Cloning and Characterization of Drosophila melanogaster Juvenile Hormone Epoxide Hydrolases (JHEH) and Their Promoters

Borovsky, Dov; Breyssens, Hilde; Buytaert, Esther; Peeters, Tom; Laroye, Carole; Stoffels, Karolien; Rougé, Pierre

doi:10.3390/biom12070991

Cited by 5 publications

(22 citation statements)

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“…The common catalytic amino acids triad for Ae. aegypti JHEH 1, 2, and 3 is D, E and H (Figure 1a−c), in comparison the JHEH 1, and 3 of D. melanogaster have amino acids catalytic triad of D, D, H, and JHEH 2 exhibits an amino acids catalytic triad of D, E, H (Borovsky et al, 2022). The jheh of Ae.…”

Section: Discussionmentioning

confidence: 97%

“…aegypti (Tusun et al, 2017;. The cDNAs code for proteins of 461, 462, and 458 amino acids, melanogaster (Borovsky et al, 2022;Reetz et al, 2009;Tusun et al, 2017;Wen et al, 2018). Ae.…”

Section: Discussionmentioning

confidence: 99%

“…These authors, however, never tested JH IIIA to find out if JHEH-r3, r4 and r5 prefer JH IIIA as their specific substrate. The jheh of D. melanogaster was reported to exhibit three related genes jheh 1, 2, and 3 with 3, 3, and 2 introns, respectively (Borovsky et al, 2022) The jheh 2 of D.…”

Section: Discussionmentioning

confidence: 99%

“…JH III acid (JH IIIA) and [12-3 H]JH IIIA were synthesized by hydrolyzing the methyl ester of JH III, converting it into JH IIIA by incubating JH III with 0.5 N NaOH in ethanol for 24 h at room temperature. The JH IIIA was purified by reversed phase C 18 HPLC and stored in hexane (Borovsky et al, 1992(Borovsky et al, , 2022. Jheh dsRNA was synthesized using a HiScribe RNAi transcription kit (New England BioLabs) and the double-stranded DNA(dsDNA) was cloned into pLitmus 28i between EcoRI and BamHI.…”

Section: Methodsmentioning

confidence: 99%

“…aegypti λ ZAP cDNA library that was constructed using a commercial kit (Stratagene) following manufacturer's guidelines from 200 guts removed from female A. aegypti 30 to 48 h after a blood meal. The PCR steps were similar to what was described before (Borovsky et al, 2022). Briefly, a DNA denaturation for 3 min at 95°C (1 cycle), followed by annealing for 4 min at 48°C, and extension for 40 min at 60°C (1 cycle each).…”

Section: Cloning and Sequencing Of Jheh 1 2 And 3 Cdnasmentioning

confidence: 99%

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Cloning and characterization of Aedes aegypti juvenile hormone epoxide hydrolases (JHEHs)

Borovsky

Ekert

Buytaert

et al. 2022

Arch Insect Biochem Physiol

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Juvenile hormone epoxide hydrolase (JHEH) plays an important role in the metabolism of juvenile hormone III (JH III) in insects. To study the role that JHEH plays in female Aedes aegypti JHEH 1, 2, and 3 complementary DNA (cDNAs) were cloned and sequenced. Northern blot analyses show that the three transcripts are expressed in the head thorax, the gut, the ovaries, and the fat body of females. Molecular modeling shows that the enzyme is a homodimer that binds JH III acid (JH IIIA) at the catalytic groove better than JH III. The cDNA of JHEH 1 and 2 are very similar indicating close relationship. Knocking down of jheh 1, 2, and 3 in adult female and larval Ae. aegypti using double‐stranded RNA (dsRNA) did not affect egg development or caused adult mortality. Larvae that were fed bacterial cells expressing dsRNA against jheh 1, 2, and 3 grew normally. Treating blood‐fed female Ae. aegypti with [12‐3H](10R) JH III and analyzing the metabolites by C18 reversed phase chromatography showed that JHEH preferred substrate is not JH III but JH IIIA. Genomic analysis of jheh 1, 2, and 3 indicate that jheh 1 and 2 are transcribed from a 1.53 kb DNA whereas jheh 3 is transcribed from a 10.9 kb DNA. All three genes are found on chromosome two at distinct locations. JHEH 2 was expressed in bacterial cells and purified by Ni affinity chromatography. Sequencing of the recombinant protein by MS/MS identified JHEH 2 as the expressed recombinant protein.

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Section: Discussionmentioning

confidence: 97%

“…aegypti (Tusun et al, 2017;. The cDNAs code for proteins of 461, 462, and 458 amino acids, melanogaster (Borovsky et al, 2022;Reetz et al, 2009;Tusun et al, 2017;Wen et al, 2018). Ae.…”

Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Cloning and Sequencing Of Jheh 1 2 And 3 Cdnasmentioning

confidence: 99%

See 3 more Smart Citations

Cloning and characterization of Aedes aegypti juvenile hormone epoxide hydrolases (JHEHs)

Borovsky

Ekert

Buytaert

et al. 2022

Arch Insect Biochem Physiol

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Cloning and characterization of Aedes aegypti blood downregulated chymotrypsin II

Borovsky

Rougé

2023

Arch Insect Biochem Physiol

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Aedes aegypti adult and larval blood downregulated chymotrypsin II was cloned, sequenced and its 3D conformation modeled. Cloning of the enzymes from adult and larval guts indicated that both genes sit at the same location on Chromosome 2. Genomic analyses showed that larval and adult genes are the same and both have four exons and three introns that are located on an 8.32 Kb DNA in direction with the Ae. aegypti genome. The adult and larval transcript synthesis is controlled by alternative splicing explaining small difference in the amino acids sequences. Chymotrypsin II that was extracted from guts of sugar‐fed and at 48 after blood feeding showed a pH optimum of 4–5 with a broad shoulder of activity from pH 6 to 10. Dot blot analyses show that the enzyme's transcript is downregulated after females take a blood meal and upregulated at 48 h after the blood meal. A Chymotrypsin II transcript was also detected in the larval gut during different times of larval developmental stages, indication that Ae. aegypti chymotrypsin II is synthesized by adults and larval guts. The possibility that JH III and 20HE play an active role in the regulation is discussed.

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The moulting arthropod: a complete genetic toolkit review

Campli,

Volovych,

Kim

et al. 2024

Biological Reviews

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Exoskeletons are a defining character of all arthropods that provide physical support for their segmented bodies and appendages as well as protection from the environment and predation. This ubiquitous yet evolutionarily variable feature has been instrumental in facilitating the adoption of a variety of lifestyles and the exploitation of ecological niches across all environments. Throughout the radiation that produced the more than one million described modern species, adaptability afforded by segmentation and exoskeletons has led to a diversity that is unrivalled amongst animals. However, because of the limited extensibility of exoskeleton chitin and cuticle components, they must be periodically shed and replaced with new larger ones, notably to accommodate the growing individuals encased within. Therefore, arthropods grow discontinuously by undergoing periodic moulting events, which follow a series of steps from the preparatory pre‐moult phase to ecdysis itself and post‐moult maturation of new exoskeletons. Each event represents a particularly vulnerable period in an arthropod's life cycle, so processes must be tightly regulated and meticulously executed to ensure successful transitions for normal growth and development. Decades of research in representative arthropods provide a foundation of understanding of the mechanisms involved. Building on this, studies continue to develop and test hypotheses on the presence and function of molecular components, including neuropeptides, hormones, and receptors, as well as the so‐called early, late, and fate genes, across arthropod diversity. Here, we review the literature to develop a comprehensive overview of the status of accumulated knowledge of the genetic toolkit governing arthropod moulting. From biosynthesis and regulation of ecdysteroid and sesquiterpenoid hormones, to factors involved in hormonal stimulation responses and exoskeleton remodelling, we identify commonalities and differences, as well as highlighting major knowledge gaps, across arthropod groups. We examine the available evidence supporting current models of how components operate together to prepare for, execute, and recover from ecdysis, comparing reports from Chelicerata, Myriapoda, Crustacea, and Hexapoda. Evidence is generally highly taxonomically imbalanced, with most reports based on insect study systems. Biases are also evident in research on different moulting phases and processes, with the early triggers and late effectors generally being the least well explored. Our synthesis contrasts knowledge based on reported observations with reasonably plausible assumptions given current taxonomic sampling, and exposes weak assumptions or major gaps that need addressing. Encouragingly, advances in genomics are driving a diversification of tractable study systems by facilitating the cataloguing of putative genetic toolkits in previously under‐explored taxa. Analysis of genome and transcriptome data supported by experimental investigations have validated the presence of an “ultra‐conserved” core of arthropod genes involved in moulting processes. The molecular machinery has likely evolved with elaborations on this conserved pathway backbone, but more taxonomic exploration is needed to characterise lineage‐specific changes and novelties. Furthermore, linking these to transformative innovations in moulting processes across Arthropoda remains hampered by knowledge gaps and hypotheses based on untested assumptions. Promisingly however, emerging from the synthesis is a framework that highlights research avenues from the underlying genetics to the dynamic molecular biology through to the complex physiology of moulting.

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Cloning and Characterization of Drosophila melanogaster Juvenile Hormone Epoxide Hydrolases (JHEH) and Their Promoters

Cited by 5 publications

References 54 publications

Cloning and characterization of Aedes aegypti juvenile hormone epoxide hydrolases (JHEHs)

Cloning and characterization of Aedes aegypti juvenile hormone epoxide hydrolases (JHEHs)

Cloning and characterization of Aedes aegypti blood downregulated chymotrypsin II

The moulting arthropod: a complete genetic toolkit review

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