Molecular and biochemical characterization of two P450 enzymes in the ecdysteroidogenic pathway of Drosophila melanogaster

Abstract

Five different enzymatic activities, catalyzed by both microsomal and mitochondrial cytochrome P450 monooxygenases (CYPs), are strongly implicated in the biosynthesis of ecdysone (E) from cholesterol. However, none of these enzymes have been characterized completely. The present data show that the wild-type genes of two members of the Halloween family of embryonic lethals, disembodied (dib) and shadow (sad), code for mitochondrial cytochromes P450 that mediate the last two hydroxylation reactions in the ecdysteroidogenic pathway in Drosophila, namely the C 22-and C 2-hydroxylases. When sad (CYP315A1) is transfected into Drosophila S2 cells, the cells metabolize 2-deoxyecdysone (2dE) H]E in high yield. The expression of sad and dib is concentrated within the individual segments of the developing epidermis when there is a surge of ecdysteroid midway through embryogenesis. This result occurs before the ring gland has developed and suggests that the embryonic epidermis is a site of ecdysteroid biosynthesis. This pattern then diminishes, and, during late embryogenesis, expression of both genes is concentrated in the prothoracic gland cells of the developing ring gland. Expression of dib and sad continues to be localized in this endocrine compartment during larval development, being maximal in both the late second and third instar larvae, about the time of the premolt peaks in the ecdysteroid titer.

Discrete pulses of molting hormone, 20‐hydroxyecdysone, during late larval development of Drosophila melanogaster: Correlations with changes in gene activity

“…Overall, these data are consistent with prior semi-quantitative RT-PCR and immunohistochemical data, indicating relatively constant phm and dib expression during the early third instar, although the expression of both phm and dib does appear later in the instar to be under the control of the nuclear receptor ␤Ftz-F1 in the ring gland (Parvy et al, 2005). Only the analysis of specific Phm and Dib enzymatic activities during this period, along with that of the 2-hydroxylase, Shadow (Warren et al, 2002) and the 20-hydroxylase, Shade (Petryk et al, 2003), will clarify the importance of CYP enzyme regulation to the changing titers of active molting hormone during Drosophila ecdysteroidogenesis (Fig. 4).…”

“…To address this issue, we studied the expression of Phm and Dib, two cytochrome P450 enzymes specific to the prothoracic gland cells of the larval ring gland that are required for E biosynthesis (Warren et al, 2002(Warren et al, , 2004Niwa et al, 2004). By Western blot analysis, the significant, although low, expression of both proteins in these same early third instar larval samples is indicative of a rather constant capacity to synthesize E during this period (Fig.…”

“…4) (Warren et al, 2002(Warren et al, , 2004Petryk et al, 2003;Niwa et al, 2004). Quantitative Western blot analysis, performed on the insoluble protein remaining after ecdysteroid extraction (see Experimental Procedures section), reveals significant expression throughout the third instar of both Phantom (Phm, the terminal 25-hydroxylase) and Disembodied (Dib, the subsequent 22-hydroxylase) (Fig.…”

“…Both the 3-dehydro metabolites 3-dehydroecdysone (3DE) and 3-dehydro-20-hydroxyecdysone (3D20E), and their 3-␣-epimeric derivatives, 3-␣-epi-ecdysone (E') and 3-␣-epi-20-hydroxyecdysone (20E'), were obtained by chemical oxidation and reduction of the parent ecdysteroid (Spindler, et al, 1977;Dinan and Rees, 1978). Rabbit antiPhantom (Phm) peptide antibodies (Warren et al, 2004) and rabbit antiDisembodied (Dib) antibodies, similarly made from an N-terminal Dib peptide (Warren et al, 2002), were obtained from FabGennix International (Shreveport, LA).…”

“…Twenty wild type brain-ring gland complexes were treated the same, yielding 100 l in 1ϫ sample buffer. S2R protein samples were made by transfection of Schneider S2R cells with phantom or disembodied cDNA in pBRAcPA, using DDAB as the transfection reagent (Warren et al, 2002(Warren et al, , 2004. After 5 days, the cells were collected, suspended in 1ϫ sample buffer, boiled 5 min, and centrifuged at 14,000 rpm for 10 min.…”