13 research outputs found
Pyrazolopyrimidines in âall-naturalâ products for erectile dysfunction treatment: the unreliable quality of dietary supplements
<div><p>A herbal food supplement advertised as a potency pill was screened for the presence of PDE5 inhibitors. The resulting signals were characterised by UV, LC-MS in ESI-negative mode, and NMR spectroscopy using 1D and 2D experiments. Several substances were identified, bearing the basic chemical structure of sildenafil, but were not supposed to exhibit PDE5 inhibition. These compounds may be process-related impurities or by-products of different reaction steps in the synthesis of PDE5 analogues. As they were found to be present in different capsules at different concentrations, this is an example of the unreliable quality of dietary supplements.</p></div
Biosynthesis of panaxynol and panaxydol in Panax ginseng
The natural formation of the bioactive C17-polyacetylenes (â)-(R)-panaxynol and panaxydol was analyzed by 13C-labeling experiments. For this purpose, plants of Panax ginseng were supplied with 13CO2 under field conditions or, alternatively, sterile root cultures of P. ginseng were supplemented with [U-13C6]glucose. The polyynes were isolated from the labeled roots or hairy root cultures, respectively, and analyzed by quantitative NMR spectroscopy. The same mixtures of eight doubly 13C-labeled isotopologues and one single labeled isotopologue were observed in the C17-polyacetylenes obtained from the two experiments. The polyketide-type labeling pattern is in line with the biosynthetic origin of the compounds via decarboxylation of fatty acids, probably of crepenynic acid. The 13C-study now provides experimental evidence for the biosynthesis of panaxynol and related polyacetylenes in P. ginseng under in planta conditions as well as in root cultures. The data also show that 13CO2 experiments under field conditions are useful to elucidate the biosynthetic pathways of metabolites, including those from roots
Biosynthesis of pteridines. Reaction mechanism of GTP cyclohydrolase I
GTP cyclohydrolase I catalyses the hydrolytic release of formate from GTP followed by cyclization to dihydroneopterin triphosphate. The enzymes from bacteria and animals are homodecamers containing one zinc ion per subunit. Replacement of Cys110, Cys181, His112 or His113 of the enzyme from Escherichia coli by serine affords catalytically inactive mutant proteins with reduced capacity to bind zinc. These mutant proteins are unable to convert GTP or the committed reaction intermediate, 2-amino-5-formylamino-6-(β-ribosylamino)-4(3H)-pyrimidinone 5â˛-triphosphate, to dihydroneopterin triphosphate.
The crystal structures of GTP complexes of the His113Ser, His112Ser and Cys181Ser mutant proteins determined at resolutions of 2.5 Ă
, 2.8 Ă
and 3.2 Ă
, respectively, revealed the conformation of substrate GTP in the active site cavity. The carboxylic group of the highly conserved residue Glu152 anchors the substrate GTP, by hydrogen bonding to N-3 and to the position 2 amino group. Several basic amino acid residues interact with the triphosphate moiety of the substrate. The structure of the His112Ser mutant in complex with an undefined mixture of nucleotides determined at a resolution of 2.1 Ă
afforded additional details of the peptide folding.
Comparison between the wild-type and mutant enzyme structures indicates that the catalytically active zinc ion is directly coordinated to Cys110, Cys181 and His113. Moreover, the zinc ion is complexed to a water molecule, which is in close hydrogen bond contact to His112. In close analogy to zinc proteases, the zinc-coordinated water molecule is suggested to attack C-8 of the substrate affording a zinc-bound 8R hydrate of GTP. Opening of the hydrated imidazole ring affords a formamide derivative, which remains coordinated to zinc. The subsequent hydrolysis of the formamide motif has an absolute requirement for zinc ion catalysis. The hydrolysis of the formamide bond shows close mechanistic similarity with peptide hydrolysis by zinc proteases
Biosynthesis of panaxynol and panaxydol in Panax ginseng
The natural formation of the bioactive C17-polyacetylenes (â)-(R)-panaxynol and panaxydol was analyzed by 13C-labeling experiments. For this purpose, plants of Panax ginseng were supplied with 13CO2 under field conditions or, alternatively, sterile root cultures of P. ginseng were supplemented with [U-13C6]glucose. The polyynes were isolated from the labeled roots or hairy root cultures, respectively, and analyzed by quantitative NMR spectroscopy. The same mixtures of eight doubly 13C-labeled isotopologues and one single labeled isotopologue were observed in the C17-polyacetylenes obtained from the two experiments. The polyketide-type labeling pattern is in line with the biosynthetic origin of the compounds via decarboxylation of fatty acids, probably of crepenynic acid. The 13C-study now provides experimental evidence for the biosynthesis of panaxynol and related polyacetylenes in P. ginseng under in planta conditions as well as in root cultures. The data also show that 13CO2 experiments under field conditions are useful to elucidate the biosynthetic pathways of metabolites, including those from roots
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In Vivo Screening Unveils Pervasive RNA-Binding Protein Dependencies in Leukemic Stem Cells and Identifies ELAVL1 as a Therapeutic Target.
UNLABELLED: Acute myeloid leukemia (AML) is fueled by leukemic stem cells (LSC) whose determinants are challenging to discern from hematopoietic stem cells (HSC) or uncover by approaches focused on general cell properties. We have identified a set of RNA-binding proteins (RBP) selectively enriched in human AML LSCs. Using an in vivo two-step CRISPR-Cas9 screen to assay stem cell functionality, we found 32 RBPs essential for LSCs in MLL-AF9;NrasG12D AML. Loss-of-function approaches targeting key hit RBP ELAVL1 compromised LSC-driven in vivo leukemic reconstitution, and selectively depleted primitive malignant versus healthy cells. Integrative multiomics revealed differentiation, splicing, and mitochondrial metabolism as key features defining the leukemic ELAVL1-mRNA interactome with mitochondrial import protein, TOMM34, being a direct ELAVL1-stabilized target whose repression impairs AML propagation. Altogether, using a stem cell-adapted in vivo CRISPR screen, this work demonstrates pervasive reliance on RBPs as regulators of LSCs and highlights their potential as therapeutic targets in AML. SIGNIFICANCE: LSC-targeted therapies remain a significant unmet need in AML. We developed a stem-cell-adapted in vivo CRISPR screen to identify key LSC drivers. We uncover widespread RNA-binding protein dependencies in LSCs, including ELAVL1, which we identify as a novel therapeutic vulnerability through its regulation of mitochondrial metabolism. This article is highlighted in the In This Issue feature, p. 171
Isotopologue Profiling of Triterpene Formation under Physiological Conditions. Biosynthesis of Lupeol-3-(3â˛â<i>R</i>âhydroxy)-stearate in <i>Pentalinon andrieuxii</i>
The
biosynthesis of lupeol-3-(3â˛<i>R</i>-hydroxy)-stearate
(procrim b, <b>1</b>) was investigated in the Mexican medicinal
plant <i>Pentalinon andrieuxii</i> by <sup>13</sup>CO<sub>2</sub> pulse-chase experiments. NMR analyses revealed positional
enrichments of <sup>13</sup>C<sub>2</sub>-isotopologues in both the
triterpenoid and the hydroxystearate moieties of <b>1</b>. Five
of the six isoprene units reflected a pattern with [1,2-<sup>13</sup>C<sub>2</sub>]- and [3,5-<sup>13</sup>C<sub>2</sub>]-isotopologues
from the respective C<sub>5</sub>-precursors, IPP and DMAPP, whereas
one isoprene unit in the ring E of <b>1</b> showed only the
[3,5-<sup>13</sup>C<sub>2</sub>]-connectivity of the original C<sub>5</sub>-precursor, due to rearrangement of the dammarenyl cation
intermediate during the cyclization process. The presence of <sup>13</sup>C<sub>2</sub>-isotopologues was indicative of [<sup>13</sup>C<sub>2</sub>]Âacetyl-CoA being the precursor units in the formation
of the fatty acid moiety and of the triterpene via the mevalonate
route. The observed labeling pattern was in agreement with a chair-chair-chair-boat
conformation of the (<i>S</i>)-2,3-oxidosqualene precursor
during the cyclization process, suggesting that the lupeol synthase
from <i>P. andrieuxii</i> is of the same type as that from <i>Olea europea</i> and <i>Taraxacum officinale</i>,
but different from that of <i>Arabidopsis thaliana</i>.
The study shows that <sup>13</sup>CO<sub>2</sub> pulse-chase experiments
are powerful in elucidating, under <i>in vivo</i> conditions
and in a single experiment, the biosynthesis of complex plant products
including higher terpenes
Translation from unconventional 5Ⲡstart sites drives tumour initiation
We are just beginning to understand how translational control affects tumour initiation and malignancy. Here we use an
epidermis-specific, in vivo ribosome profiling strategy to investigate the translational landscape during the transition from
normal homeostasis to malignancy. Using a mouse model of inducible SOX2, which is broadly expressed in oncogenic
RAS-associated cancers, we show that despite widespread reductions in translation and protein synthesis, certain
oncogenic mRNAs are spared. During tumour initiation, the translational apparatus is redirected towards unconventional
upstream initiation sites, enhancing the translational efficiency of oncogenic mRNAs. An in vivo RNA interference screen
of translational regulators revealed that depletion of conventional eIF2 complexes has adverse effects on normal but not
oncogenic growth. Conversely, the alternative initiation factor eIF2A is essential for cancer progression, during which
it mediates initiation at these upstream sites, differentially skewing translation and protein expression. Our findings
unveil a role for the translation of 5Ⲡuntranslated regions in cancer, and expose new targets for therapeutic intervention