Studies on the black box: Incorporation of 3-oxo-7-dehydrocholesterol into ecdysteroids by Drosophila melanogaster and Manduca sexta

Warren, James T.; O’Connor, Michael B.; Gilbert, Lawrence I.

doi:10.1016/j.ibmb.2009.08.004

Cited by 36 publications

(28 citation statements)

References 39 publications

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“…In A. gambiae adults, cyp307a1 is highly up regulated in active steroidogenic tissues and this highlights that this gene must encode one of the early steps of steroidogenesis which are known to be more tightly regulated than the last steps leading to 20E [4]. This is consistent with results obtained in D. melanogaster indicating that they could act in the currently uncharacterized black box, from which one or multiple steps are believed to limit the production of ecdysone, in that no stable intermediate has been yet identified [4], [17]. No conversion of C or 7dC was observed in S2 cells transfected with cyp307a1 but since the black box is supposed to contain several oxidative transformations, unless this gene catalyzes the initial reaction, expressing cyp307a1 alone with 7dC would not be expected to produce a product [18]–[19], [48].…”

Section: Discussionsupporting

confidence: 85%

“…This includes the oxidation of 3beta-alcohol to ketone, the oxidation of carbon 6 with concomitant loss of the 4beta- and 6-hydrogens to form the 6-keto group, and 14alpha-hydroxylation. Δ 4 -diketol would then be converted by a 5ß-reductase to 5ß-diketol further transformed in 5ß-ketodiol by a 3ß-reductase [4], [17]. The black box, and more particularly the oxidative steps, is thought to involve one or more P450 enzymes that still remain uncharacterized [6], [18].…”

Section: Introductionmentioning

confidence: 99%

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Microarray and RNAi Analysis of P450s in Anopheles gambiae Male and Female Steroidogenic Tissues: CYP307A1 Is Required for Ecdysteroid Synthesis

et al. 2013

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In insects, the steroid hormone 20-hydroxyecdysone (20E) coordinates major developmental transitions. While the first and the final steps of 20E biosynthesis are characterized, the pathway from 7-dehydrocholesterol to 5β-ketodiol, commonly referred as the “black box”, remains hypothetical and whether there are still unidentified enzymes is unknown. The black box would include some oxidative steps, which are believed to be mediated by P450 enzymes. To identify new enzyme(s) involved in steroid synthesis, we analyzed by small-scale microarray the expression of all the genes encoding P450 enzymes of the malaria mosquito Anopheles gambiae in active steroidogenic organs of adults, ovaries from blood-fed females and male reproductive tracts, compared to inactive steroidogenic organs, ovaries from non-blood-fed females. Some genes encoding P450 enzymes were specifically overexpressed in female ovaries after a blood-meal or in male reproductive tracts but only three genes were found to be overexpressed in active steroidogenic organs of both females and males: cyp307a1, cyp4g16 and cyp6n1. Among these genes, only cyp307a1 has an expression pattern similar to other mosquito steroidogenic genes. Moreover, loss-of-function by transient RNAi targeting cyp307a1 disrupted ecdysteroid production demonstrating that this gene is required for ecdysteroid biosynthesis in Anopheles gambiae.

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Section: Discussionsupporting

confidence: 85%

Section: Introductionmentioning

confidence: 99%

Microarray and RNAi Analysis of P450s in Anopheles gambiae Male and Female Steroidogenic Tissues: CYP307A1 Is Required for Ecdysteroid Synthesis

et al. 2013

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“…Thus far, we have not assigned a specific enzymatic activity to any substrate for Nm-g/Sro, as no intermediates between 7dC and 5-ketodiol have been precisely identified in insects (Warren and Hetru, 1990;Gilbert et al, 2002). One possibility is that Nm-g/Sro catalyzes the 3-dehydrogenation of 7dC to 3-oxo-7dC (cholesta-5,7-diene-3-one), which is hypothesized to be involved in the initial step in the Black Box (Dauphin-Villemant et al, 1997;Warren et al, 2009). An alternative possibility is that Nm-g/Sro might act as an ecdysteroid 5-reductase converting  4 -diketol to diketol.…”

Section: Discussionmentioning

confidence: 98%

Non-molting glossy/shroud encodes a short-chain dehydrogenase/reductase that functions in the ‘Black Box’ of the ecdysteroid biosynthesis pathway

et al. 2010

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SUMMARYIn insects, the precise timing of molting and metamorphosis is strictly guided by a principal steroid hormone, ecdysone. Among the multiple conversion steps for synthesizing ecdysone from dietary cholesterol, the conversion of 7-dehydrocholesterol to 5-ketodiol, the so-called 'Black Box', is thought to be the important rate-limiting step. Although a number of genes essential for ecdysone synthesis have recently been revealed, much less is known about the genes that are crucial for functioning in the Black Box. Here we report on a novel ecdysteroidgenic gene, non-molting glossy (nm-g)/shroud (sro), which encodes a short-chain dehydrogenase/reductase. This gene was first isolated by positional cloning of the nm-g mutant of the silkworm Bombyx mori, which exhibits a low ecdysteroid titer and consequently causes a larval arrest phenotype. In the fruit fly, Drosophila melanogaster, the closest gene to nm-g is encoded by the sro locus, one of the Halloween mutant members that are characterized by embryonic ecdysone deficiency. The lethality of the sro mutant is rescued by the overexpression of either sro or nm-g genes, indicating that these two genes are orthologous. Both the nm-g and the sro genes are predominantly expressed in tissues producing ecdysone, such as the prothoracic glands and the ovaries. Furthermore, the phenotypes caused by the loss of function of these genes are restored by the application of ecdysteroids and their precursor 5-ketodiol, but not by cholesterol or 7-dehydrocholesterol. Altogether, we conclude that the Nm-g/Sro family protein is an essential enzyme for ecdysteroidogenesis working in the Black Box.

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“…The 7DC undergoes a series of reactions involving 3-oxo-4 intermediates [36], the so-called "Black Box", the details of which are becoming clearer [39]. YO cells from C. maenas convert a 25-deoxy-3-oxo-4 intermediate to 3DE and 25dE; no ecdysone was detected [36].…”

Section: Cholesterolmentioning

confidence: 99%

Ecdysteroid metabolism in crustaceans

Mykles

2011

The Journal of Steroid Biochemistry and Molecular Biology

166

121

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Studies on the black box: Incorporation of 3-oxo-7-dehydrocholesterol into ecdysteroids by Drosophila melanogaster and Manduca sexta

Cited by 36 publications

References 39 publications

Microarray and RNAi Analysis of P450s in Anopheles gambiae Male and Female Steroidogenic Tissues: CYP307A1 Is Required for Ecdysteroid Synthesis

Microarray and RNAi Analysis of P450s in Anopheles gambiae Male and Female Steroidogenic Tissues: CYP307A1 Is Required for Ecdysteroid Synthesis

Non-molting glossy/shroud encodes a short-chain dehydrogenase/reductase that functions in the ‘Black Box’ of the ecdysteroid biosynthesis pathway

Ecdysteroid metabolism in crustaceans

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