Browsing by Author "Haubrich, Brad A. (TTU)"
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Item Discovery of an ergosterol-signaling factor that regulates trypanosoma brucei growth(2015) Haubrich, Brad A. (TTU); Singha, Ujjal K.; Miller, Matthew B. (TTU); Nes, Craigen R. (TTU); Anyatonwu, Hosanna (TTU); Lecordier, Laurence; Patkar, Presheet (TTU); Leaver, David J. (TTU); Villalta, Fernando; Vanhollebeke, Benoit; Chaudhuri, Minu; Nes, W. David (TTU)Ergosterol biosynthesis and homeostasis in the parasitic protozoan Trypanosoma brucei was analyzed by RNAi silencing and inhibition of sterol C24β -methyltransferase ( TbSMT) and sterol 14α-demethylase [ TbSDM ( TbCYP51)] to explore the functions of sterols in T. brucei growth. Inhibition of the amount or activity of these enzymes depletes ergosterol from cells at <6 fg/cell for procyclic form (PCF) cells or <0.01 fg/cell for bloodstream form (BSF) cells and reduces infectivity in a mouse model of infection. Silencing of Tb SMT expression by RNAi in PCF or BSF in combination with 25-azalanosterol (AZA) inhibited parasite growth and this inhibition was restored completely by adding synergistic cholesterol (7.8 μM from lipid-depleted media) with small amounts of ergosterol (1.2 μM) to the medium. These observations are consistent with the proposed requirement for ergosterol as a signaling factor to spark cell proliferation while imported cholesterol or the endogenously formed cholesta-5,7,24-trienol act as bulk membrane components. To test the potential chemotherapeutic importance of disrupting ergosterol biosynthesis using pairs of mechanismbased inhibitors that block two enzymes in the post-squalene segment, parasites were treated with AZA and itraconazole at 1 μM each (ED50 values) resulting in parasite death . Taken together, our results demonstrate that the ergosterol pathway is a prime drug target for intervention in T. brucei infection. -Haubrich, B. A., U. K. Singha, M. B. Miller, C. R. Nes, H. Anyatonwu, L. Lecordier, P. Patkar, D. J. Leaver, F. Villalta, B. Vanhollebeke, M. Chaudhuri, and W. D. Nes. Discovery of an ergosterol-signaling factor that regulates Trypanosoma brucei growth. J. Lipid Res. 2015.Item Evolutionarily conserved Δ25(27)-olefin ergosterol biosynthesis pathway in the alga Chlamydomonas reinhardtii(2012) Miller, Matthew B. (TTU); Haubrich, Brad A. (TTU); Wang, Qian; Snell, William J.; Nes, W. David (TTU)Ergosterol is the predominant sterol of fungi and green algae. Although the biosynthetic pathway for sterol synthesis in fungi is well established and is known to use C24-methylation-C24 (28)-reduction (Δ24(28)-olefin pathway) steps, little is known about the sterol pathway in green algae. Previous work has raised the possibility that these algae might use a novel pathway because the green alga Chlamydomonas reinhardtii was shown to possess a mevalonate-independent methylerythritol 4-phosphate not present in fungi. Here, we report that C. reinhardtii synthesizes the protosterol cycloartenol and converts it to ergosterol (C24β-methyl) and 7-dehydroporiferasterol (C24β-ethyl) through a highly conserved sterol C24- methylation-C25- reduction (Δ25(27)-olefin) pathway that is distinct from the well-described acetate-mevalonate pathway to fungal lanosterol and its conversion to ergosterol by the Δ24 (28)-olefin pathway. We isolated and characterized 23 sterols by a combination of GC-MS and proton nuclear magnetic resonance spectroscopy analysis from a set of mutant, wild-type, and 25-thialanosterol-treated cells. The structure and stereochemistry of the final C24-alkyl sterol side chains possessed different combinations of 24β-methyl/ethyl groups and Δ22(23) E and Δ25 (27)-double bond constructions. When incubated with [methyl-2H3]methionine, cells incorporated three (into ergosterol) or five (into 7-dehydroporiferasterol) deuterium atoms into the newly biosynthesized 24β-alkyl sterols, consistent only with a Δ25 (27)-olefin pathway. Thus, our findings demonstrate that two separate isoprenoid-24-alkyl sterol pathways evolved in fungi and green algae, both of which converge to yield a common membrane insert ergosterol. Copyright © 2012 by the American Society for Biochemistry and Molecular Biology, Inc.