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Hedgehog (Hh) signaling ligands undergo carboxy terminal sterylation through specialized autoprocessing, called cholesterolysis. Sterylation is brought about intramolecularly in a single turn-over by an enzymatic domain, called HhC. HhC is found in precursor Hh proteins only. Through cholesterolysis, HhC is cleaved from the precursor. Attempts to identify molecules that inhibit intramolecular cleavage/sterylation activity of HhC have resulted in antagonists that bind HhC irreversibly through covalent mechanisms, as is commonplace for protein autoprocessing inhibitors. Here we report an exception to the “irreversibility rule” for protein autoprocessing inhibition. Using a FRET-based activity assay for HhC, we screened a focused library of sterol-like analogs for HhC cholesterolysis inhibitors. We identified and validated four structurally related noncovalent inhibitors, which were then used for SAR studies. The most effective derivative, tBT-HBT, binds HhC reversibly with an IC50 of 300 nM. An allosteric binding site for tBT-HBT, encompassing interactions from the two subdomains of HhC, is suggested by kinetic analysis, mutagenesis studies, and photoaffinity labeling. A striking resemblance is found between the inhibitors described here and a family of noncovalent, allosteric activators of HhC, which we described previously. The inhibitor/activator duality appears to be mediated by the same allosteric site, which displays sensitivity to subtle differences in the structure of a heterocycle substituent on the effector molecule.

The Sonic hedgehog (SHh) precursor protein undergoes biosynthetic autoprocessing to cleave off and cholesterylate the SHh signaling ligand, a vital morphogen and oncogenic effector protein. Autoprocessing is self-catalyzed by SHhC, the SHh precursor’s enzymatic domain. Here we describe the development and validation of the first cellular reporter to monitor human SHhC autoprocessing non-invasively in high-throughput compatible plates. The assay couples intracellular SHhC autoprocessing to the extracellular secretion of the bioluminescent nanoluciferase enzyme. We developed a wild-type (WT) SHhC reporter line for evaluating potential autoprocessing inhibitors by concentration response-dependent suppression of extracellular bioluminescence. A conditional mutant SHhC (D46A) reporter line was developed for identifying potential autoprocessing activators by a concentration response-dependent gain of extracellular bioluminescence. The D46A mutation removes a conserved general base that is critical for the substrate activity of cholesterol. Inducibility of the D46A reporter was established using a synthetic sterol, 2-α carboxy cholestanol, designed to bypass the defect through intra-molecular general base catalysis. To facilitate direct nanoluciferase detection in the cell culture media of 1536-well plates, we designed a novel membrane-impermeable nanoluciferase substrate, CLZ-2P. This new reporter system offers a long-awaited resource for small molecule discovery for cancer and for developmental disorders where SHh ligand biosynthesis is dysregulated.Abstract Figure

Sterol lipids are required by most eukaryotes and are readily preserved as sterane molecular fossils. These geologic steranes are broadly interpreted as biomarkers for ancient eukaryotes1,2 although diverse bacteria also produce sterols3. Steranes with side-chain methylations can act as more specific biomarkers4 if their sterol precursors are limited to particular extant eukaryotes and are absent in bacteria. An abundance of one such sterane, 24-isopropylcholestane, in late Neoproterozoic rocks has been attributed to marine demosponges and potentially represents the earliest evidence for animals on Earth5. However, debates over this interpretation6–14 continue given the potential for alternative sources of 24-isopropylcholestane and the lack of experimental evidence demonstrating the function of enzymes that methylate sterols to give the 24-isopropyl side-chain. Here we show that sterol methyltransferases from both sponges and bacteria are functional and identify three bacterial methyltransferases each capable of sequential methylations resulting in the 24-isopropyl sterol side-chain. We identified two of these propylating enzymes in a demosponge metagenome suggesting bacterial symbionts contribute to 24-isopropyl sterol biosynthesis in demosponges. Our results demonstrate yet-uncultured bacteria have the genomic capacity to synthesize side-chain alkylated sterols and should therefore be considered when interpreting side-chain alkylated sterane biomarkers in the rock record.

Hedgehog proteins, a family of vital cell signaling factors, are expressed in precursor form, which requires specialized autoprocessing, called cholesterolysis, for full biological activity. Cholesterolysis occurs in cis through the action of the precursor’s C-terminal enzymatic domain, HhC. In this work, we describe HhC activator compounds (HACs), a novel class of noncovalent modulators that induce autoprocessing infidelity, diminishing native cholesterolysis in favor of precursor autoproteolysis, an otherwise minor and apparently nonphysiological side reaction. HAC-induced autoproteolysis generates hedgehog protein that is cholesterol free and hence signaling deficient. The most effective HAC has an AC(50) of 9 μM, accelerates HhC autoproteolytic activity by 225-fold, and functions in the presence and absence of cholesterol, the native substrate. HACs join a rare class of “antagonists” that suppress native enzymatic activity by subverting mechanistic fidelity.

The Sonic Hedgehog (SHh) precursor protein undergoes biosynthetic autoprocessing to cleave off and covalently attach cholesterol to the SHh signaling ligand, a vital morphogen and oncogenic effector protein. Autoprocessing is self-catalyzed by SHhC, the SHh precursor's C-terminal enzymatic domain. A method to screen for small molecule regulators of this process may be of therapeutic value. Here, we describe the development and validation of the first cellular reporter to monitor human SHhC autoprocessing noninvasively in high-throughput compatible plates. The assay couples intracellular SHhC autoprocessing using endogenous cholesterol to the extracellular secretion of the bioluminescent nanoluciferase enzyme. We developed a WT SHhC reporter line for evaluating potential autoprocessing inhibitors by concentration response-dependent suppression of extracellular bioluminescence. Additionally, a conditional mutant SHhC (D46A) reporter line was developed for identifying potential autoprocessing activators by a concentration response-dependent gain of extracellular bioluminescence. The D46A mutation removes a conserved general base that is critical for the activation of the cholesterol substrate. Inducibility of the D46A reporter was established using a synthetic sterol, 2-α carboxy cholestanol, designed to bypass the defect through intramolecular general base catalysis. To facilitate direct nanoluciferase detection in the cell culture media of 1536-well plates, we designed a novel anionic phosphonylated coelenterazine, CLZ-2P, as the nanoluciferase substrate. This new reporter system offers a long-awaited resource for small molecule discovery for cancer and for developmental disorders where SHh ligand biosynthesis is dysregulated.

Hedgehog (Hh) precursor proteins contain an autoprocessing domain called HhC whose native function is protein cleavage and C-terminal glycine sterylation. The transformation catalyzed by HhC occurs in cis from a precursor protein and exhibits wide tolerance toward both sterol and protein substrates. Here, we repurpose HhC as a 1:1 protein−nucleic acid ligase, with the sterol serving as a molecular linker. A procedure is described for preparing HhC-active sterylated DNA, called steramers, using aqueous compatible chemistry and commercial reagents. Steramers have K(M) values of 7−11 μM and reaction t(1/2) values of ∼10 min. Modularity of the HhC/steramer method is demonstrated using four different proteins along with structured and unstructured sterylated nucleic acids. The resulting protein−DNA conjugates retain the native solution properties and biochemical function. Unlike self-tagging domains, HhC does not remain fused to the conjugate; rather, enzymatic activity is mechanistically coupled to conjugate release. That unique feature of HhC, coupled with efficient kinetics and substrate tolerance, may ease access and open new applications for these suprabiological chimeras.

Summary In order to improve interpretations of the demosponge biomarker 24-isopropylcholestane (24-ipc), we examine the biosynthesis of side chain alkylated sterols, including 24-isopropenylcholesterol, through functional analysis of sterol methyltransferases (SMTs). We experimentally demonstrate sponge SMTs are capable of multiple rounds of methylation as previously hypothesized. We also identify functional SMTs of probable bacterial origin capable of producing 24-methyl, 24-ethyl, and 24-isopropyl sterols, suggesting bacteria may be an overlooked source of side chain alkylated steranes in the geologic record, including 24-ipc.

The "red tide" organism Karenia brevis (Davis) HansenMoestrup (=Gymnodinium breve Davis) produces a mixture of brevetoxins, potent neurotoxins responsible for neurotoxic shellfish poisoning in humans and massive fish kills in the Gulf of Mexico and the southern Atlantic coast of the United States. The sterol composition of K. brevis was found to be a mixture of six novel and rare Δ

Cyclic triterpenoids are a broad class of polycyclic lipids produced by bacteria and eukaryotes. They are biologically relevant for their roles in cellular physiology, including membrane structure and function, and biochemically relevant for their exquisite enzymatic cyclization mechanism. Cyclic triterpenoids are also geobiologically significant as they are readily preserved in sediments and are used as biomarkers for ancient life throughout Earth's history. Isoarborinol is one such triterpenoid whose only known biological sources are certain angiosperms and whose diagenetic derivatives (arboranes) are often used as indicators of terrestrial input into aquatic environments. However, the occurrence of arborane biomarkers in Permian and Triassic sediments, which predates the accepted origin of angiosperms, suggests that microbial sources of these lipids may also exist. In this study, we identify two isoarborinol-like lipids, eudoraenol and adriaticol, produced by the aerobic marine heterotrophic bacterium Eudoraea adriatica Phylogenetic analysis demonstrates that the E. adriatica eudoraenol synthase is an oxidosqualene cyclase homologous to bacterial lanosterol synthases and distinct from plant triterpenoid synthases. Using an Escherichia coli heterologous sterol expression system, we demonstrate that substitution of four amino acid residues in a bacterial lanosterol synthase enabled synthesis of pentacyclic arborinols in addition to tetracyclic sterols. This variant provides valuable mechanistic insight into triterpenoid synthesis and reveals diagnostic amino acid residues to differentiate between sterol and arborinol synthases in genomic and metagenomic datasets. Our data suggest that there may be additional bacterial arborinol producers in marine and freshwater environments that could expand our understanding of these geologically informative lipids.

Hedgehog (Hh) autoprocessing converts Hh precursor protein to cholesterylated Hh ligand for downstream signaling. A conserved active-site aspartate residue, D46, plays a key catalytic role in Hh autoprocessing by serving as a general base to activate substrate cholesterol. Here we report that a charge-altering Asp-to-His mutant (D46H) expands native cholesterylation activity and retains active-site conformation. Native activity toward cholesterol was established for D46H in vitro using a continuous FRET-based autoprocessing assay and in cellulo with stable expression in human 293T cells. The catalytic efficiency of cholesterylation with D46H is similar to that with wild type (WT), with k(max)/K(M) = 2.1 × 10(3) and 3.7 × 10(3) M(−1) s(−1), respectively, and an identical pK(a) = 5.8 is obtained for both residues by NMR. To our knowledge this is the first example where a general base substitution of an Asp for His preserves both the structure and activity as a general base. Surprisingly, D46H exhibits increased catalytic efficiency toward non-native substrates, especially coprostanol (>200-fold) and epicoprostanol (>300-fold). Expanded substrate tolerance is likely due to stabilization by H46 of the negatively charged tetrahedral intermediate using electrostatic interactions, which are less constrained by geometry than H-bond stabilization by D46. In addition to providing fundamental insights into Hh autoprocessing, our findings have important implications for protein engineering and enzyme design.

As a first line of defense against insect herbivores many plants store high concentrations of toxic and deterrent secondary metabolites in glandular trichomes. Plant Pleiotropic Drug Resistance (PDR)-type ABC transporters are known secondary metabolite transporters, and several have been implicated in pathogen or herbivore defense. Here, we report on Petunia hybrida PhPDR2 as a major contributor to trichome-related chemical defense. PhPDR2 was found to localize to the plasma membrane and be predominantly expressed in multicellular glandular trichomes of leaves and stems. Down-regulation of PhPDR2 via RNA interference (pdr2) resulted in a markedly higher susceptibility of the transgenic plants to the generalist foliage feeder Spodoptera littoralis. Untargeted screening of pdr2 trichome metabolite contents showed a significant decrease in petuniasterone and petuniolide content, compounds, which had previously been shown to act as potent toxins against various insects. Our findings suggest that PhPDR2 plays a leading role in controlling petuniasterone levels in leaves and trichomes of petunia, thus contributing to herbivory resistance.

Bivalve mollusks lack de novo cholesterol biosynthesis capabilities and therefore rely upon dietary sources of sterols for rapid growth. Microalgae that constitute the main source of nutrition for suspension-feeding bivalves contain a diverse array of phytosterols, in most cases lacking cholesterol. Rapid growth of bivalves on microalgal diets with no cholesterol implies that some phytosterols can satisfy the dietary requirement for cholesterol through metabolic conversion to cholesterol, but such metabolic pathways have not been rigorously demonstrated. In the present study, stable isotope-labeled phytosterols were used to supplement a unialgal diet of Rhodomonas sp. and their biological transformation to cholesterol within scallop tissues was determined using (13)C-NMR spectroscopy. Scallops efficiently dealkylated ∆(5) C29 (24-ethyl) sterols to cholesterol, and the only C28 sterol that was dealkylated efficiently possessed the 24(28)-double bond. Non-metabolized dietary phytosterols accumulated in the soft tissues. Observed formation of ∆(5,7) sterols (provitamin D) from ∆(5) sterols may represent initiation of steroid hormone (possibly ecdysone) biosynthesis. These findings provide a key component necessary for formulation of nutritionally complete microalgal diets for hatchery production of seed for molluscan aquaculture.

Sweet potatoes (the tuber of Ipomoea batatas) are a major food crop globally. The sweet potato weevil (Cylas formicarius elegantulus) is a serious pest of this important crop. The triterpenol, boehmerol, has previously been found in the skin of the tuber where, as its acetate ester, it has been shown to signal oviposition by the weevil. A new triterpenol, batatasenol, was identified in two varieties of sweet potatoes, 'Covington' and 'Purple Stokes'. In the 'Covington' variety, batatasenol was practically the only triterpenol present in the skins. In the 'Purple Stokes' variety, batatasenol was present along with boehmerol and several minor triterpenols. Based on the structures of the co-occurring compounds, it is proposed that their biosynthesis involves an epoxysqualene cyclase which can carry out both all-chair and B-boat cyclizations.

Sterols are essential eukaryotic lipids that are required for a variety of physiological roles. The diagenetic products of sterol lipids, sterane hydrocarbons, are preserved in ancient sedimentary rocks and are utilized as geological biomarkers, indicating the presence of both eukaryotes and oxic environments throughout Earth’s history. However, a few bacterial species are also known to produce sterols, bringing into question the significance of bacterial sterol synthesis for our interpretation of sterane biomarkers. Recent studies suggest that bacterial sterol synthesis may be distinct from what is observed in eukaryotes. In particular, phylogenomic analyses of sterol-producing bacteria have failed to identify homologs of several key eukaryotic sterol synthesis enzymes, most notably those required for demethylation at the C-4 position. In this study, we identified two genes of previously unknown function in the aerobic methanotrophic γ-Proteobacterium Methylococcus capsulatus that encode sterol demethylase proteins (Sdm). We show that a Rieske-type oxygenase (SdmA) and an NAD(P)-dependent reductase (SdmB) are responsible for converting 4,4-dimethylsterols to 4α-methylsterols. Identification of intermediate products synthesized during heterologous expression of SdmA-SdmB along with 13C-labeling studies support a sterol C-4 demethylation mechanism distinct from that of eukaryotes. SdmA-SdmB homologs were identified in several other sterol-producing bacterial genomes but not in any eukaryotic genomes, indicating that these proteins are unrelated to the eukaryotic C-4 sterol demethylase enzymes. These findings reveal a separate pathway for sterol synthesis exclusive to bacteria and show that demethylation of sterols evolved at least twice—once in bacteria and once in eukaryotes.

Proteins in the hedgehog family undergo self-catalyzed endoproteolysis involving nucleophilic attack by a molecule of cholesterol. Recently, a conserved aspartate residue (D303, or D46) of hedgehog was identified as the general base that activates cholesterol during this unusual autoprocessing event; mutation of the catalyzing functional group (D303A) reduces activity by >104-fold. Here we report near total rescue of this ostensibly dead general base mutant by a synthetic substrate, 3β-hydroperoxycholestane (3HPC) in which the sterol −OH group is replaced by the hyper nucleophilic −OOH group. Other hedgehog point mutants at D303, also unreactive with cholesterol, accepted 3HPC as a substrate with the rank order: WT > D303A ≈ D303N ≫ D303R, D303E. We attribute the revived activity with 3-HPC to the α-effect, where tandem electronegative atoms exhibit exceptionally high nucleo- philicity despite relatively low basicity.

The AIDS-associated lung pathogen Pneumocystis is classified as a fungus although Pneumocystis has several distinct features such as the absence of ergosterol, the major sterol of most fungi. The Pneumocystis carinii S-adenosylmethionine:sterol C24-methyltransferase (SAM:SMT) enzyme, coded by the erg6 gene, transfers either one or two methyl groups to the C-24 position of the sterol side chain producing both C28 and C29 24-alkylsterols in approximately the same proportions, whereas most fungal SAM:SMT transfer only one methyl group to the side chain. The sterol compositions of wild-type Sacchromyces cerevisiae, the erg6 knockout mutant (Δerg6), and Δerg6 expressing the P. carinii or the S. cerevisiae erg6 gene were analyzed by a variety of chromatographic and spectroscopic procedures to examine functional complementation in the yeast expression system. Detailed sterol analyses were obtained using high performance liquid chromatography and proton nuclear magnetic resonance spectroscopy ((1)H-NMR). The P. carinii SAM:SMT in the Δerg6 restored its ability to produce the C28 sterol ergosterol as the major sterol, and also resulted in low levels of C29 sterols. This indicates that while the P. carinii SAM:SMT in the yeast Δerg6 cells was able to transfer a second methyl group to the side chain, the action of Δ(24(28)) -sterol reductase (coded by the erg4 gene) in the yeast cells prevented the formation and accumulation of as many C29 sterols as that found in P. carinii.

We report a study of the sterols produced by the thecate allogromid foraminiferan, Allogromia laticollaris, grown in culture. Gas chromatographic retention time, together with mass spectrometric fragmentation patterns of trimethysilyl and acetate derivatives enabled us to assign a suite of C27–C30 sterols. Two C30 sterols were identified as (24E)-n-propylidenecholesterol and its 24Z isomer by direct comparison with an authentic standard. The C30 sterols were not detected in the two algae, Isochrysis galbana and Dunaliella tertiolecta, used as a food source for the A. laticollaris, consistent with previous analysis of these organisms. The assignment of (24E)-n-propylidenecholesterol was confirmed using 600 MHz 1H nuclear magnetic resonance (NMR) spectroscopy. The result has relevance as to how we might interpret fossil sterane distributions.

The sugar subunits of natural glycosides can be conveniently determined by acid hydrolysis and (1)H NMR spectroscopy without isolation or derivatization. The chemical shifts, coupling constants, and integral ratios of the anomeric signals allow each monosaccharide to be identified and its molar ratio to other monosaccharides to be quantified. The NMR data for the anomeric signals of 28 monosaccharides and three disaccharides are reported. Application of the method is demonstrated with the flavonoid glycoside naringin (1), the aminoglycoside antibiotics kanamycin (2) and tobramycin (3), and the saponin digitonin (4).

Human Vγ2Vδ2 T cells monitor isoprenoid metabolism by recognizing (E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate (HMBPP), an intermediate in the 2-C-methyl-d-erythritol-4-phosphate pathway used by microbes, and isopentenyl pyrophosphate (IPP), an intermediate in the mevalonate pathway used by humans. Aminobisphosphonates and alkylamines indirectly stimulate Vγ2Vδ2 cells by inhibiting farnesyl diphosphate synthase (FDPS) in the mevalonate pathway, thereby increasing IPP/triphosphoric acid 1-adenosin-5′-yl ester 3-(3-methylbut-3-enyl) ester that directly stimulate. In this study, we further characterize stimulation by these compounds and define pathways used by new classes of compounds. Consistent with FDPS inhibition, stimulation of Vγ2Vδ2 cells by aminobisphosphonates and alkylamines was much more sensitive to statin inhibition than stimulation by prenyl pyrophosphates; however, the continuous presence of aminobisphosphonates was toxic for T cells and blocked their proliferation. Aminobisphosphonate stimulation was rapid and prolonged, independent of known Ag-presenting molecules, and resistant to fixation. New classes of stimulatory compounds—mevalonate, the alcohol of HMBPP, and alkenyl phosphonates—likely stimulate differently. Mevalonate, a rate-limiting metabolite, appears to enter cells to increase IPP levels, whereas the alcohol of HMBPP and alkenyl phosphonates are directly recognized. The critical chemical feature of bisphosphonates is the amino moiety, because its loss switched aminobisphosphonates to direct Ags. Transfection of APCs with small interfering RNA downregulating FDPS rendered them stimulatory for Vγ2Vδ2 cells and increased cellular IPP. Small interfering RNAs for isopentenyl diphosphate isomerase functioned similarly. Our results show that a variety of manipulations affecting isoprenoid metabolism lead to stimulation of Vγ2Vδ2 T cells and that pulsing aminobisphosphonates would be more effective for the ex vivo expansion of Vγ2Vδ2 T cells for adoptive cancer immunotherapy.

Sterol compositions for three diatom species, recently shown to contain sterols with side chains typically found in dinoflagellates, were determined by HPLC and ¹H NMR spectroscopic analyses. The centric diatom Triceratium dubium (=Biddulphia sp., CCMP 147) contained the highest percentage of 23-methylated sterols (37.2% (24R)-23-methylergosta-5,22-dienol), whereas the pennate diatom Delphineis sp. (CCMP 1095) contained the cyclopropyl sterol gorgosterol, as well as the 27-norsterol occelasterol. The sterol composition of Ditylum brightwellii (CCMP 358) was the most complex, containing Δ⁰- and Δ⁷-sterols, in addition to the predominant Δ⁵-sterols. A pair of previously unknown sterols, stigmasta-5,24,28-trienol and stigmasta-24,28-dienol, were detected in D. brightwellii and their structures were determined by NMR spectroscopic analysis and by synthesis of the former sterol from saringosterol. Also detected in D. brightwellii was the previously unknown 23-methylcholesta-7,22-dienol.

Pneumocystis carinii is an unusual fungus that can cause pneumonitis in immunosuppressed laboratory rats. Reactions in sterol biosynthesis are attractive targets for development of antimycotic drugs. A key enzyme in sterol biosynthesis is sterol 14α-demethylase (14DM), which is coded by the erg11 gene. Here we describe detailed sterol analysis of wild-type Saccharomyces cerevisiae and in an erg11 knockout mutant expressing either P. carinii or S. cerevisiae 14DM from a plasmid-borne cDNA. Sterols of the three strains were qualitatively and quantitatively analyzed using thin-layer chromatography, high-performance liquid chromatography, and gas-liquid chromatography and mass spectrometry and nuclear magnetic resonance spectroscopy. Biochemical evidence for functional complementation was provided by detecting the same major sterols in all three strains with ergosterol being by far the most abundant. A total of 25 sterols was identified, 16 of which were identified in all three strains. The ratios of lanosterol:14-desmethyllanosterol in the three strains indicate that the mutant transformed with erg11 showed more 14DM activity than wild-type yeast. The sterol analyses also indicated that the P. carinii 14DM can utilize the sterol substrates used by the S. cerevisiae 14DM and suggested that the yeast 14DM in the yeast cell utilizes 4α-methyl sterols better than the P. carinii enzyme.

Human Vgamma2Vdelta2 T cells play important role in immunity to infection and cancer by monitoring self and foreign isoprenoid metabolites with their gammadelta T cell antigen receptors. Like CD4 and CD8 alphabeta T cells, adult peripheral Vgamma2Vdelta2 T cells represent a pool of heterogeneous cells with distinct functional capabilities.The aim of this study was to characterize the phenotypes and functions of various Vgamma2Vdelta2 T cell subsets in patients with nasopharyngeal carcinoma (NPC). We sought to develop a better understanding of the role of these cells during the course of disease and to facilitate the development of immunotherapeutic strategies against NPC.Although similar total percentages of peripheral blood Vgamma2Vdelta2 T cells were found in both NPC patients and normal donors, Vgamma2Vdelta2 T cells from NPC patients showed decreased cytotoxicity against tumor cells whereas Vgamma2Vdelta2 T cells from normal donors showed potent cytotoxicity. To investigate further, we compared the phenotypic characteristics of Vgamma2Vdelta2 T cells from 96 patients with NPC and 54 healthy controls. The fraction of late effector memory Vgamma2Vdelta2 T cells (T(EM RA)) was significantly increased in NPC patients with corresponding decreases in the fraction of early memory Vgamma2Vdelta2 T cells (T(CM)) compared with those in healthy controls. Moreover, T(EM RA) and T(CM) Vgamma2Vdelta2 cells from NPC patients produced significantly less IFN-gamma and TNF-alpha, potentially contributing to their impaired cytotoxicity. Radiotherapy or concurrent chemo-radiotherapy further increased the T(EM RA) Vgamma2Vdelta2 T cell population but did not correct the impaired production of IFN-gamma and TNF-alpha observed for T(EM RA) Vgamma2Vdelta2 T cells.We have identified distinct alterations in the Vgamma2Vdelta2 T cell subsets of patients with NPC. Moreover, the overall cellular effector function of gammadelta T cells is compromised in these patients. Our data suggest that the contribution of Vgamma2Vdelta2 T cells to control NPC may depend on the activation state and differentiation of these cells.

Sterol and fatty acid compositions were determined for three harmful algal species previously classified in the genus Chattonella (Raphidophyceae): the new genus Chloromorum toxicum (ex Chattonella cf. verruculosa), Verrucophora farcimen (Dictyochophyceae), previously Chattonella aff. verruculosa, and Verrucophora verruculosa (=Pseudochattonella verruculosa) previously Chattonella verruculosa. The major fatty acids of C. toxicum were 14:0, 16:0, 18:1n−9, 18:4n−3 and 20:5n−3, and those of the Verrucophora strains were. 14:0, 16:0, 18:0, 18:4n−3, 18:5n−3 and 22:6n−3. C. toxicum contained the 24β-ethyl sterols, poriferasterol and clionasterol, as its major sterols. For comparison, the stereochemistry of the 24-ethyl sterols of two raphidophytes, Chattonella marina and Heterosigma akashiwo, was determined to be 24α and 24β, respectively. Both Verrucophora strains contained the 27-nor sterol occelasterol as the only detected sterol. This was the first time occelasterol has been found in algae.

Human Vg2Vd2 T cells are stimulated by prenyl pyrophosphates, such as isopentenyl pyrophosphate (IPP), and play important roles in mediating immunity against microbial pathogens and have potent anti-tumor activity. (E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate (HMBPP) has been identified as a metabolite in the 2-C-methyl-D-erythritol-4 phosphate (MEP) pathway for isoprenoid biosynthesis that is used by many bacteria and protozoan parasites. We find that HMBPP is the major Vg2Vd2 T-cell antigen for many bacteria, including Mycobacterium tuberculosis, Yersinia enterocolitica and Escherichia coli. HMBPP was a 30 000-fold more potent antigen than IPP. Using mutant bacteria, we show that bacterial antigen levels for Vg2Vd2 T cells are controlled by MEP pathway enzymes and find no evidence for the production of 3-formyl-1-butyl pyrophosphate. Moreover, HMBPP reactivity required only germ line-encoded Vg2Vd2 TCR elements and is present at birth. Importantly, we show that bacterial HMBPP levels correlated with their ability to expand Vg2Vd2 T cells in vivo upon engraftment into severe combined immunodeficiency–beige mice. Thus, the production of HMBPP by a microbial-specific isoprenoid pathway plays a major role in determining whether bacteria will stimulate Vg2Vd2 T cells in vivo. This preferential stimulation by a common microbial isoprenoid metabolite allows Vg2Vd2 T cells to respond to a broad array of pathogens using this pathway.

Bacteriovorax stolpii is a predator of larger gram-negative bacteria and lives as a parasite in the intraperiplasmic space of the host cell. This bacterium is unusual among prokaryotes in that sphingolipids comprise a large proportion of its lipids. We here report the presence of 18 molecular species of B. stolpii UKi2 sphingophosphonolipids (SPNLs). (31)P NMR spectroscopy and analysis of P(i) released by a differential hydrolysis protocol confirmed the phosphonyl nature of these lipids. The SPNLs were dominated by those with 1-hydroxy-2-aminoethane phosphonate (hydroxy-aminoethylphosphonate) polar head groups; aminoethylphosphonate was also detected in minor SPNL components. The long-chain bases (LCBs) were dominated by C(17) iso-branched phytosphingosine; C(17) iso-branched dihydrosphingosine was also present in some SPNLs. The N-linked fatty acids were predominantly iso-branched and most contained an alpha-hydroxy group (C(15) alpha-hydroxy fatty acid was the major fatty acid). Minor molecular species containing nonhydroxy fatty acids were also detected. The definitive iso-structures of the predominant fatty acids and LCBs present in the B. stolpii SPNLs were established using (13)C and (3)H nuclear magnetic resonance spectroscopy; less than 20% were unbranched. Detection and analyses of intact compounds by MS-MS were performed by a hybrid quadrupole time-of-flight (Q-TOF-II) MS equipped with an electrospray ionization source. Analyses of peracetylated derivatives verified the structural assignments of these lipids.

The complete assignments of the 1H and 13C NMR spectra of the hallucinogenic neoclerodane diterpenoid salvinorin A were determined in three different NMR solvents using HSQC, HMBC and COSY. Solvent systems are described that allow the resolution of all 1H signals. Virtual coupling was observed for the protons at C-2, C-3 and C-4 in the 600 MHz 1H spectrum in CDCl3. The complete assignments of the 1H and 13C NMR spectra of salvinorin B are also reported. Copyright © 2007 John Wiley & Sons, Ltd.

The genus Euphorbia contains over 2000 species which exhibit a considerable diversity of di- and triterpenes in their latex. The North American species Euphorbia polygonifolia is a low growing plant of Atlantic and Great Lake beaches. The composition of its free and esterified triterpene alcohols was determined by HPLC and (1) H-NMR analyses. An unreported triterpene alcohol was found as 2.6% and 10.3% of the free and esterified fractions, respectively. The structure of the new triterpene alcohol was determined using HMBC, and its configurational assignment was secured by acid-catalyzed isomerization to isotirucallol. The new compound, polygonifoliol, was shown to be Δ(12) -isotirucallol.

Sterol and fatty acid compositions were determined for Cochlodinium polykrikoides, a toxic, bloom-forming dinoflagellate of global significance. The major sterols were dinosterol (40% of total sterols), dihydrodinosterol (32%), and the rare 4α-methyl Δ(8(14)) sterol, amphisterol (23%). A minor sterol, 4α-methylergost-24(28)-enol was also detected (5.0%). The fatty acids had a high proportion of PUFAs (47%), consisting mainly of EPA (20%) and the relatively uncommon octadecapentaenoic acid (18 : 5, 22%). While unlikely to be responsible for toxicity to fish, these lipids may contribute to the deleterious effects of this alga to invertebrates.

Theoretical investigation of cyclopropane-to-cyclopropane rearrangements of sterols indicates a role for highly delocalized bicyclobutonium ions in biosynthesis.

Fluorescent analogs were synthesized of the potent PI 3-kinase inhibitors, wortmannin and demethoxyviridin. The esterification of 11-deacetylwortmannin, 17-hydroxywortmannin, and demethoxyviridin with the fluorescent carboxylic acids NBD-sarcosine and 7-dimethylaminocoumarin-4-acetic acid generated six novel fluorescent esters. Potent inhibition of PI 3-kinase-alpha was observed for the derivatives of 11-desacetylwortmannin and demethoxyviridin.

The relative rates of acid-catalyzed rearrangements of epoxy esters to [3.2.1]bicyclic orthoesters, the subsequent rearrangements of these ortho esters to substituted tetrahydrofurans, and the rates of orthoester hydrolysis at pH 4.75 were measured in NMR kinetics experiments. The ease of formation and stabilities of these orthoesters compared favorably with the OBO-type [2.2.2]bicyclic orthoesters typically used as protecting groups of carboxylic acids. Studies with 13C NMR-detected 18O-labeling show that epoxy ester rearrangement takes place preferentially via 6-exo cyclization, although the 7-endo process competes when the distal center of the epoxide is disubstituted. The ortho ester-cyclic ether rearrangement was shown by 18O-labeling to occur exclusively via intermediacy of a five-membered dioxonium ion. The structures of the hydrolysis products also indicate the intermediacy of a dioxolanium ion during hydrolysis. The implications for a hypothetical biosynthesis of marine polyether toxins are discussed.

A steroidal oxetanyl ester was synthesized in eight steps as a biomimetic model of taxol oxetane. The model compound was surprisingly reactive under acidic conditions, rearranging in the absence of H2O to a [2.2.1]-bicyclic orthoester. Both the oxetanyl ester and the orthoester readily hydrolyze to produce the same triol monoacetate. On the basis of the oxetanyl ester/orthoester rearrangement, a novel biochemical function is suggested for the epoxy esters and oxetanyl esters found in taxoids whereby dioxonium ions, generated from these functional groups, react with cellular proteins to form mixed orthoesters or ethers. A similar process could be involved in the mechanism of action of natural orthoesters such as resiniferatoxin.

The "red tide" organism Karenia brevis (Davis) Hansen & Moestrup (= Gymnodinium breve Davis) produces a mixture of brevetoxins, potent neurotoxins responsible for neurotoxic shellfish poisoning in humans and massive fish kills in the Gulf of Mexico and the southern Atlantic coast of the United States. The sterol composition of K. brevis was found to be a mixture of six novel and rare < 8 ( 1 4 ) sterols. The two predominant sterols, (24R)-4α-methylergosta-8(14), 22-dienol and (24R)-4α-methyl-27-norergosta-8(14), 22-dienol, were named gymnodinosterol and brevesterol and represent potentially useful biomarkers for K. brevis. A possible function for such unusual marine sterols is proposed whereby structural modifications render the sterols non-nutritious to marine invertebrates, reducing predation and thereby enhancing the ability of the dinoflagellates to form massive blooms.

The biomimetic epoxy ester[bond]orthoester rearrangement has been applied to a new synthesis of 2-methyl-D-erythritol, a branched five-carbon sugar of importance to the deoxyxylulose pathway of isoprenoid biosynthesis. The intermediate orthoacetate is one of the few [2.2.1]-orthoesters to have been reported. Labeling studies with O-18 indicated that this reaction proceeds exclusively via a 5-exo cyclization. NMR analysis of chiral esters indicated an ee of 87% for the starting epoxide and an ee of 86% for the product. This route represents a rapid and convenient method for the synthesis of 2-methyl-D-erythritol and is expected to be useful for generating isotopically labeled intermediates for biochemical studies.

Pneumocystis is an opportunistic pathogen that can cause pneumonitis in immunodeficient people such as AIDS patients. Pneumocystis remains difficult to study in the absence of culture methods for luxuriant growth. Recombinant protein technology now makes it possible to avoid some major obstacles. The P. carinii expressed sequence tag (EST) database contains 11 entries of a sequence encoding a protein homologous to S-adenosyl-L-methionine (SAM):C-24 sterol methyl transferase (SMT), suggesting high activity of this enzyme in the organism. We sequenced the erg6 cDNA, identified the putative peptide motifs for the sterol and SAM binding sites in the deduced amino acid sequence and expressed the protein in Escherichia coli. Unlike SAM:SMT from other organisms, the P. carinii enzyme had higher affinities for lanosterol and 24-methylenelanosterol than for zymosterol, the preferred substrate in other fungi. Cycloartenol was not a productive substrate. With lanosterol and 24-methylenelanosterol as substrates, the major reaction products were 24-methylenelanosterol and pneumocysterol respectively. Thus, the P. carinii SAM:SMT catalysed the transfer of both the first and the second methyl groups to the sterol C-24 position, and the substrate preference was found to be a unique property of the P. carinii SAM:SMT. These observations, together with the absence of SAM:SMT among mammals, further support the identification of sterol C-24 alkylation reactions as excellent targets for the development of drugs specifically directed against this pathogen.

The genus Euphorbia contains over 2000 species which exhibit a considerable diversity of di- and triterpenes in their latex. The North American species Euphorbia polygonifolia is a low growing plant of Atlantic and Great Lake beaches. The composition of its free and esterified triterpene alcohols was determined by HPLC and (1) H-NMR analyses. An unreported triterpene alcohol was found as 2.6% and 10.3% of the free and esterified fractions, respectively. The structure of the new triterpene alcohol was determined using HMBC, and its configurational assignment was secured by acid-catalyzed isomerization to isotirucallol. The new compound, polygonifoliol, was shown to be Δ(12) -isotirucallol.

This organic laboratory experiment introduces students to stereoselective enzyme reactions, resolution of enantiomers, and NMR analysis of diastereomers. The reaction between racemic 1-phenylethanol and vinyl acetate in hexane to form an ester is catalyzed by acylase I. The unreacted alcohol is then treated with a chiral acid and the resulting ester diastereomer is analyzed by NMR. This experiment is suitable for group work in the laboratory as several diastereomers are synthesized and compared to determine which enantiomer of 1-phenylethanol reacts with the enzyme.

The biochemically important deoxysugar 1-deoxy-D-xylulose was synthesized by improved methods. D-Tartaric acid is the starting material for a synthesis which proceeds via the intermediacy of 2,3,4-tribenzyl-D-threitol. Another, highly efficient route used the Sharpless asymmetric dihydroxylation of 5-benzyloxy-3-penten-2-one as its key step. These syntheses are especially useful for isotopic labeling.

The important regulatory enzyme protein kinase C is physiologically activated by the interaction of (S)-diglycerides with its regulatory domain. This interaction can be mimicked by the structurally diverse tumor promoters, which share, along with the diglycerides, the common structural feature of three hydrophilic atoms at the vertices of a triangle with sides of approximately 6 A. It is shown in this article that molecules with the same triangular arrangement of hydrophillic atoms but with shorter sides can also activate PKC. S-Farnesylthiotriazole (FTT) is a heterocyclic molecule previously shown to specifically activate PKC. In the work reported here, structure-activity studies in the FTT series reveal that three hydrophilic atoms are required for activation, and that the minimal activation unit is close to an equilateral triangle with sides of between 2.4 – 2.7 A. This demonstrates that there is an unanticipated flexibility at the PKC regulatory site. The intermolecular activation model based on structural analysis of the tumor promoters may represent the maximum distances allowed between the hydrophilic atoms of a PKC activator. © 1997 Elsevier Science Ltd.

[reaction: see text] Conversion of a carboxylic acid to an orthoester provides protection toward nucleophiles and strong bases. The addition of methyl substituents to the oxetane precursor of the commonly used [2.2.2]-bicyclic OBO orthoester significantly increased the ease of orthoester formation and its resistance to hydrolysis. NMR kinetics show the DMOBO protecting group is formed 85 times faster than the OBO group, and that its stability toward aqueous hydrolysis is 36 times greater. Nucleophilic attack of the ester carbonyl on the oxetane ring was shown by 18O-labeling to take place at the most substituted position.

A concise preparation of the pheromone secreted by the male Colorado potato beetle [viz. (3S)-1,3-dihydroxy-3,7-dimethyl-6-octen-2-one] was accomplished in 4 steps starting from 2-fluoronerol or 2-fluorogeraniol. The key step in the synthesis involves a 6-endo epoxide ring opening with ester participation that simultaneously inverts the 3R-configuration of the (3R)-2,3-epoxy-2-fluoroprenyl acetate intermediate, and installs the ketone functionality of the semiochemical. Extensive NMR studies validate the proposed 6-endo mechanism of the featured rearrangement, which under anhydrous conditions, resulted in the formation of two bicyclic 1,3-dioxan-5-ones via an unprecedented intramolecular Prins cyclization. “In memory of Robert Milton Silverstein (1917–2007), pioneer of insect pheromone chemistry and organic spectrometry”

The cariogenic bacterium Streptococcus mutans is an important dental pathogen that forms biofilms on tooth surfaces, which provide a protective niche for the bacterium where it secretes organic acids leading to the demineralization of tooth enamel. Lipids, especially glycolipids are likely to be key components of these biofilm matrices. The UA159 strain of S. mutans was among the earliest microorganisms to have its genome sequenced. While the lipids of other S. mutans strains have been identified and characterized, lipid analyses of UA159 have been limited to a few studies on its fatty acids. Here we report the structures of the four major glycolipids from stationary-phase S. mutans UA159 cells grown in standing cultures. These were shown to be monoglucosyldiacylglycerol (MGDAG), diglucosyldiacylglycerol (DGDAG), diglucosylmonoacylglycerol (DGMAG) and, glycerophosphoryldiglucosyldiacylglycerol (GPDGDAG). The structures were determined by high performance thin-layer chromatography, mass spectrometry and nuclear magnetic resonance spectroscopy. The glycolipids were identified by accurate, high resolution, and tandem mass spectrometry. The identities of the sugar units in the glycolipids were determined by a novel and highly efficient NMR method. All sugars were shown to have alpha-glycosidic linkages and DGMAG was shown to be acylated in the sn-1 position by NMR. This is the first observation of unsubstituted DGMAG in any organism and the first mass spectrometry data for GPDGDAG.

Quantum chemical calculations (B3LYP and MP2) are described for the formation and rearrangements of carbocations derived from the biological methylation reaction that produces 24-propyl sterols in pelagophyte algae. Previous mechanistic proposals are discussed in light of the results of these calculations. Of particular note is the prediction of a new triple-shift rearrangement that is inherently preferred for the biosynthetically relevant carbocations. Our calculations also reveal how these reactions may be affected by intermolecular interactions with S-adenosylmethionine.

Proteins containing CX3, CXC, and CC (where C is cysteine and X is undefined) undergo posttranslational isoprenylation at their cysteine residues. In the case of proteins which terminate in CX3, proteolytic removal of X3 is followed by the carboxymethylation of the isoprenylated cysteine residue. CXC proteins also undergo C-terminal methylation. The present study addresses the question of whether this methylation is catalyzed by a different isoprenylated protein methyltransferase than that previously described for CX3 proteins. The S-adenosylmethionine (AdoMet) dependent methylation of a small peptide-N-acetyl-S-geranylgeranyl-L-cysteinyl-L-alanyl-S-geranylgeranyl- L- cysteine (Ac(GG)CysAla(GG)Cys)--was investigated using membranes from a variety of bovine tissues as sources of enzyme. Ac(GG)CysAla(GG)Cys was a substrate for methylation, while Ac(GG)Cys(GG)Cys was not. Reciprocal inhibition studies on the methylation reactions of the CXC peptide and of N-acetyl-S-farnesyl-L-cysteine (AFC), a previously described methyltransferase substrate, suggested that these reactions are catalyzed by distinct enzymatic activities. Farnesylthioacetic acid (FTA), a potent competitive inhibitor of the methylation of AFC, did not inhibit the methylation of the CXC peptide. Moreover the KI values for S-adenosylhomocysteine and S-adenosylethionine inhibition differed for the two enzymatic activities. These data indicate that more than one AdoMet-dependent methyltransferase is involved in the carboxymethylation of isoprenylated proteins.

The synthesis of ( E )-4-hydroxy-3-methyl-2-butenyl pyrophosphate, an intermediate in the deoxyxylulose pathway of isoprenoid biosynthesis, was accomplished by pyrophosphorylation of ( E )-4-chloro-2-methyl-2-buten-1-ol. This route enables convenient access to isotopically labeled products, as demonstrated through the preparation of [4- 13 C]-( E )-4-hydroxy-3-methyl-2-butenyl pyrophosphate in 28% overall yield from [1- 13 C]-2-bromopropionic acid.