Showing papers on "Lanosterol synthase published in 2014"

A Review Article on Hyperlipidemia: Types, Treatments and New Drug Targets

Abstract: Hyperlipidemia is a medical condition characterized by an increase in one or more of the plasma lipids, including triglycerides, cholesterol, cholesterol esters, phospholipids and or plasma lipoproteins including very low-density lipoprotein and low-density lipoprotein along with reduced high-density lipoprotein levels. This elevation of plasma lipids is among the leading risk factors associated with cardiovascular diseases. In the meantime, statins and fibrates remain the major anti-hyperlipidemic agents for the treatment of elevated plasma cholesterol and triglycerides respectively, with the price of severe side effects on the muscles and the liver. The present review focuses mainly on the types of hyperlipidemias, lipid metabolism, treatments and new drug targets for the treatment of elevated lipid profile. Many agents such as lanosterol synthase inhibitors, squalene epoxidase inhibitors, diacyl glycerol acyl transferase inhibitors, ATP citrate lyase inhibitors have shown a promising potential in the treatment of hyperlipidemia in clinical trials.

Plant Oxidosqualene Metabolism: Cycloartenol Synthase–Dependent Sterol Biosynthesis in Nicotiana benthamiana

Abstract: The plant sterol pathway exhibits a major biosynthetic difference as compared with that of metazoans. The committed sterol precursor is the pentacyclic cycloartenol (9β,19-cyclolanost-24-en-3β-ol) and not lanosterol (lanosta-8,24-dien-3β-ol), as it was shown in the late sixties. However, plant genome mining over the last years revealed the general presence of lanosterol synthases encoding sequences (LAS1) in the oxidosqualene cyclase repertoire, in addition to cycloartenol synthases (CAS1) and to non-steroidal triterpene synthases that contribute to the metabolic diversity of C30H50O compounds on earth. Furthermore, plant LAS1 proteins have been unambiguously identified by peptidic signatures and by their capacity to complement the yeast lanosterol synthase deficiency. A dual pathway for the synthesis of sterols through lanosterol and cycloartenol was reported in the model Arabidopsis thaliana, though the contribution of a lanosterol pathway to the production of 24-alkyl-Δ5-sterols was quite marginal (Ohyama et al. (2009) PNAS 106, 725). To investigate further the physiological relevance of CAS1 and LAS1 genes in plants, we have silenced their expression in Nicotiana benthamiana. We used virus induced gene silencing (VIGS) based on gene specific sequences from a Nicotiana tabacum CAS1 or derived from the solgenomics initiative (http://solgenomics.net/) to challenge the respective roles of CAS1 and LAS1. In this report, we show a CAS1-specific functional sterol pathway in engineered yeast, and a strict dependence on CAS1 of tobacco sterol biosynthesis.

β-Amyrin synthase from Euphorbia tirucalli. Steric bulk, not the π-electrons of Phe, at position 474 has a key role in affording the correct folding of the substrate to complete the normal polycyclization cascade

Abstract: β-Amyrin, a triterpene, is widely distributed in plants and its glycosides confer important biological activities. Mutagenesis studies on β-amyrin synthase are very limited as compared with those of squalene-hopene cyclase and lanosterol synthase. This study was conducted to elucidate the function of the F474 residue of Euphorbia tirucalli β-amyrin cyclase, which is highly conserved in the superfamily of oxidosqualene cyclases. Nine site-specific variants with Gly, Ala, Val, Leu, Met, Tyr, Trp, His, and Thr were constructed. We isolated 9 products from these mutants in addition to β-amyrin and determined the chemical structures. The Gly and Ala mutants produced significantly larger amounts of the bicyclic products and a decreased amount of β-amyrin, indicating that the F474 residue was located near the B-ring formation site. Surprisingly, the Ala variant produced (9βH)-polypoda-7,13,17,21-tetraen-3β-ol and (9βH)-polypoda-8(26),13,17,21-tetraen-3β-ol, which are generated from a chair–boat folding conformation. This is the first report describing the conformational change from the chair–chair into the chair–boat folding conformation among the reported mutagenesis studies of oxidosqualene cyclases. Substitution with aliphatic amino acids lacking π-electrons such as Val, Leu, and Met led to a significantly decreased production of bicyclic compounds, and in turn exhibited a higher production of β-amyrin. Furthermore, the Leu and Met variants exhibited high enzymatic activities: ca. 74% for Leu and ca. 91% for Met variants as compared to the wild-type. These facts unambiguously demonstrate that the major role of Phe474 is not to stabilize the transient cation via cation–π interaction, but is to confer the appropriate steric bulk near the B-ring formation site, leading to the completion of the normal polycyclization pathway without accumulation of abortive cyclization products.

Structural and functional analyses of a sterol carrier protein in Spodoptera litura.

Abstract: Backgrounds In insects, cholesterol is one of the membrane components in cells and a precursor of ecdysteroid biosynthesis. Because insects lack two key enzymes, squalene synthase and lanosterol synthase, in the cholesterol biosynthesis pathway, they cannot autonomously synthesize cholesterol de novo from simple compounds and therefore have to obtain sterols from their diet. Sterol carrier protein (SCP) is a cholesterol-binding protein responsible for cholesterol absorption and transport. Results In this study, a model of the three-dimensional structure of SlSCPx-2 in Spodoptera litura, a destructive polyphagous agricultural pest insect in tropical and subtropical areas, was constructed. Docking of sterol and fatty acid ligands to SlSCPx-2 and ANS fluorescent replacement assay showed that SlSCPx-2 was able to bind with relatively high affinities to cholesterol, stearic acid, linoleic acid, stigmasterol, oleic acid, palmitic acid and arachidonate, implying that SlSCPx may play an important role in absorption and transport of these cholesterol and fatty acids from host plants. Site-directed mutation assay of SlSCPx-2 suggests that amino acid residues F53, W66, F89, F110, I115, T128 and Q131 are critical for the ligand-binding activity of the SlSCPx-2 protein. Virtual ligand screening resulted in identification of several lead compounds which are potential inhibitors of SlSCPx-2. Bioassay for inhibitory effect of five selected compounds showed that AH-487/41731687, AG-664/14117324, AG-205/36813059 and AG-205/07775053 inhibited the growth of S. litura larvae. Conclusions Compounds AH-487/41731687, AG-664/14117324, AG-205/36813059 and AG-205/07775053 selected based on structural modeling showed binding affinity to SlSCPx-2 protein and inhibitory effect on the growth of S. litura larvae.

Construction of Saccharomyces cerevisiae haploid mutant deficient in lanosterol synthase gene

Abstract: Lanosterol synthase is encoded by the erg7 gene and catalyzes the cyclization of 2, 3-oxidosqualene, which is a rate-limiting step of the inherent mevalonate (MVA)/ergosterol metabolic pathway in Saccharomyces cerevisiae. The intermediate 2, 3-oxidosqualene is also the precursor of triterpenoids. Therefore, the cyclization of 2, 3-oxidosqualene is the key branch point of ergosterol and triterpenoids biosynthesis. Down-regulation of 2, 3-oxidosqualene metabolic flux to ergosterol in S. cerevisiae may redirect the metabolic flux toward the triterpenoid synthetic pathway reconstructed by the synthetic biology approach. To construct erg7 knockout cassette harboring the loxP-Marker-loxP element, long primers were designed, which were homologous to the sequences of both erg7 ORF and plasmid pUG66. The cassette was transformed into diploid wild strain INVSc1 by LiAc/SS Carrier DNA/PEG method and then erg7 gene haploid deficient mutant was obtained by homologous recombination. The results of semiquantitative PCR and real-time quantitative PCR revealed that erg7 expression level in erg7 gene haploid deficient mutant is one time lower than that in wild strain. The results of TLC and HPLC showed that the ergosterol content in deficient mutant decreased to 42% of that in wild strain.

Investigations of the Specificity of Oxidosqualene Cyclization: Errors are the Rule, Not the Exception

Abstract: Investigations of the Specificity of Oxidosqualene Cyclization: Errors are the Rule, not the Exception By Paul G. Bodager This thesis describes the characterization of oxidosqualene cyclases from numerous organisms, through heterologous expression in Saccharomyces cerevisiae, and extraction from organismal tissue. Oxidosqualene cyclases are a family of proteins which catalyze the cyclization of the linear substrate oxidosqualene into cyclic compounds known as triterpene alcohols, acting in both the primary and secondary metabolism of organisms. Detailed analyses of cyclase product profiles in both primary and secondary metabolism are used herein to develop a comprehensive discussion of cyclase product specificity. First, the characterization of two oxidosqualene cyclases of secondary metabolism from the plant Arabidopsis thaliana, LUP4 and LUP5, by heterologous expression, is described. While demonstrating quite different product specificity, both cyclases make a mixture of nearly 20 triterpene alcohols. The isolation of a novel triterpene alcohol, (20S)-dammara-12,24-dienol, is reported. Next, the characterization of six oxidosqualene cyclases of primary metabolism are detailed, including lanosterol synthases from S. cerevisiae, Trypanosoma cruzi, Trypanosoma brucei, Homo sapiens, Bos taurus, and cycloartenol synthase from A. thaliana. Despite no reports of minor product generation by lanosterol synthases prior to this work, each cyclase is shown to make minor “errors”. These cyclases make different sets of minor products, and produce the major product with varying accuracy. This work demonstrates that minor product formation is characteristic of oxidosqualene cyclization, and leads to the conclusion that no cyclase produces only a single product. Finally, lanosterol synthase product profiles are extended to in vivo systems, via the analysis of triterpene alcohols present in yeast culture, as well as in mammalian tissue. This analysis demonstrates that S. cerevisiae lanosterol synthase produces at least 16 products, including three generated through B-ring-chair intermediates, the first evidence of a non-mutant cyclase accessing B-ring-boat and B-ring-chair intermediates. Analysis of bovine brain extracts led to the discovery that 18 lanosterol synthase minor products are detectable in mammalian tissue, including two novel triterpene alcohols, protosta-20(22)E-dienol and CB-thalianol A. Finally, this analysis demonstrated that one lanosterol synthase minor product, parkeol, is metabolized by enzymes in the sterol biosynthetic pathway, demonstrating that enzymatic errors generate a previously hidden level of chemical diversity in primary metabolism. Acknowledgements I would first like to thank my advisor, Professor Seiichi P. T. Matsuda, for providing me with the environment and resources necessary to complete the work contained in this thesis. More importantly, I would also like to thank him for the freedom and trust he gave me to work on projects that I thought were interesting, and for very thought-provoking discussions regarding not just the details of experimental results, but also what those results meant in terms of a more comprehensive picture. I would also like to thank Professors Zachary Ball and Joff Silberg for agreeing to be members of my thesis committee, and for their valuable input. I am grateful to the members of the Matsuda Lab who have been my colleagues for the last several years, especially for putting up with my occasionally unpleasant experiments. I would especially like to thank Dr. Bill Wilson for his enormous amount of help with not only the acquisition and analysis of NMR spectra, but also for valuable scientific discussions, and for his continued desire to help everyone in the lab succeed. I thank Dr. Hui Shan for her kindness, and for her assistance in helping me become expert in the operation of HPLC instrumentation. I would also like to thank my graduate student co-workers, Dr. Dorianne Castillo Rivera, Melisa Moreno Garcia, Matias Kinzurik, and Jing Jin for helping to create a friendly working environment. Finally, I would like to thank the undergraduates I supervised, Blair Lunceford and Carrie Levinn, as well as Aparna Bhaduri, an undergraduate who trained me in cell culturing and molecular biology. v I would not be where I am now without an excellent educational background; thank you to the professors whom I had the pleasure of learning from here at Rice, especially Professors Paul Engel and Ron Parry. Additionally, I would like to thank my professors at Marietta College, Professors Kevin Pate, Debra Egolf, Robert Walker, and Jim Jeitler. The education I received at Marietta provided a solid foundation for my future chemical endeavors. I am also deeply indebted to my family for their love and support throughout graduate school and life in general. Thank you to my siblings, Jacob and Haley, for always being able to make me laugh; I could not ask for a better brother and sister. I would especially like to thank my parents, Greg and Sheila, for always believing in me, and always encouraging me to do my best, in all phases of my life. I would also like to thank my dad for teaching me from a young age that “Chemistry is Life”, which is a lesson that definitely seems to have stuck. Finally, and most importantly, I would like to thank my wife and best friend Britt, for her steadfast support and encouragement throughout my time in graduate school. She was always there through the highs and the lows of research, and she was always able to help me forget about science when I needed to clear my head. She has been incredibly understanding of the long hours in the lab (and especially in the preparation of this thesis), and tremendously supportive of my work. Thank you Britt for being such a good teammate; I can’t imagine being able to have done this without you.

In-silico screening of potential inhibitors of HMGCoA reductase and Lanosterol synthase, key enzymes in cholesterol biosynthesis pathway

Abstract: Hyperlipidemia is elevated level of lipids or lipoproteins in our body. Cholesterol is a major lipid particle circulating in our body. Cholesterol synthesis takes place in liver and this biosynthesis pathway is called Mevalonate pathway. Cholesterol is the end product of this Mevalonate pathway. Several enzymes play a role in the Cholesterol biosynthesis. Enzymes like HMGCoA synthase, HMGCoA reductase, Farnesyl PP synthase, Lanosterol synthase, Squalene synthase. This cholesterol is mainly responsible for several health effects especially Coronary heart diseases, atherosclerosis etc. So now in order to prevent the cholesterol synthesis in our body, we need to find potent inhibitors against the above enzymes and there after modify them as drugs. In this study, two enzymes HMGCoA reductase and Lanosterol synthase were targeted. For our docking study, we used Arguslab, Mgl tools, Autodock vina softwares for obtaining binding energies of protein and ligands. We search for already available drugs acting against HMGCoA reductase called Statins. We obtained binding energy results with NADH, PRAVASTATIN, LOVASTATIN, CERIVASTATIN, SIMVASTATIN, FLUVASTATIN, BEZAFIBRATE and note them as POSITIVE CONTROL 1. Then we dock HMGCoA reductase with natural molecules and compare them with positive control 1. In the next step, we targeted Lanosterol Synthase with available Quinuclidine inhibitors and results obtained was noted as POSITIVE CONTROL 2. Now we cross docked Lanosterol synthase with previous natural ligands and compared them with Positive control 2. We later found the toxicity and druglikeness of all inhibitors used against both the targets. We found two natural molecules APIGENIN and NARINGENIN which showed best binding energy results against both the targets. Both Apigenin and Naringenin act as suitable inhibitors against HMGCoA reductase and Lanosterol synthase inhibiting at two places along the Mevalonate pathway.