino acid metabolites were positively correlated, and sphingolipids and long-chain fatty acids were negatively correlated with serum fTLI, respectively. These results suggest that EPI in cats is associated with increased lipolysis of peripheral adipose stores, dysfunction of the mevalonate pathway, and altered amino acid metabolism. Differences in microbial metabolites indicate that feline EPI is also associated with enteric microbial dysbiosis. Targeted studies of the metabolome of cats with EPI are warranted to further elucidate the mechanisms of these metabolic derangements and their influence on the pathogenesis and pathophysiology of EPI in cats.

Further engineering of R. toruloides for the production of terpenes from lignocellulosic biomass

BackgroundMitigation of climate change requires that new routes for the production of fuels and chemicals be as oil-independent as possible. The microbial conversion of lignocellulosic feedstocks into terpene-based biofuels and bioproducts represents one such route. This work builds upon previous demonstrations that the single-celled carotenogenic basidiomycete, Rhodosporidium toruloides, is a promising host for the production of terpenes from lignocellulosic hydrolysates.ResultsThis study focuses on the optimization of production of the monoterpene 1,8-cineole and the sesquiterpene α-bisabolene in R. toruloides. The α-bisabolene titer attained in R. toruloides was found to be proportional to the copy number of the bisabolene synthase (BIS) expression cassette, which in turn influenced the expression level of several native mevalonate pathway genes. The addition of more copies of BIS under a stronger promoter resulted in production of α-bisabolene at 2.2 g/L from lignocellulosic hydrolysate in a 2-L fermenter. Production of 1,8-cineole was found to be limited by availability of the precursor geranylgeranyl pyrophosphate (GPP) and expression of an appropriate GPP synthase increased the monoterpene titer fourfold to 143 mg/L at bench scale. Targeted mevalonate pathway metabolite analysis suggested that 3-hydroxy-3-methyl-glutaryl-coenzyme A reductase (HMGR), mevalonate kinase (MK) and phosphomevalonate kinase (PMK) may be pathway bottlenecks are were therefore selected as targets for overexpression. Expression of HMGR, MK, and PMK orthologs and growth in an optimized lignocellulosic hydrolysate medium increased the 1,8-cineole titer an additional tenfold to 1.4 g/L. Expression of the same mevalonate pathway genes did not have as large an impact on α-bisabolene production, although the final titer was higher at 2.6 g/L. Furthermore, mevalonate pathway intermediates accumulated in the mevalonate-engineered strains, suggesting room for further improvement.ConclusionsThis work brings R. toruloides closer to being able to make industrially relevant quantities of terpene from lignocellulosic biomass.

Valproic acid enhances pamidronate-sensitized cytotoxicity of Vδ2+ T cells against EBV-related lymphoproliferative cells

Therapeutic options for Epstein–Barr virus (EBV)-associated post-transplantation lymphoproliferative diseases (PTLD) are currently limited, accompanying with some off-target toxicities. We previously demonstrated that early recovery of Vδ2+ T cells inversely correlated to EBV reactivation after allogeneic hematopoietic cell transplantation. Studies in vitro and in the mouse models showed the cytotoxic activity of Vδ2+ T cells on EBV-transformed lymphoproliferative cells, but the efficacy was moderate. Bisphosphonate, such as pamidronate (PAM), have been reported as a sensitizer to trigger tumor cells for Vδ2+ T cells recognition. Valproic acid (VPA) has attracted attentions due to its adjuvant anti-tumor effect with chemotherapy or immunotherapy. Whether PAM and VPA facilitate the immunogenicity of EBV-infected cells towards Vδ2+ T cells cytotoxicity remains unknown. Herein, we demonstrated that lower dosage of VPA and/or PAM did not induce apoptosis of EBV-transformed B lymphoblastoid cell lines (EBV-LCLs) or Vδ2+ T cells. Notably, pre-treatment with PAM significantly increased the cell death of EBV-LCLs after co-culture with Vδ2+ T cells at different ratios. Combining treatment with VPA reinforced the sensitizing effect of PAM. This efficacy was through inducing the accumulation of mevalonate pathway intermediates and dependent on the γδ T cell receptor of Vδ2+ T cells. Similar sensitizing effects of PAM and PAM plus VPA were also demonstrated on the primary PTLD cells. These results highlight the roles of PAM and VPA in the enhancement of immune surveillance and expand the fields of these two drugs in the treatment of different types of malignancies.

Application of Stable Isotope Tracing to Elucidate Metabolic Dynamics During Yarrowia lipolytica α-Ionone Fermentation.

SummaryTargeted metabolite analysis in combination with 13C-tracing is a convenient strategy to determine pathway activity in biological systems; however, metabolite analysis is limited by challenges in separating and detecting pathway intermediates with current chromatographic methods. Here, a hydrophilic interaction chromatography tandem mass spectrometry approach was developed for improved metabolite separation, isotopologue analysis, and quantification. The physiological responses of a Yarrowia lipolytica strain engineered to produce ∼400 mg/L α-ionone and temporal changes in metabolism were quantified (e.g., mevalonate secretion, then uptake) indicating bottleneck shifts in the engineered pathway over the course of fermentation. Dynamic labeling results indicated limited tricarboxylic acid cycle label incorporation and, combined with a measurable ATP shortage during the high ionone production phase, suggested that electron transport and oxidative phosphorylation may limit energy supply and strain performance. The results provide insights into terpenoid pathway metabolic dynamics of non-model yeasts and offer guidelines for sensor development and modular engineering.

Abstract C50: Dissecting vulnerabilities of pancreatic tumors’ silent fraction unravels secrets of an alternative cell state

Abstract Tumors contain a heterogeneous mixture of live cells that are either undergoing proliferation or reside in a nonproliferative state. Cells that do not divide are often associated with increased chemoresistance, challenging therapies that target rapidly proliferating cells. In pancreatic cancer, paucity of blood vessels frequently generates hypoperfused, “arid” regions that constrain tumor cell metabolism and proliferation. Nonetheless, experimental conditions for studying tumor cells in vitro typically mimic well-nourished, “fertile” environments optimized for supporting proliferating cells rather than the more prevalent quiescent state. Characterization of the poorly vascularized KPC model confirmed that the quiescent phenotype is common and is strongly correlated to reduced tissue perfusion. To formulate culture conditions that will emulate the arid microenvironment associated with the nonproliferative state in vitro, we systematically deprived various nutrients from tumor cells and established the necessary components for attaining cell quiescence. Deprivation of amino acids on top of glucose, oxygen, and serum levels was essential for obtaining a complete and reversible cell cycle arrest without loss of viability. Cell cycle arrest was accompanied by a number of physiologic adaptations. Metabolomic analysis indicated reduction in the levels of TCA cycle and mevalonate pathway intermediates indicative of increased reliance on nonglycolytic metabolism. In addition, free amino acid pools were diminished and tumor cells exhibited a significant increase in autophagic flux and micropinocytosis, suggesting that amino acid availability is limited under arid conditions. Concomitant with reduced proliferation and metabolic rewiring, tumor cells under arid conditions exhibited remarkable resistance to gemcitabine. To explore alternative strategies to target these nonproliferating pancreatic tumor cells, we performed a comprehensive genome-wide CRISPR/Cas9-based genetic screen and compared vulnerabilities under ”fertile” and “arid” conditions. Functional annotation of sgRNAs depleted under arid conditions indicated a striking dependence on oxidative phosphorylation for survival. Remarkably, our analysis also revealed that a number of targetable genetic and epigenetic programs driving cell cycle progression were not essential and, in some instances, even hazardous for cells under arid conditions. These results suggest that pancreatic tumor cells that cease proliferation in a nutrient-depleted environment are physiologically rewired and may exhibit a distinct drug response profile from rapidly proliferating cells. The experimental model we developed allows investigation of this dominant yet understudied cell population and may serve as a platform for identification of agents targeting the quiescent fraction of tumors. Citation Format: Yogev Sela, Jinyang Li, Miriam Doepner, Shivahamy Maheswaran, Clementina Mesaros, Ian Blair, Ophir Shalem, Ben Stanger. Dissecting vulnerabilities of pancreatic tumors’ silent fraction unravels secrets of an alternative cell state [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Advances in Science and Clinical Care; 2019 Sept 6-9; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2019;79(24 Suppl):Abstract nr C50.

Statins Promote Interleukin-1β-Dependent Adipocyte Insulin Resistance Through Lower Prenylation, Not Cholesterol.

Statins lower cholesterol and adverse cardiovascular outcomes, but this drug class increases diabetes risk. Statins are generally anti-inflammatory. However, statins can promote inflammasome-mediated adipose tissue inflammation and insulin resistance through an unidentified immune effector. Statins lower mevalonate pathway intermediates beyond cholesterol, but it is unknown whether lower cholesterol underpins statin-mediated insulin resistance. We sought to define the mevalonate pathway metabolites and immune effectors that propagate statin-induced adipose insulin resistance. We found that LDL cholesterol lowering was dispensable, but statin-induced lowering of isoprenoids required for protein prenylation triggered NLRP3/caspase-1 inflammasome activation and interleukin-1β (IL-1β)-dependent insulin resistance in adipose tissue. Multiple statins impaired insulin action at the level of Akt/protein kinase B signaling in mouse adipose tissue. Providing geranylgeranyl isoprenoids or inhibiting caspase-1 prevented statin-induced defects in insulin signaling. Atorvastatin (Lipitor) impaired insulin signaling in adipose tissue from wild-type and IL-18-/- mice, but not IL-1β-/- mice. Atorvastatin decreased cell-autonomous insulin-stimulated lipogenesis but did not alter lipolysis or glucose uptake in 3T3-L1 adipocytes. Our results show that statin lowering of prenylation isoprenoids activates caspase-1/IL-1β inflammasome responses that impair endocrine control of adipocyte lipogenesis. This may allow the targeting of cholesterol-independent statin side effects on adipose lipid handling without compromising the blood lipid/cholesterol-lowering effects of statins.

Detection of Small GTPase Prenylation and GTP Binding Using Membrane Fractionation and GTPase-linked Immunosorbent Assay.

The Rho GTPase family belongs to the Ras superfamily and includes approximately 20 members in humans. Rho GTPases are important in the regulation of diverse cellular functions, including cytoskeletal dynamics, cell motility, cell polarity, axonal guidance, vesicular trafficking, and cell cycle control. Changes in Rho GTPase signaling play an essential regulatory role in many pathological conditions, such ascancer, central nervous system diseases, and immune system-dependent diseases. The posttranslational modification of Rho GTPases (i.e., prenylation by mevalonate pathway intermediates) and GTP binding are key factors which affect the activation of this protein. In this paper, two essential and simple methods are provided to detect a broad range of Rho GTPase prenylation and GTP binding activities. Details of the technical procedures that have been used are explained step by step in this manuscript.

Valproic Acid Combined with Zoledronate Enhance γδ T Cell-Mediated Cytotoxicity against Osteosarcoma Cells via the Accumulation of Mevalonate Pathway Intermediates.

The long-term survival of osteosarcoma has remained unchanged in the last several decades. Immunotherapy is proved to be a promising therapeutic strategy against osteosarcoma, especially for those with metastasis. Our previous study explored the sensibilization of zoledronate (ZOL) in γδ T cell-mediated cytotoxicity against osteosarcoma, but we have not yet elucidated the specific mechanism. Besides, high concentration is required to achieve these effects, whereas plasma ZOL concentration declines rapidly in the circulation. Valproic acid (VPA), a histone deacetylase inhibitor commonly used as the antiepileptic drug, has attracted much attention due to its synergistic antitumor efficacy with chemotherapy or immunotherapy. Here, we demonstrated that VPA combined with ZOL revealed the synergistic effect in enhancing antitumor efficacy of γδ T cells against osteosarcoma cells. This enhancement was mainly TCR-mediated and largely dependent on granule exocytose pathway. Of note, our findings indicated that ZOL sensitized osteosarcoma cells to γδ T cells by increasing the accumulation of the mevalonate pathway intermediates, which could be facilitated by VPA. We also found that this combination had similar effects on primary osteosarcoma cells. All the results suggested that VPA combined with ZOL could reduce the dose required to achieve a significant antitumor effect of γδ T cells, promoting it to be a novel therapy against osteosarcoma.

Toward industrial production of isoprenoids in Escherichia coli: Lessons learned from CRISPR-Cas9 based optimization of a chromosomally integrated mevalonate pathway.

Escherichia coli has been the organism of choice for the production of different chemicals by engineering native and heterologous pathways. In the present study, we simultaneously address some of the main issues associated with E. coli as an industrial platform for isoprenoids, including an inability to grow on sucrose, a lack of endogenous control over toxic mevalonate (MVA) pathway intermediates, and the limited pathway engineering into the chromosome. As a proof of concept, we generated an E. coli DH1 strain able to produce the isoprenoid bisabolene from sucrose by integrating the cscAKB operon into the chromosome and by expressing a heterologous MVA pathway under stress-responsive control. Production levels dropped dramatically relative to plasmid-mediated expression when the entire pathway was integrated into the chromosome. In order to optimize the chromosomally integrated MVA pathway, we established a CRISPR-Cas9 system to rapidly and systematically replace promoter sequences. This strategy led to higher pathway expression and a fivefold improvement in bisabolene production. More interestingly, we analyzed proteomics data sets to understand and address some of the challenges associated with metabolic engineering of the chromosomally integrated pathway. This report shows that integrating plasmid-optimized operons into the genome and making them work optimally is not a straightforward task and any poor engineering choices on the chromosome may lead to cell death rather than just resulting in low titers. Based on these results, we also propose directions for chromosomal metabolic engineering.

Geranylgeraniol Prevents Statin‐Dependent Myotoxicity in C2C12 Muscle Cells through RAP1 GTPase Prenylation and Cytoprotective Autophagy

The present study investigated the cytotoxic effects of statins (atorvastatin (ATR) and simvastatin (SIM), resp.) and methyl-beta-cyclodextrin (MβCD), at their respective IC50 concentrations, on muscle regeneration in the in vitro model of murine C2C12 myoblasts. Cotreatment with mevalonate (MEV), farnesol (FOH), geranylgeraniol (GGOH), or water-soluble cholesterol (Chol-PEG) was employed to determine whether the statin-dependent myotoxicity resulted from the lower cholesterol levels or the attenuated synthesis of intermediates of mevalonate pathway. Our findings demonstrated that while GGOH fully reverted the statin-mediated cell viability in proliferating myoblasts, Chol-PEG exclusively rescued MβCD-induced toxicity in myocytes. Statins caused loss of prenylated RAP1, whereas the GGOH-dependent positive effect was accompanied by loss of nonprenylated RAP1. Geranylgeranyltransferases are essential for muscle cell survival as inhibition with GGTI-286 could not be reversed by GGOH cotreatment. The increase in cell viability correlated with elevated AKT 1(S463) and GSK-3β(S9) phosphorylations. Slight increase in the levels of autophagy markers (Beclin 1, MAP LC-3IIb) was found in response to GGOH cotreatment. Autophagy rose time-dependently during myogenesis and was inhibited by statins and MβCD. Statins and MβCD also suppressed myogenesis and neither nonsterol isoprenoids nor Chol-PEG could reverse this effect. These results point to GGOH as the principal target of statin-dependent myotoxicity, whereas plasma membrane cholesterol deposit is ultimately essential to restore viability of MβCD-treated myocytes. Overall, this study unveils for the first time a link found between the GGOH- and Chol-PEG-dependent reversal of statin- or MβCD-mediated myotoxicity and cytoprotective autophagy, respectively.

Statin Therapy Negatively Impacts Skeletal Muscle Regeneration and Cutaneous Wound Repair in Type 1 Diabetic Mice.

Those with diabetes invariably develop complications including cardiovascular disease (CVD). To reduce their CVD risk, diabetics are generally prescribed cholesterol-lowering 3-hydroxy-methylglutaryl coenzyme A reductase inhibitors (i.e., statins). Statins inhibit cholesterol biosynthesis, but also reduce the synthesis of a number of mevalonate pathway intermediates, leading to several cholesterol-independent effects. One of the pleiotropic effects of statins is the reduction of the anti-fibrinolytic hormone plasminogen activator inhibitor-1 (PAI-1). We have previously demonstrated that a PAI-1 specific inhibitor alleviated diabetes-induced delays in skin and muscle repair. Here we tested if statin administration, through its pleiotropic effects on PAI-1, could improve skin and muscle repair in a diabetic rodent model. Six weeks after diabetes onset, adult male streptozotocin-induced diabetic (STZ), and WT mice were assigned to receive control chow or a diet enriched with 600 mg/kg Fluvastatin. Tibialis anterior muscles were injured via Cardiotoxin injection to induce skeletal muscle injury. Punch biopsies were administered on the dorsal scapular region to induce injury of skin. Twenty-four days after the onset of statin therapy (10 days post-injury), tissues were harvested and analyzed. PAI-1 levels were attenuated in statin-treated diabetic tissue when compared to control-treated tissue, however no differences were observed in non-diabetic tissue as a result of treatment. Muscle and skin repair were significantly attenuated in Fluvastatin-treated STZ-diabetic mice as demonstrated by larger wound areas, less mature granulation tissue, and an increased presence of smaller regenerating muscle fibers. Despite attenuating PAI-1 levels in diabetic tissue, Fluvastatin treatment impaired cutaneous healing and skeletal muscle repair in STZ-diabetic mice.

Insights into the mevalonate pathway in the anticancer effect of a platinum complex on human gastric cancer cells

A platinum(II) complex [Pt(en)]2ZL [en = ethylenediamine; ZL = 1-hydroxy-3(1H-imidazol-1-yl)ethane-1,1-diylbisphosphonic acid, commonly called as zoledronic acid] has been designed and synthesized recently in order to look for new anticancer drugs with high efficacy and low side effects. It exhibited cytotoxic effects on the human cancer cells SGC7901, HepG2, MCF-7, MDA-MB-231, HCT116, and U2OS, and the cytotoxicity against SGC7901 is particularly remarkable. It also showed higher cytotoxicity and better selectivity than the corresponding ligand ZL in inhibiting cancer cells SGC7901 and HepG2 rather than normal cells GES-1 and LO2. To investigate the role of mevalonate pathway involved in the mechanism of anticancer action of [Pt(en)]2ZL, the effects of farnesol (FOH) and geranylgeraniol (GGOH), precursors of important mevalonate pathway intermediates farnesyl pyrophosphate (FPP) and geranylgeranyl pyrophosphate (GGPP), on the cytotoxic effects of [Pt(en)]2ZL against the human gastric cancer cells SGC7901 were investigated systematically, since inhibition of the key enzyme farnesyl pyrophosphate synthase (FPPS) in the mevalonate pathway was acknowledged as the mechanism of most anticancer actions of the ligand ZL. The experiments revealed that FOH and GGOH both rescued the SGC7901 cells, especially FOH. The cell cycle arrest and apoptosis of SGC7901 cells induced by [Pt(en)]2ZL was decreased by the addition of FOH, and the prenylation of small guanine-nucleotide-binding regulatory proteins (small G proteins) down-regulated by [Pt(en)]2ZL was recovered by the addition of FOH, demonstrating that [Pt(en)]2ZL exerted anticancer effects on SGC7901 via inhibiting the mevalonate pathway. This will provide deep insights into the mechanism of action of [Pt(en)]2ZL.

ATP citrate lyase mediated cytosolic acetyl-CoA biosynthesis increases mevalonate production in Saccharomyces cerevisiae

BackgroundWith increasing concern about the environmental impact of a petroleum based economy, focus has shifted towards greener production strategies including metabolic engineering of microbes for the conversion of plant-based feedstocks to second generation biofuels and industrial chemicals. Saccharomyces cerevisiae is an attractive host for this purpose as it has been extensively engineered for production of various fuels and chemicals. Many of the target molecules are derived from the central metabolite and molecular building block, acetyl-CoA. To date, it has been difficult to engineer S. cerevisiae to continuously convert sugars present in biomass-based feedstocks to acetyl-CoA derived products due to intrinsic physiological constraints—in respiring cells, the precursor pyruvate is directed away from the endogenous cytosolic acetyl-CoA biosynthesis pathway towards the mitochondria, and in fermenting cells pyruvate is directed towards the byproduct ethanol. In this study we incorporated an alternative mode of acetyl-CoA biosynthesis mediated by ATP citrate lyase (ACL) that may obviate such constraints.ResultsWe characterized the activity of several heterologously expressed ACLs in crude cell lysates, and found that ACL from Aspergillus nidulans demonstrated the highest activity. We employed a push/pull strategy to shunt citrate towards ACL by deletion of the mitochondrial NAD+-dependent isocitrate dehydrogenase (IDH1) and engineering higher flux through the upper mevalonate pathway. We demonstrated that combining the two modifications increases accumulation of mevalonate pathway intermediates, and that both modifications are required to substantially increase production. Finally, we incorporated a block strategy by replacing the native ERG12 (mevalonate kinase) promoter with the copper-repressible CTR3 promoter to maximize accumulation of the commercially important molecule mevalonate.ConclusionBy combining the push/pull/block strategies, we significantly improved mevalonate production. We anticipate that this strategy can be used to improve the efficiency with which industrial strains of S. cerevisiae convert feedstocks to acetyl-CoA derived fuels and chemicals.Electronic supplementary materialThe online version of this article (doi:10.1186/s12934-016-0447-1) contains supplementary material, which is available to authorized users.

Mevalonate Biosynthesis Intermediates Are Key Regulators of Innate Immunity in Bovine Endometritis.

Metabolic changes can influence inflammatory responses to bacteria. To examine whether localized manipulation of the mevalonate pathway impacts innate immunity, we exploited a unique mucosal disease model, endometritis, where inflammation is a consequence of innate immunity. IL responses to pathogenic bacteria and LPS were modulated in bovine endometrial cell and organ cultures by small molecules that target the mevalonate pathway. Treatment with multiple statins, bisphosphonates, squalene synthase inhibitors, and small interfering RNA showed that inhibition of farnesyl-diphosphate farnesyl transferase (squalene synthase), but not 3-hydroxy-3-methylglutaryl-CoA reductase or farnesyl diphosphate synthase, reduced endometrial organ and cellular inflammatory responses to pathogenic bacteria and LPS. Although manipulation of the mevalonate pathway reduced cellular cholesterol, impacts on inflammation were independent of cholesterol concentration as cholesterol depletion using cyclodextrins did not alter inflammatory responses. Treatment with the isoprenoid mevalonate pathway-intermediates, farnesyl diphosphate and geranylgeranyl diphosphate, also reduced endometrial cellular inflammatory responses to LPS. These data imply that manipulating the mevalonate pathway regulates innate immunity within the endometrium, and that isoprenoids are regulatory molecules in this process, knowledge that could be exploited for novel therapeutic strategies.

Inhibition of prenyltransferase activity by statins in both liver and muscle cell lines is not causative of cytotoxicity.

As inhibitors of 3-hydroxy-3-methylglutaryl-CoA reductase, statins are an important first-line treatment for hypercholesterolemia. However, a recognized side-effect of statin therapy is myopathy, which in severe cases can present as potentially fatal rhabdomyolysis. This represents an important impediment to successful statin therapy, and despite decades of research the molecular mechanisms underlying this side-effect remain unclear. Current evidence supports a role for reduced levels of mevalonate pathway intermediates, with the most accepted hypothesis being a reduction in isoprenoids formation, leading to faulty post-translational modifications of membrane-associated proteins. We have undertaken a comprehensive analysis of the impact of nine statins on two human cell lines; Huh7 hepatoma and RD rhabdomyosarcoma. In both cell lines, concentration-dependent inhibition of prenylation was observed for cerivastatin and simvastatin, which could be rescued with the pathway intermediate mevalonate; in general, muscle cells were more sensitive to this effect, as measured by the levels of unprenylated Rap1A, a marker for prenylation by geranylgeranyl transferase I. Concentration-dependent toxicity was observed in both cell lines, with muscle cells again being more sensitive. Importantly, there was no correlation between inhibition of prenylation and cell toxicity, suggesting they are not causally linked. The lack of a causal relationship was confirmed by the absence of cytotoxicity in all cell lines following exposure to specific inhibitors of geranylgeranyl transferases I and II, and farnesyl transferase. As such, we provide strong evidence against the commonly accepted hypothesis linking inhibition of prenylation and statin-mediated toxicity, with the two processes likely to be simultaneous but independent.

Action of fluvastatin on signal transduction pathways of an aggressive natural killer cell leukemia cell line (LB227)

Aggressive natural killer cell leukemia is an acute neoplastic proliferation of natural killer cells, resulting in progressive disease and poor prognosis. Death ensues within months and a successful treatment plan for aggressive natural killer cell leukemia has not been established. Typically, statins are utilized to lower cholesterol by inhibiting the production of mevalonate via competitive inhibition of HMG‐CoA reductase. Statins have also been shown to exhibit anti‐cancer effects.Previous research in our lab has shown combination therapy with a chemotherapy agent and statin decreased cell proliferation and cytotoxicity. Statins alone were also shown to inhibit cytotoxicity in concentrations from 3.1uM‐5uM. Based on these previous results, we began to investigate the specific mechanism of action of statins. In this study, the effect of fluvastatin on signal transduction was examined using the aggressive natural killer cell leukemia cell line, known as YT‐INDY. YT‐INDY cells were treated with 10uM of fluvastatin every 24 hours, for a total of 72 hours. Cell lysates were prepared for both western blot analysis and human phospho‐MAP‐K analysis. Western blots demonstrated decreased phosphorylation of ERK1 and ERK2, upon statin treatment.We followed that up with human‐phospho‐ MAP‐K antibody arrays. Preliminary results indicate that 10uM fluvastatin significantly increases the phosphorylation, and activation of AKT, P38, and HSP27, suggesting an increased stress response, as expected in cancers. Significant decreases in the phosphorylation of ERK, RSK, and CREB were evident in the signal transduction pathways; thereby indicating a decrease in cell survival, proliferation, and tumorigenesis of aggressive natural killer cell leukemia cells. Current studies are investigating the use of mevalonate pathway inhibitors and pathway intermediates to more specifically identify the parts of the pathway that are responsible for the inhibition of proliferation and cytotoxicity. It is the hope that the outcome of this line of research will result in more effective treatment strategies for aggressive natural killer cell leukemia.Grant Funding Source: Iowa Osteopathic Education and Research Foundation

Synergistic antiproliferative and anticholesterogenic effects of linalool, 1,8-cineole, and simvastatin on human cell lines

Monoterpenes are naturally occurring plant hydrocarbons with multiple effects on the mevalonate pathway (MP), while statins competitively inhibit hydroxymethylglutarylcoenzyme-A reductase (HMGCR), the rate-limiting enzyme in the MP. Monoterpenes and statins proved capable of inhibiting both proliferation and cholesterogenesis. In the present study we assess the in vitro antiproliferative and anticholesterogenic effects of two monoterpenes: linalool and 1,8-cineole—either alone, in combination with each other, or combined individually with simvastatin—on liver-derived (HepG2) and extrahepatic (A549) cell lines. The three compounds alone inhibited cell proliferation in a dose-dependent fashion, while their pairwise combination produced synergistic antiproliferative effects in both cell lines. Incorporation experiments with [14C]acetate revealed that linalool and 1,8-cineole inhibited the MP, probably at different points, resulting in a reduction in cholesterogenesis and an accumulation of other MP intermediates and products. Linalool or 1,8-cineole, either together or individually with simvastatin, synergistically inhibited cholesterol synthesis. At low concentrations both monoterpenes inhibited steps specifically involved in cholesterol synthesis, whereas at higher concentrations HMGCR levels became down-regulated. Added exogenous mevalonate failed to reverse the inhibition of proliferation exerted by linalool and 1,8-cineole, suggesting that HMGCR inhibition alone is not responsible for the antiproliferative activity of those agents. This work demonstrates that monoterpenes in combination with each other, or individually in combination with simvastatin synergistically inhibits proliferation and cholesterogenesis in the human cell lines investigated, thus contributing to a clearer understanding of the action of essential-oil components, and their combination with the statins, in the targeting of specific points within a complex metabolic pathway.

Cholesterol-Induced Impact on Murine Macrophage Responsiveness Involves down-Regulation of Mevalonate Pathway

Free cholesterol (Ch) and its oxidative derivatives, oxysterols, are often accumulated in macrophages during chronic inflammation and atherogenesis. The effects of Ch and oxysterols on the balance of pro- and anti-inflammatory cytokines in inflammatory response and the role of mevalonate pathway in the effects of these sterols are studied poorly. We studied the effects of cholesterol, oxysterols, atorvastatin, and mevalonic acid on the LPS-induced TNF-α, IL-10, and TGF-β1 production in macrophage cell culture. The study was carried out in murine peritoneal macrophages preincubated for 4 h with Ch (5 µg/mL), 25-hydroxycholesterol (25-OH-Ch) (5 µg/mL), 7-keto-Ch (5 µg/mL), farnesol (10 µM), or atorvastatin (5 µmol/mL) in the presence or absence of 1 mM of mevalonate. The cells were further incubated in the presence or absence of E. coli 0111:B4 lipopolysaccharide (LPS) for 24 h, and cytokine concentrations in incubation media were determined. Macrophages preincubation with Ch, 25-OH-Ch, or atorvastatin decreased LPS-induced TNF-α production in cell cultures, while supplementation of preincubation medium with mevalonic acid abrogated the effects of atorvastatin and Ch. The Ch, 25-OH-Ch, 7-keto-Ch and atorvastatin significantly reduced IL-10 production by LPS–stimulated macrophages, while farnesol had no effect. Supplementation of Ch or atorvastatin-containing preincubation medium with mevalonate restored IL-10 production. The TGF-β1 production was significantly enhanced by the presence of Ch or atorvastatin in preincubation medium as compared to the control level in non-treated macrophages, while 25-OH-Ch or farnesol decreased profoundly TGF-β1 production. Mevalonate abrogated the effect of Ch or atorvastatin but not of 25-OH-Ch or farnesol. These results allow to conclude, that Ch is able to promote anti-inflammatory and fibrogenic macrophage response, which is connected, at least in part, with the deficiency of mevalonate pathway intermediates, particularly to the deficiency of farnesol, whereas hydroxysterols produce tolerogenic, but not fibrogenic effect, independently of mevalonate pathway.