FR171456 is a specific inhibitor of mammalian NSDHL and yeast Erg26p.

FR171456 is a natural product with cholesterol-lowering properties in animal models, but its molecular target is unknown, which hinders further drug development. Here we show that FR171456 specifically targets the sterol-4-alpha-carboxylate-3-dehydrogenase (Saccharomyces cerevisiae--Erg26p, Homo sapiens--NSDHL (NAD(P) dependent steroid dehydrogenase-like)), an essential enzyme in the ergosterol/cholesterol biosynthesis pathway. FR171456 significantly alters the levels of cholesterol pathway intermediates in human and yeast cells.

Identification of natural RORγ ligands that regulate the development of lymphoid cells.

Mice deficient in the nuclear hormone receptor RORγt have defective development of thymocytes, lymphoid organs, Th17 cells, and type 3 innate lymphoid cells. RORγt binds to oxysterols derived from cholesterol catabolism, but it is not clear whether these are its natural ligands. Here, we show that sterol lipids are necessary and sufficient to drive RORγt-dependent transcription.

Plant cyclopropylsterol-cycloisomerase: key amino acids affecting activity and substrate specificity.

The enzyme CPI (cyclopropylsterol-cycloisomerase) from the plant sterol pathway catalyses the cleavage of the 9β,19-cyclopropane ring of the 4α-methyl-cyclopropylsterol cycloeucalenol to produce the Δ8-sterol obtusifoliol. Randomly mutated plasmids carrying the Arabidopsis thaliana cpi gene were screened for inactive CPI mutant enzymes on the basis of their ability to genetically complement a Saccharomyces cerevisiae erg7 (defective in oxidosqualene cyclase) ergosterol auxotroph grown in the presence of exogenous cycloeucalenol, and led to the identification of four catalytically important residues. Site-directed mutagenesis experiments confirmed the role of the identified residues, and demonstrated the importance of selected acidic residues and a conserved G108NYFWTHYFF117 motif.

Arabidopsis ERG28 tethers the sterol C4-demethylation complex to prevent accumulation of a biosynthetic intermediate that interferes with polar auxin transport.

Sterols are vital for cellular functions and eukaryotic development because of their essential role as membrane constituents. Sterol biosynthetic intermediates (SBIs) represent a potential reservoir of signaling molecules in mammals and fungi, but little is known about their functions in plants. SBIs are derived from the sterol C4-demethylation enzyme complex that is tethered to the membrane by Ergosterol biosynthetic protein28 (ERG28).

DOLICHOL PHOSPHATE MANNOSE SYNTHASE1 mediates the biogenesis of isoprenyl-linked glycans and influences development, stress response, and ammonium hypersensitivity in Arabidopsis.

The most abundant posttranslational modification in nature is the attachment of preassembled high-mannose-type glycans, which determines the fate and localization of the modified protein and modulates the biological functions of glycosylphosphatidylinositol-anchored and N-glycosylated proteins. In eukaryotes, all mannose residues attached to glycoproteins from the luminal side of the endoplasmic reticulum (ER) derive from the polyprenyl monosaccharide carrier, dolichol P-mannose (Dol-P-Man), which is flipped across the ER membrane to the lumen. We show that in plants, Dol-P-Man is synthesized when Dol-P-Man synthase1 (DPMS1), the catalytic core, interacts with two binding proteins, DPMS2 and DPMS3, that may serve as membrane anchors for DPMS1 or provide catalytic assistance.

Dissecting the sterol C-4 demethylation process in higher plants. From structures and genes to catalytic mechanism.

Sterols become functional only after removal of the two methyl groups at C-4. This review focuses on the sterol C-4 demethylation process in higher plants. An intriguing aspect in the removal of the two C-4 methyl groups of sterol precursors in plants is that it does not occur consecutively as it does in yeast and animals, but is interrupted by several enzymatic steps.

Characterization of plant carotenoid cyclases as members of the flavoprotein family functioning with no net redox change.

The later steps of carotenoid biosynthesis involve the formation of cyclic carotenoids. The reaction is catalyzed by lycopene beta-cyclase (LCY-B), which converts lycopene into beta-carotene, and by capsanthin-capsorubin synthase (CCS), which is mainly dedicated to the synthesis of kappa-cyclic carotenoids (capsanthin and capsorubin) but also has LCY-B activity. Although the peptide sequences of plant LCY-Bs and CCS contain a putative dinucleotide-binding motif, it is believed that these two carotenoid cyclases proceed via protic activation and stabilization of resulting carbocation intermediates.

Abscisic acid negatively regulates elicitor-induced synthesis of capsidiol in wild tobacco.

In the Solanaceae, biotic and abiotic elicitors induce de novo synthesis of sesquiterpenoid stress metabolites known as phytoalexins. Because plant hormones play critical roles in the induction of defense-responsive genes, we have explored the effect of abscisic acid (ABA) on the synthesis of capsidiol, the major wild tobacco (Nicotiana plumbaginifolia) sesquiterpenoid phytoalexin, using wild-type plants versus nonallelic mutants Npaba2 and Npaba1 that are deficient in ABA synthesis. Npaba2 and Npaba1 mutants exhibited a 2-fold higher synthesis of capsidiol than wild-type plants when elicited with either cellulase or arachidonic acid or when infected by Botrytis cinerea.

Homology modeling and site-directed mutagenesis reveal catalytic key amino acids of 3beta-hydroxysteroid-dehydrogenase/C4-decarboxylase from Arabidopsis.

Sterols become functional only after removal of the two methyl groups at C4 by a membrane-bound multienzyme complex including a 3beta-hydroxysteroid-dehydrogenase/C4-decarboxylase (3betaHSD/D). We recently identified Arabidopsis (Arabidopsis thaliana) 3betaHSD/D as a bifunctional short-chain dehydrogenase/reductase protein. We made use of three-dimensional homology modeling to identify key amino acids involved in 4alpha-carboxy-sterol and NAD binding and catalysis.

Identification of essential amino acid residues in a sterol 8,7-isomerase from Zea mays reveals functional homology and diversity with the isomerases of animal and fungal origin.

A putative 8,7SI (sterol 8,7-isomerase) from Zea mays, termed Zm8,7SI, has been isolated from an EST (expressed sequence tag) library and subcloned into the yeast erg2 mutant lacking 8,7SI activity. Zm8,7SI restored endogenous ergosterol synthesis. An in vitro enzymatic assay in the corresponding yeast microsomal extract indicated that the preferred Delta(8)-sterol substrate possesses a single C4alpha methyl group, in contrast with 8,7SIs from animals and fungi, thus reflecting the diversity in the structure of their active site in relation to the distinct sterol biosynthetic pathways.

Molecular and enzymatic characterizations of novel bifunctional 3beta-hydroxysteroid dehydrogenases/C-4 decarboxylases from Arabidopsis thaliana.

We have isolated two cDNAs from Arabidopsis thaliana encoding bifunctional 3beta-hydroxysteroid dehydrogenase/C-4 decarboxylases (3betaHSD/D) involved in sterol synthesis, termed At3betaHSD/D1 and At3betaHSD/D2. Transformation of the yeast ergosterol auxotroph erg26 mutant, which lacks 3betaHSD/D activity, with the At3betaHSD/D1 isoform or with At3betaHSD/D2 isoform containing a C-terminal At3betaHSD/D1 endoplasmic reticulum-retrieval sequence restored growth and ergosterol synthesis in erg26. An in vitro enzymatic assay revealed high 3betaHSD/D activity for both isoenzymes in the corresponding microsomal extracts.

Biogenesis, molecular regulation and function of plant isoprenoids.

Isoprenoids represent the oldest class of known low molecular-mass natural products synthesized by plants. Their biogenesis in plastids, mitochondria and the endoplasmic reticulum-cytosol proceed invariably from the C5 building blocks, isopentenyl diphosphate and/or dimethylallyl diphosphate according to complex and reiterated mechanisms. Compounds derived from the pathway exhibit a diverse spectrum of biological functions.

Lipid signaling in plants. Cloning and expression analysis of the obtusifoliol 14alpha-demethylase from Solanum chacoense Bitt., a pollination- and fertilization-induced gene with both obtusifoliol and lanosterol demethylase activity.

The sterol 14alpha-demethylase (CYP51) is the most widely distributed cytochrome P450 gene family being found in all biological kingdoms. It catalyzes the first step following cyclization in sterol biosynthesis, leading to the formation of precursors of steroid hormones, including brassinosteroids, in plants. Most enzymes involved in the plant sterol biosynthesis pathway have been characterized biochemically and the corresponding genes cloned.

Plant sterol biosynthesis: identification of two distinct families of sterol 4alpha-methyl oxidases.

In plants, the conversion of cycloartenol into functional phytosterols requires the removal of the two methyl groups at C-4 by an enzymic complex including a sterol 4alpha-methyl oxidase (SMO). We report the cloning of candidate genes for SMOs in Arabidopsis thaliana, belonging to two distinct families termed SMO1 and SMO2 and containing three and two isoforms respectively. SMO1 and SMO2 shared low sequence identity with each other and were orthologous to the ERG25 gene from Saccharomyces cerevisiae which encodes the SMO.

Enzymological properties of sterol-C4-methyl-oxidase of yeast sterol biosynthesis.

Despite genes of the sterol methyl-oxidase component (SMO) of the sterol-C4-demethylation multienzymatic complex have been identified in a variety of organisms and the key role played by SMO in yeast sterol biosynthesis, the enzymological properties of yeast SMO have not been investigated. An enzymatic assay for measuring specifically sterol 4alpha-methyl-oxidase activity in Saccharomyces cerevisiae has been developed for the first time by using [14C]-4,4-dimethyl-zymosterol as substrate. It allowed enzymatically formed C4 mono- and di-demethylated products to be characterized as well as two novel C4-hydroxymethyl-zymosterol derivatives to be identified as immediate oxidative metabolites by the yeast 4,4-dimethyl-zymosterol 4alpha-methyl-oxidase (ScSMO).