Synthesis and biological activity of the (25R)-cholesten-26-oic acids—ligands for the hormonal receptor DAF-12 in Caenorhabditis elegans

Martin, René; Schmidt, Arndt W.; Theumer, Gabriele; Krause, Tilo; Entchev, Eugeni V.; Kurzchalia, Teymuras V.; Knölker, Hans‐Joachim

doi:10.1039/b817358c

Cited by 32 publications

(32 citation statements)

References 35 publications

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“…The following day, cells were stimulated with 0.5 µM (25R)-Δ7-DA [70] or DMSO for 16 hr. At this concentration, no difference in activity was observed between the (25R)- Δ7-DA and (25S)- Δ7-DA [71] (not shown). Luciferase was measured using Dual-Luciferase Reporter Assay System (Promega).…”

Section: Methodsmentioning

confidence: 85%

DAF-12 Regulates a Connected Network of Genes to Ensure Robust Developmental Decisions

et al. 2011

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The nuclear receptor DAF-12 has roles in normal development, the decision to pursue dauer development in unfavorable conditions, and the modulation of adult aging. Despite the biologic importance of DAF-12, target genes for this receptor are largely unknown. To identify DAF-12 targets, we performed chromatin immunoprecipitation followed by hybridization to whole-genome tiling arrays. We identified 1,175 genomic regions to be bound in vivo by DAF-12, and these regions are enriched in known DAF-12 binding motifs and act as DAF-12 response elements in transfected cells and in transgenic worms. The DAF-12 target genes near these binding sites include an extensive network of interconnected heterochronic and microRNA genes. We also identify the genes encoding components of the miRISC, which is required for the control of target genes by microRNA, as a target of DAF-12 regulation. During reproductive development, many of these target genes are misregulated in daf-12(0) mutants, but this only infrequently results in developmental phenotypes. In contrast, we and others have found that null daf-12 mutations enhance the phenotypes of many miRISC and heterochronic target genes. We also find that environmental fluctuations significantly strengthen the weak heterochronic phenotypes of null daf-12 alleles. During diapause, DAF-12 represses the expression of many heterochronic and miRISC target genes, and prior work has demonstrated that dauer formation can suppress the heterochronic phenotypes of many of these target genes in post-dauer development. Together these data are consistent with daf-12 acting to ensure developmental robustness by committing the animal to adult or dauer developmental programs despite variable internal or external conditions.

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Section: Methodsmentioning

confidence: 85%

DAF-12 Regulates a Connected Network of Genes to Ensure Robust Developmental Decisions

et al. 2011

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“…3731-51-9) were purchased from J&K Scientific Ltd (Beijing, China). (25S)-Δ 4 -dafachronic acid 15 and (25R)-Δ 7 -dafachronic acid 16 3 ]-DA will be published elsewhere. The identities of the synthesized molecules were confirmed by NMR and high-resolution mass spectrometry on a LTQ-Orbitrap mass spectrometer (Thermo Fisher Scientific).…”

Section: ■ Experimental Sectionmentioning

confidence: 99%

Absolute Quantification of a Steroid Hormone that Regulates Development in Caenorhabditis elegans

Chen

et al. 2013

Anal. Chem.

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Under favorable conditions, Caenorhabditis elegans larvae grow into reproductive adults after a series of molting cycles. When environmental conditions are harsh, they arrest as dauer larvae. Dafachronic acid (DA), a C. elegans steroid hormone, is required for reproductive development. Here, we report a mass spectrometry (MS) method for absolute quantitation of DA in C. elegans. The extraction of DA from C. elegans was optimized to achieve a recovery rate of greater than 83%. The MS sensitivity to DA increased 100-fold after carboxyl group derivatization with 2-picolylamine. Highresolution selected ion monitoring (HR-SIM) on a Q-Orbitrap mass spectrometer Q Exactive outperformed targeted-MS2 on the same instrument and selected reaction monitoring (SRM) on a triple-quadrupole mass spectrometer TSQ Quantum Discovery. With a limit of quantification as low as 1 pg of DA, the HR-SIM method enables absolute quantification of endogenous DA during the reproductive development of C. elegans. We found that in wild-type (WT) worms, DA increases from 0.04 ± 0.02 ng/mg protein in the L1 larval stage to 1.21 ± 0.67 ng/mg protein in the L2 larval stage and decreases again after the L3 stage. In comparison, four genetic mutants that have a constitutive dauer-formation phenotype due to disrupted insulin, TGF-β, or cGMP signaling all have a very low DA level in the L2 stage (below 15% of the WT). These mutants are able to escape the dauer fate and most of them grow into fertile adults when supplied with exogenous DA. Therefore, a DA spike in the L2 stage is critical for the reproductive development of C. elegans.S teroid hormones are signaling molecules that are synthesized from cholesterol. They are found in many organisms, from worms to mammals, and regulate a variety of vital physiological functions, including development, metabolism, and reproduction. 1 The first steroid hormone identified in Caenorhabditis elegans is dafachronic acid (DA), 2 a key molecule that helps determine whether C. elegans larvae grow into reproductive adults.Under favorable conditions, C. elegans undergo four larval stages (L1-L4) after hatching and develop into adults in 3 days. However, when environmental conditions are harsh, such as high temperature, starvation, or high population density, worms arrest at an alternative L3 stage in the form of dauer larvae 3 (Figure 1). Dauer larvae exhibit many adaptive changes in morphology, behavior, and metabolism that help them survive under stressful conditions. 3,4 Dauer larvae can live up to several months before recovering and developing into normal L4 larvae and subsequently adults when the environmental conditions improve. 3,4 Before the chemical structure of DA was determined, its receptor DAF-12 was identified as a common target downstream of several dauer regulatory pathways. 5−7 Mutations in the daf-9 or daf-36 gene, each encoding an enzyme involved in DA synthesis (Figure 1), cause a constitutive dauer-formation phenotype (Daf-c) and impairment in gonad development. 8−10 These data indi...

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“…Cholest-4-en-3-one, androst-4-en-3,17-dione (AD), (25R)-cholesten-26-oic acid with purity of 99% were synthesized and characterized using 1 HNMR, 13 C NMR, IR, and TOF–MS as it was done in our previous studies (Liu et al 2011; Wu et al 2015). (25R)-cholesten-26-oic acid was prepared as recommended (Martin et al 2009). Restriction enzymes, dNTPs, Taq polymerase were purchased from TaKaRa Co. (Dalian, China).…”

Section: Methodsmentioning

confidence: 99%

Heterologous expression and characterization of a 3-ketosteroid-∆1-dehydrogenase from Gordonia neofelifaecis and its utilization in the bioconversion of androst-4,9(11)-dien-3,17-dione

Wang

et al. 2017

3 Biotech

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3-Ketosteroid-∆1-dehydrogenase (KstD), a key enzyme in microbial steroid catabolism, catalyzes the trans-axial elimination of the C1 and C2 hydrogen atoms of the A-ring from the polycyclic ring structure of 3-ketosteroids, and it was usually used to transform androst-4-ene-3,17-dione (AD) to produce androsta-1,4-diene-3,17-dione. Here, the KstD from Gordonia neofelifaecis was expressed efficiently in Escherichia coli. E. coli cells expressing KstD3gor were subjected to the investigation of dehydrogenation activity for different steroids. The results showed that KstD3gor has a clear preference for steroid substrates with 3-keto-4-ene configuration, and it exhibits higher activity towards steroid substrates carrying a small or no aliphatic side chain than towards substrates having a bulky side chain at the C-17 atom. The recombinant strain could efficiently convert androst-4,9(11)-dien-3,17-dione into androst-1,4,9(11)-trien-3,17-dione (with conversion rate of 96%). 1(2)-Dehydrogenation of androst-4,9(11)-dien-3,17-dione is one of the key steps in glucocorticoid production. To the best of our knowledge, this is the first study reporting on the conversion of androst-4,9(11)-dien-3,17-dione catalyzed by recombinant KstD; the expression system of KstD3gor reported here would have an impact in the industrial production of glucocorticoid in the future.

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Synthesis and biological activity of the (25R)-cholesten-26-oic acids—ligands for the hormonal receptor DAF-12 in Caenorhabditis elegans

Cited by 32 publications

References 35 publications

DAF-12 Regulates a Connected Network of Genes to Ensure Robust Developmental Decisions

DAF-12 Regulates a Connected Network of Genes to Ensure Robust Developmental Decisions

Absolute Quantification of a Steroid Hormone that Regulates Development in Caenorhabditis elegans

Heterologous expression and characterization of a 3-ketosteroid-∆1-dehydrogenase from Gordonia neofelifaecis and its utilization in the bioconversion of androst-4,9(11)-dien-3,17-dione

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