Enzyme In Mevalonate Pathway Research Articles

Effects of statins on mitochondrial pathways.

Statins are a family of drugs that are used for treating hyperlipidaemia with a recognized capacity to prevent cardiovascular disease events. They inhibit β‐hydroxy β‐methylglutaryl‐coenzyme A reductase, i.e. the rate‐limiting enzyme in mevalonate pathway, reduce endogenous cholesterol synthesis, and increase low‐density lipoprotein clearance by promoting low‐density lipoprotein receptor expression mainly in the hepatocytes. Statins have pleiotropic effects including stabilization of atherosclerotic plaques, immunomodulation, anti‐inflammatory properties, improvement of endothelial function, antioxidant, and anti‐thrombotic action. Despite all beneficial effects, statins may elicit adverse reactions such as myopathy. Studies have shown that mitochondria play an important role in statin‐induced myopathies. In this review, we aim to report the mechanisms of action of statins on mitochondrial function. Results have shown that statins have several effects on mitochondria including reduction of coenzyme Q10 level, inhibition of respiratory chain complexes, induction of mitochondrial apoptosis, dysregulation of Ca2+ metabolism, and carnitine palmitoyltransferase‐2 expression. The use of statins has been associated with the onset of additional pathological conditions like diabetes and dementia as a result of interference with mitochondrial pathways by various mechanisms, such as reduction in mitochondrial oxidative phosphorylation, increase in oxidative stress, decrease in uncoupling protein 3 concentration, and interference in amyloid‐β metabolism.Overall, data reported in this review suggest that statins may have major effects on mitochondrial function, and some of their adverse effects might be mediated through mitochondrial pathways.

Deficiency and absence of endogenous isoprene in adults, disqualified its putative origin

BackgroundIsoprene (C5H8) is a clinically important breath metabolite. Although, hundreds of studies have reported differential expressions in isoprene exhalation as breath biomarker for diverse diseases, the substance couldn't enter to clinical practice as diagnostic marker. Moreover, many experimental/basic observations upon breath isoprene remained unrelated to the corresponding pathophysiological effects on its putative metabolic origin (i.e. mevalonate pathway). Here, we investigated the fundamental reason that hindered the rational interpretation and translation of this marker from basic to clinical science. MethodsVia high-resolution mass-spectrometry based breathomics in 1026 human subjects, we discovered adults with significant deficiency (order of magnitude lower than the normal) and complete absence of breath isoprene. We prospectively applied real-time breathomics, quantitative gene expression analysis of the mevalonate pathway enzymes, lipid-profiling and hemodynamic monitoring on those isoprene deficient subjects and controls. Additionally, the subject with absence of isoprene was followed up throughout different phases of her womanhood. ResultsIn contrast to convention, we witnessed that adults can live healthy without exhaling isoprene or with significant deficiency. This rare phenotype represents a recessive inheritance. Despite physio-metabolic changes during menstrual cycle (that is known to profoundly affect isoprene exhalation) and profoundly increased plasma cholesterol during pregnancy and after childbirth, isoprene remained absent. All genes of mevalonate pathway enzymes were normally expressed in all participants, without any down-regulation or compensatory up-regulation. ConclusionsAbsence/deficiency of isoprene despite normal lipid profiles and no mevalonate pathway malfunction disqualifies the long-believed metabolic origin of isoprene from cholesterol biosynthesis. Thus, clinical translation of breath isoprene expressions should not be generally attributed to corresponding pathophysiological effects onto mevalonate/cholesterol pathway. Our finding has refined and optimized the clinical interpretation of isoprene as biomarker in volatile metabolomics and breathomics. Future studies will address the correct metabolic origin of isoprene to imply this important marker to routine practice.

Mevalonate Pathway Provides Ubiquinone to Maintain Pyrimidine Synthesis and Survival in p53-Deficient Cancer Cells Exposed to Metabolic Stress.

Oncogene activation and loss of tumor suppressor function changes the metabolic activity of cancer cells to drive unrestricted proliferation. Moreover, cancer cells adapt their metabolism to sustain growth and survival when access to oxygen and nutrients is restricted, such as in poorly vascularized tumor areas. We show here that p53-deficient colon cancer cells exposed to tumor-like metabolic stress in spheroid culture activated the mevalonate pathway to promote the synthesis of ubiquinone. This was essential to maintain mitochondrial electron transport for respiration and pyrimidine synthesis in metabolically compromised environments. Induction of mevalonate pathway enzyme expression in the absence of p53 was mediated by accumulation and stabilization of mature SREBP2. Mevalonate pathway inhibition by statins blocked pyrimidine nucleotide biosynthesis and induced oxidative stress and apoptosis in p53-deficient cancer cells in spheroid culture. Moreover, ubiquinone produced by the mevalonate pathway was essential for the growth of p53-deficient tumor organoids. In contrast, inhibition of intestinal hyperproliferation by statins in an Apc/KrasG12D-mutant mouse model was independent of de novo pyrimidine synthesis. Our results highlight the importance of the mevalonate pathway for maintaining mitochondrial electron transfer and biosynthetic activity in cancer cells exposed to metabolic stress. They also demonstrate that the metabolic output of this pathway depends on both genetic and environmental context. SIGNIFICANCE: These findings suggest that p53-deficient cancer cells activate the mevalonate pathway via SREBP2 and promote the synthesis of ubiquinone that plays an essential role in reducing oxidative stress and supports the synthesis of pyrimidine nucleotide.

Cloning and expression analysis of 5-phosphomevalonate kinase gene (CcPMK) in Cinnamomum camphora

The 5-phosphomevalonate kinase(PMK) is a key enzyme in mevalonate(MVA) pathway which reversibly catalyzes the phosphorylation of mevalonate 5-phosphate(MVAP) to form mevalonate-5-diphosphate(MVAPP) in the presence of ATP and divalent metal ion such as Mg~(2+). In this research, on the basis of the transciptome database of Cinnamomum camphora, the PMK was cloned by cDNA from C. camphora, and was named CcPMK(GenBank number KU886266). The ORF of CcPMK was composed of 1 545 bp, encoding 514 amino acids. The bioinformatics analysis of CcPMK indicated that the molecular weight of the encoded protein was 56.14 kDa, with a theoretically isoelectric point of 7.64, and there was no signal peptide and transmembrane structure in putative protein. By multiple sequence alignment and phylogenetic tree analysis, we found that similarity between CcPMK and PMK amino acid sequence of other plants was as high as 75%. Among the similar sequences, 45% of them belonged to the alpha helix, while 16% belonged to the beta strand. CcPMK obtained 3 PMK protein family motifs and 1 ATP binding site Gly-Leu-Gly-Ser-Ser-Ala-Ala, and its 3 D structure contained a catalytic pocket structure, proving CcPMK as a member of PMK gene family. The result of phylogenetic tree showed that CcPMK was closely related to monocotyledon plants such as Phonenix dactylifera. The results of the Real-time PCR indicated that the expression level of CcPMK in borneol type was higher than that in linalool type, cineol type, iso-nerolidol type and camphor type. CcPMK expressed highest in roots and lowest in branches. Our results revealed that the expression level of CcPMK was different among five chemical types and different plant tissues, and the research provides foundation for further study of the terpenoids biosynthetic pathway in C. camphora.

Isolation, characterization and in silico analysis of 3-Hydroxy-3-methylglutaryl-coenzyme A reductase (HMGR) gene from Andrographis paniculata (Burm. f) Nees.

3-Hydroxy-3-methylglutaryl-coenzymeA reductase (HMGR), the first rate-limiting enzyme of Mevalonate (MVA) pathway was isolated from Andrographis paniculata (ApHMGR) and expressed in bacterial cells. Full length ApHMGR (1937bp) was submitted to NCBI with accession number MG271748.1. The open reading frame (ORF) was flanked by a 31-bp 5'-UTR, 118-bp 3'-UTR and ApHMGR contained a 1787bp ORF encoding protein of 595 amino acids. ApHMGR protein was approximately 64kDa, with isoelectric point of 5.75. Isolated ApHMGR was cloned into pET102 vector and expressed in E. coli BL21 (DE 3) cells, and characterized by SDS-PAGE. HPLC analysis for andrographolide content in leaf, stem and root of A. paniculata revealed highest in leaf tissue. The expression patterns of ApHMGR in different plant tissues using qRT-PCR revealed high in root tissue correlating with HPLC data. Three dimensional (3D) structural model of ApHMGR displayed 90% of the amino acids in most favored regions of the Ramachandran plot with 93% overall quality factor. ApHMGR was highly conserved with plant specific N-terminal membrane domains and C-terminal catalytic regions. Phylogenetic analysis showed A. paniculata sharing common ancestor with Handroanthus impetiginosus. 3D model of ApHMGR was screened for the interaction with substrates NADPH, HMG CoA and inhibitor using Auto Dock Vina. In silico analysis revealed that full length ApHMGR had extensive similarities to other plant HMGRs. The present communication reports the isolation of full length HMGR from A. paniculata, its heterologous expression in bacterial cells and in silico structural and functional characterization providing valuable genomic information for future molecular interventions.

ASPP2 inhibits tumor growth by repressing the mevalonate pathway in hepatocellular carcinoma

Cancer is, fundamentally, a disorder of cell growth and proliferation, which requires adequate supplies of energy and nutrients. In this study, we report that the haplo-insufficient tumor suppressor ASPP2, a p53 activator, negatively regulates the mevalonate pathway to mediate its inhibitory effect on tumor growth in hepatocellular carcinoma (HCC). Gene expression profile analysis revealed that the expression of key enzymes in the mevalonate pathway were increased when ASPP2 was downregulated. HCC cells gained higher cholesterol levels and enhanced tumor-initiating capability in response to the depletion of ASPP2. Simvastatin, a mevalonate pathway inhibitor, efficiently abrogated ASPP2 depletion-induced anchorage-independent cell proliferation, resistance to chemotherapy drugs in vitro, and tumor growth in xenografted nude mice. Mechanistically, ASPP2 interacts with SREBP-2 in the nucleus and restricts the transcriptional activity of SREBP-2 on its target genes, which include key enzymes involved in the mevalonate pathway. Moreover, clinical data revealed better prognosis in patients with high levels of ASPP2 and low levels of the mevalonate pathway enzyme HMGCR. Our findings provide functional and mechanistic insights into the critical role of ASPP2 in the regulation of the mevalonate pathway and the importance of this pathway in tumor initiation and tumor growth, which may provide a new therapeutic opportunity for HCC.

Modular enzyme assembly for enhanced cascade biocatalysis and metabolic flux

Enzymatic reactions in living cells are highly dynamic but simultaneously tightly regulated. Enzyme engineers seek to construct multienzyme complexes to prevent intermediate diffusion, to improve product yield, and to control the flux of metabolites. Here we choose a pair of short peptide tags (RIAD and RIDD) to create scaffold-free enzyme assemblies to achieve these goals. In vitro, assembling enzymes in the menaquinone biosynthetic pathway through RIAD–RIDD interaction yields protein nanoparticles with varying stoichiometries, sizes, geometries, and catalytic efficiency. In Escherichia coli, assembling the last enzyme of the upstream mevalonate pathway with the first enzyme of the downstream carotenoid pathway leads to the formation of a pathway node, which increases carotenoid production by 5.7 folds. The same strategy results in a 58% increase in lycopene production in engineered Saccharomyces cerevisiae. This work presents a simple strategy to impose metabolic control in biosynthetic microbe factories.

Effects of 1-MCP and storage temperature on transcription of mevalonate (MVA) enzyme genes of α-farnesene in ‘White Winter Pearmain’ apples fruit

The effects of 1-methylcylopropene (1-MCP) and storage temperature on the transcription of key mevalonate pathway enzyme genes in relation to α-farnesene and the conjugated trienols of apples (cv. White Winter Pearmain) were studied. Fruit samples were treated with 0.7 M 1-MCP and stored at 0 °C along with a control fruit were stored at 0 °C (Control@0 °C) and another group stored at 5 °C (Temp@5 °C) for 113 d. Results indicated that Temp@5 °C improved the α-farnesene and CTols content, ethylene production rate, and fruit scald compared with Control@0 °C, and 1-MCP@0 °C was significantly lower than Control@0 °C during storage. Flesh firmness and soluble solid contents of fruit remained unchanged by either treatment in the later period of storage. In response to 1-MCP treatment and storage temperature, qRT-PCR analysis revealed changes in the transcript level of the α-farnesene-related genes (HMGR family genes, MdMVK, MdFPPS, and MdAFS) during storage. Thus, the results of this study indicated that 1-MCP treatment and storage temperature could facilitate fruit storage by decreasing the expression of α-farnesene-related genes.

Redesign and reconstruction of a mevalonate pathway and its application in terpene production in Escherichia coli

Metabolic engineering of heterologous biosynthetic pathways in microbial hosts is an alternative way for producing high-value biochemicals. Here, heterologous mevalonate (MVA) and prenyl pyrophosphate (IPP) pathways of a new isolate Enterococcus kingsejongensis Gf1 were redesigned and reconstructed in Escherichia coli. Two MVA upstream enzymes of E. kingsejongensis Gf1 were functionally expressed and overexpression of the two enzymes led to a yield of 52.5 g/L MVA after 65 h of fed-batch fermentation. Next, the functions of three MVA downstream enzymes were verified by addition of exogenous MVA. Finally, whole MVA pathway enzymes and IPP pathway enzymes were combined in E. coli in order to overproduce squalene and β-carotene. The E. coli strains coexpressing entire MVA and IPP pathway enzymes enhanced squalene production by 15-fold and β-carotene production by 2.5-fold.

Abstract 4757: Targeting the mevalonate pathway in HER2+breast cancer to overcome resistance and enhance anti-HER2 therapy efficacy

Abstract Background: Despite the advent of HER2-targeted therapies, including the monoclonal antibody trastuzumab (T) and the HER1/2 inhibitor lapatinib (L), for HER2+ breast cancer (BC), resistance still poses a major challenge. L and L+T resistant (R) HER2+ cells with inhibited HER2 signaling showed upregulation of RNA levels of the mevalonate pathway (MVA) enzymes (which were inhibited by short term treatment of parental (P) cells with L or L+T) and increased sensitivity to MVA inhibition by statins (e.g. simvastatin (Sim)), suggesting MVA’s role as an escape mechanism of resistance. Here we investigated the therapeutic potential of another MVA inhibitor Zoledronate (ZA) and the role of mTOR and YAP/TAZ (Y/T) in mediating this resistance. Lastly, we tested if co-blockade of the MVA or its downstream effectors further sensitizes HER2+ models to anti-HER2 therapies. Methods: SKBR3 and AU565 P cells and their LR and LTR derivatives were used. The effects of MVA perturbations on cell growth and HER2 or MVA signaling were studied by methylene blue staining and Western blot (WB), respectively. Y/T activity was tested by a luciferase reporter assay and functionally validated by siRNA knockdown and dominant-active (DomA) YAP overexpression. YAP target gene expression was assessed by RT PCR. SCID-Beige mice bearing HER2+ BCM-3963 PDX tumors were treated with vehicle, L, Sim, or L+Sim and monitored for time to complete response (CR). Results: ZA, like Sim, showed a selective inhibition of cell growth and mTOR signaling in R vs. P cells, which was rescued only by the downstream metabolite geranyl geranyl pyrophosphate (GGPP), but not by the upstream metabolite mevalonate, indicating the on-target effect of ZA. Increased Y/T activity in R models was confirmed, and both Sim and ZA inhibited TAZ levels and induced phospho-YAP levels, which were rescued by the corresponding downstream metabolites. Y/T knockdown inhibited growth and mTOR signaling in R vs. P cells, and DomA YAP negated the mTOR inhibition by Sim. Sim and ZA also significantly decreased levels of the Y/T target gene survivin in R vs. P cells, and the expression was rescued by the downstream metabolites. Inhibition of MVA by Sim or ZA or its downstream signaling effectors, Y/T (by siRNA) and mTOR (by everolimus), enhanced the L sensitivity in P cells. Conversely, DomA YAP reduced the sensitivity of P cells to L. In the presence of Sim or ZA, L treatment more strongly inhibited levels of phospho-S6, a downstream target of mTORC1, compared to L alone. Preliminary in vivo data showed that treatment with L+Sim vs. L alone shortened the median time to CR and numerically increased CR rates. Conclusions: The MVA pathway mediates anti-HER2 therapy resistance via Y/T, survivin, and mTOR, in some cell models and this resistance can be overcome by Sim and ZA. The potential of MVA pathway inhibition to enhance anti-HER2 therapy efficacy warrants further clinical studies. Citation Format: Vidyalakshmi Sethunath, Huizhong Hu, Carmine DeAngelis, Jamunarani Veeraraghavan, Lanfang Qin, Martin Shea, Tamika Mitchell, Sarmistha Nanda, Resel Pereira, Susan G. Hilsenbeck, Mothaffar F. Rimawi, Kent C. Osborne, Rachel Schiff. Targeting the mevalonate pathway in HER2+breast cancer to overcome resistance and enhance anti-HER2 therapy efficacy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4757.

Identification of cis-regulatory regions responsible for developmental and hormonal regulation of HbHMGS1 in transgenic Arabidopsis thaliana.

3-Hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) synthase (HMGS) is an important enzyme in mevalonate (MVA) pathway of isoprenoid biosynthesis, which regulates the rubber biosynthetic pathway in rubber tree (Hevea brasiliensis) in coordination with HMG-CoA reductase (HMGR). However, little information is available about the regulation of HMGS gene expression. To understand the mechanism controlling the HbHMGS1 gene expression, we characterized the HbHMGS1 promoter sequence in transgenic plants with the β-glucuronidase (GUS) reporter gene. GUS activity analysis of the transgenic plants showed that the HbHMGS1 promoter is active in all organs of the transgenic Arabidopsis plants during various developmental stages (from 6 to 45-day-old). Deletion of different portions of the upstream HbHMGS1 promoter identified sequences responsible for either positive or negative regulation of the GUS expression. Particularly, the - 454bp HbHMGS1 promoter resulted in a 2.19-fold increase in promoter activity compared with the CaMV 35S promoter, suggesting that the - 454bp HbHMGS1 promoter is a super-strong near-constitutive promoter. In addition, a number of promoter regions important for the responsiveness to ethylene, methyl jasmonate (MeJA) and gibberellic acid (GA) were identified. The - 454bp HbHMGS1 promoter has great application potential in plant transformation studies as an alternative to the CaMV 35S promoter. The HbHMGS1 promoter may play important roles in regulating ethylene-, MeJA- and GA-mediated gene expression. The functional complexity of cis-elements revealed by this study remains to be elucidated.

Clonal Expansion of Second-Hit Cells with Somatic Recombinations or C>T Transitions Form Porokeratosis in MVD or MVK Mutant Heterozygotes

Patients with disseminated superficial actinic porokeratosis (DSAP) and linear porokeratosis (LP) exhibit monoallelic germline mutations in genes encoding mevalonate pathway enzymes, such as MVD or MVK. Here, we showed that each skin lesion of DSAP exhibited an individual second hit genetic change in the wild-type allele of the corresponding gene specifically in the epidermis, indicating that a postnatal second hit triggering biallelic deficiency of the gene is required for porokeratosis to develop. Most skin lesions exhibited one of two principal second hits, either somatic homologous recombinations rendering the monoallelic mutation biallelic or C>T transition mutations in the wild-type allele. The second hits differed among DSAP lesions but were identical in those of congenital LP, suggesting that DSAP is attributable to sporadic postnatal second hits and congenital LP to a single second hit in the embryonic period. In the characteristic annular skin lesions of DSAP, the central epidermis featured mostly second hit keratinocytes, and that of the annular ring featured a mixture of such cells and naïve keratinocytes, implying that each lesion reflects the clonal expansion of single second hit keratinocytes. DSAP is therefore a benign intraepidermal neoplasia, which can be included in the genetic tumor disorders explicable by Knudson's two-hit hypothesis.

Gene Mutations in Ganoderma lucidum During Long-Term Preservation by Repeated Subculturing.

Subculturing is frequently used for the preservation of basidiomycetes. In this study, to assess and verify the risks of repeated subculturing on the long-term preservation of strains of culture collections, we performed single nucleotide polymorphism (SNP) analysis in genes encoding enzymes of the mevalonate pathway, 1,3-β-glucan synthesis, lignin degradation, and the tricarboxylic acid (TCA) cycle of mycelia before and after preserving for a 4-year period by the subculturing 30 times every 45 days of Ganoderma lucidum NBRC 8346. As a result of analyzing 60 genes of the strain, SNPs were found in 18 genes, and 14 of them were found in the exon region. Nonsynonymous coding SNPs were found in two genes (atoB_2, hmgr) encoding enzymes of mevalonate pathway and five genes (lcc1_9, lcc1_11, lcc1_13, dslcc6, aa5_1_9) encoding enzymes of lignin degradation after subculturing of G. lucidum NBRC 8346.

The mevalonate coordinates energy input and cell proliferation

The mevalonate pathway is known for the synthesis of cholesterol, but recent studies have reported that it also controls Hippo signaling, which is critical for the regulation of organ size and tumorigenesis. Here, we discover that the suppression of the mevalonate pathway inhibits the growth and proliferation of colon cancer cell lines. The results of transcriptomic and proteomic assays suggested that the mevalonate pathway controls multiple signaling pathways relevant to cell proliferation, and the results were further confirmed using western blot, PCR, and immunofluorescence assays. As cell proliferation is an energy-consuming process, we postulate that the mevalonate pathway may also control nutrient uptake to coordinate the processes of energy supply and cell proliferation. Here, we found that lovastatin, a mevalonate pathway inhibitor, suppresses glucose and amino acid uptake and lactate acid production. More importantly, mevalonic acid itself is sufficient to promote glucose uptake by colon cancer cells. In addition, we found that colon cancer tissues displayed a higher expression of mevalonate pathway enzymes, which may promote cell growth and stimulate energy uptake. Together, our findings establish the mevalonate pathway as a critical regulator in coordinating energy input and cell proliferation.

FDPS cooperates with PTEN loss to promote prostate cancer progression through modulation of small GTPases/AKT axis.

Farnesyl Diphosphate Synthase (FDPS), a mevalonate pathway enzyme, is highly expressed in several cancers, including prostate cancer (PCa). To date, the mechanistic, functional and clinical significance of FDPS in cancer remains unexplored. We evaluated the FDPS expression and its cancer-associated phenotypes using in vitro and in vivo methods in PTEN deficient and sufficient human and mouse PCa cells and tumors. Interestingly, FDPS overexpression synergizes with PTEN deficiency in PTEN conditionally knockout mice (P<0.05) and expressed significantly higher in human (P<0.001) PCa tissues, cell lines and murine tumoroids compared to respective controls. In silico analysis revealed that FDPS is associated with increasing Gleason score, PTEN functionally deficient status and poor survival of PCa. Ectopic overexpression of FDPS promotes oncogenic phenotypes such as colony formation (P<0.01) and proliferation (P<0.01) through activation of AKT and ERK signaling by prenylating Rho A, Rho G and CDC42 small GTPases. Of interest, knockdown of FDPS in PCa cells exhibits decreased colony growth and proliferation (P<0.001) by modulating AKT and ERK pathways. Further, genetic and pharmacological inhibition of PI3K but not AKT reduced FDPS expression. Pharmacological targeting of FDPS by zoledronic acid (ZOL), which is already in clinics exhibit reduced growth and clonogenicity of human and murine PCa cells (P<0.01) and 3D tumoroids (P<0.02) by disrupting AKT and ERK signaling through direct interference of small GTPases protein prenylation. Thus, FDPS plays an oncogenic role in PTEN-deficient PCa through GTPase/AKT axis. Identifying mevalonate pathway proteins could serve as a therapeutic target in PTEN dysregulated tumors.

Biosynthesis of cycloartenol by expression of plant and bacterial oxidosqualene cyclases in engineered Rhodobacter capsulatus

Cyclic triterpenes are a large group of secondary metabolites produced by plants, fungi and bacteria. They have diverse biological functions, and offer potential health benefits for humans. Although various terpenes from the mono-, sesqui- and diterpene classes are easy to produce in engineered bacteria, heterologous synthesis of cyclic triterpenes is more challenging. We have recently shown that the triterpene cycloartenol can be produced in Rhodobacter capsulatus SB1003 but initial titers were low with 0.34mgL−1. To assess, if this phototrophic α-proteobacterium can be engineered for enhanced triterpene production, we followed two alternative strategies by comparing the performance of the R. capsulatus SB1003 wildtype strain with two recombinant strains carrying either a mevalonate pathway implemented from Paracoccus zeaxanthinifaciens or a deletion in the intrinsic carotenoid biosynthesis gene crtE. These strains are thus engineered for an enhanced isoprenoid biosynthesis or a suppressed precursor conversion by the competing carotenoid pathway. Moreover, three different cycloartenol synthase (CAS) genes from Arabidopsis thaliana or the myxobacterial strains Stigmatella aurantiaca Sga15 and DW4/3-1 were tested for heterologous cycloartenol synthesis. We found that the heterologous expression of mevalonate pathway enzymes had little impact on cycloartenol levels irrespective of the chosen CAS. In contrast, the use of the newly constructed carotenoid-deficient crtE deletion strain showed threefold increased cycloartenol product titers. We conclude that R. capsulatus is a promising alternative host for the functional expression of triterpene biosynthetic enzymes from plants and microbes. Apparently, product titers can also be improved by suppression of competing precursor consumption.

The mevalonate pathway but not the methylerythritol phosphate pathway is critical for elaioplast and pollen coat development in &lt;i&gt;Arabidopsis&lt;/i&gt;

Pollen coat components are derived from tapetum cells, which contain elaioplasts derived from plastids and tapetosome derived from the endoplasmic reticulum. In Brassica napus, the main neutral lipids in the elaioplast and tapetosome have been reported to be sterol ester and triacylglycerol, respectively. Isopentenyl pyrophosphate, the structural component of sterol, is produced via the cytosolic mevalonate (MVA) and plastidic methylerythritol phosphate (MEP) pathways. Although these two pathways are compartmentalized, partial cross-talk between them has been reported. To investigate the contribution of these two pathways in elaioplast formation, we characterized mutant pollen of these two pathways. We observed the anthers of male sterile hmg1-1 and atipi1 atipi2 mutants ultrastructurally, which were deficient in MVA pathway enzymes. hmg1-1 and atipi1atipi2 showed a shrunken elaioplast inner granule at the bicellular pollen stage. Conversely, in the cla1-1 mutant, which showed a defective MEP pathway, elaioplast development was normal. The pollen of hmg1-1 and atipi1atipi2 was coatless, whereas cla1-1 had a pollen coat. These results indicate that the MVA pathway but not the MEP pathway is critical for elaioplast development though the organelle is derived from plastids.

Statins and the Brain: More than Lipid Lowering Agents?

Background: Statins represent a class of medications widely prescribed to efficiently treat dyslipidemia. These drugs inhibit 3-βhydroxy 3β-methylglutaryl Coenzyme A reductase (HMGR), the rate-limiting enzyme of mevalonate (MVA) pathway. Besides cholesterol, MVA pathway leads to the production of several other compounds, which are essen-tial in the regulation of a plethora of biological activities, including in the central nervous system. For these reasons, statins are able to induce pleiotropic actions, and acquire increased interest as potential and novel modulators in brain processes, es-pecially during pathological conditions.Objective: The purpose of this review is to summarize and examine the current knowledge about pharmacokinetic and phar-macodynamic properties of statins in the brain. In addition, effects of statin on brain diseases are discussed providing the most up-to-date information.Methods: Relevant scientific information was identified from PubMed database using the following keywords: statins and brain, central nervous system, neurological diseases, neurodegeneration, brain tumors, mood, stroke.Results: 315 scientific articles were selected and analyzed for the writing of this review article. Several papers highlighted that statin treatment is effective in preventing or ameliorating the symptomatology of a number of brain pathologies. Howev-er, other studies failed to demonstrate a neuroprotective effect.Conclusion: Even though considerable research studies suggest pivotal functional outcomes induced by statin therapy, addi-tional investigation is required to better determine the pharmacological effectiveness of statins in the brain, and support their clinical use in the management of different neuropathologies.

HSP90 interacts with HMGCR and promotes the progression of hepatocellular carcinoma.

Heat shock protein 90 (HSP90) has been reported to promote the growth and inhibit apoptosis of hepatocellular carcinoma (HCC) cells. However, the underlying mechanisms are not fully understood. Immunostaining of the tissue array demonstrated that HSP90 was upregulated in HCC clinical samples and was associated with clinical features. HSP90 interacted with 3‑hydroxy‑3‑methylglutaryl‑CoA reductase (HMGCR), the rate‑limiting enzyme of mevalonate pathway, in the immunoprecipitation assay and regulated its protein expression level by inhibiting protein degradation. In addition, lovastatin, an inhibitor of HMGCR, impaired the oncogenic functions of HSP90 in the cell growth, migration and colony formation assays. Taken together, this study demonstrated that HSP90 promoted the progression of HCC by positively regulating the mevalonate pathway and indicated that HSP90 may be a promising therapeutic target.

Abstract 2877: Synergistic antitumor interaction of valproic acid and simvastatin sensitizes prostate cancer to docetaxel by targeting cancer stem cells compartment via YAP-pathway modulation

Abstract Introduction: docetaxel (DTX) represents the standard of care first line treatment of castration-resistant prostate cancer (PCa). However, the onset of systemic side effects hampers patient's compliance and DTX resistance invariably emerges, leading to disease relapse, suggesting the need of novel combination strategies. Cancer stem cells (CSC) drive PCa survival and metastasis and are thought to be responsible for the development of resistance to DTX. The mevalonate pathway (MVP) plays a critical role in PCa progression and is implicated in cell stemness, proliferation, and organ size regulation through the YAP-TAZ signaling axis. Here, we evaluate the combination treatment of valproic acid (VPA), an histone deacetylase (HDAC) inhibitor and the cholesterol-lowering drug simvastatin (SIM), an inhibitor of HMGCoA reductase (HMGCR), the rate limiting enzyme in MVP, alone and plus DTX in PCa models. Method: synergistic antiproliferative effects were assessed on LNCAP, 22Rv1, DU145, PC3 and DU14580 SIM-resistant cell lines and normal epithelial prostate EPN cells, by calculating combination index (CI) according to Chou and Talalay method. Apoptosis was measured by FACS analysis and caspase assay. Tumor spheroids were obtained by low attach systems and scored with luminescence 3D-cell viability assay. Proteins and genes expression was assessed by western blot and real time PCR analysis. In vivo experiments were performed on xenograft models in athymic mice. Cholesterol content was evaluated by nuclear magnetic resonance (1H-NMR). Results: synergistic antiproliferative and proapoptotic effect of VPA-SIM was observed in all PCa cell lines tested, except in EPN cells and was confirmed in PCa spheroids. SIM-dependent cholesterol content downmodulation was potentiated by VPA and mevalonic acid, the product of HMGCR activity, reverts all the antitumor combined effect, suggesting the involvement of MVP in the synergistic interaction. Mechanistically, the combination is able to induce a reduction of HMGCR mRNA expression and the inhibitory phosphorylation of HMGCR and YAP, followed by a reduction of CTGF, BRCA5 and Cyr61 YAP-target genes, as well as a reduction of a CSC-marker gene such as NANOg. Notably, the VPA-SIM combination, sensitizes PCa cells to DTX treatment in sensitive and DTX-resistant cells, as shown by CI calculation, apoptosis, and CSC enriched-spheroid experiments. The synergistic interaction of the triple combination was also confirmed in vivo in both DU145R80 and 22Rv1 xenograft models. Conclusions: Overall, this study suggests that the combination of two safe generic drugs such as VPA and SIM, may improve the therapeutic index of DTX and revert DTX-resistance, representing an innovative and feasible antitumor strategy for the treatment of prostate cancer that warrants further clinical evaluation. Citation Format: Federica Iannelli, Maria Serena Roca, Chiara Ciardiello, Simona De Rienzo, Rita Lombardi, Angela Sorice, Susan Costantini, Tania Moccia, Maria Rita Milone, Biagio Pucci, Alfredo Budillon, Francesca Bruzzese. Synergistic antitumor interaction of valproic acid and simvastatin sensitizes prostate cancer to docetaxel by targeting cancer stem cells compartment via YAP-pathway modulation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2877.