scholarly journals Hairy and Groucho mediate the action of juvenile hormone receptor Methoprene-tolerant in gene repression

2016 ◽  
Vol 113 (6) ◽  
pp. E735-E743 ◽  
Author(s):  
Tusar T. Saha ◽  
Sang Woon Shin ◽  
Wei Dou ◽  
Sourav Roy ◽  
Bo Zhao ◽  
...  
Keyword(s):  
Juvenile Hormone ◽  
Fat Body ◽  
Gene Activation ◽  
Immunoblot Analysis ◽  
Gene Repression ◽  
Binding Motifs ◽  
Rnai Silencing ◽  
Body Culture ◽  
A Cell

The arthropod-specific juvenile hormone (JH) controls numerous essential functions. Its involvement in gene activation is known to be mediated by the transcription factor Methoprene-tolerant (Met), which turns on JH-controlled genes by directly binding to E-box–like motifs in their regulatory regions. However, it remains unclear how JH represses genes. We used the Aedes aegypti female mosquito, in which JH is necessary for reproductive maturation, to show that a repressor, Hairy, is required for the gene-repressive action of JH and Met. The RNA interference (RNAi) screen for Met and Hairy in the Aedes female fat body revealed a large cohort of Met- and Hairy-corepressed genes. Analysis of selected genes from this cohort demonstrated that they are repressed by JH, but RNAi of either Met or Hairy renders JH ineffective in repressing these genes in an in vitro fat-body culture assay. Moreover, this JH action was prevented by the addition of the translational inhibitor cycloheximide (CHX) to the culture, indicating the existence of an indirect regulatory hierarchy. The lack of Hairy protein in the CHX-treated tissue was verified using immunoblot analysis, and the upstream regions of Met/Hairy-corepressed genes were shown to contain common binding motifs that interact with Hairy. Groucho (gro) RNAi silencing phenocopied the effect of Hairy RNAi knockdown, indicating that it is involved in the JH/Met/Hairy hierarchy. Finally, the requirement of Hairy and Gro for gene repression was confirmed in a cell transfection assay. Thus, our study has established that Hairy and its cofactor Gro mediate the repressive function of JH and Met.

scholarly journals Dual binding motifs underpin the hierarchical association of perilipins1–3 with lipid droplets

2019 ◽  
Vol 30 (5) ◽  
pp. 703-716 ◽  
Author(s):  
Dalila Ajjaji ◽  
Kalthoum Ben M'barek ◽  
Michael L. Mimmack ◽  
Cheryl England ◽  
Haya Herscovitz ◽  
...  
Keyword(s):  
Lipid Droplets ◽  
Tissue Type ◽  
Binding Motifs ◽  
Amino Terminal ◽  
A Cell ◽  
Oil Water ◽  
Carboxy Terminal ◽  
Hierarchical Manner

Lipid droplets (LDs) in all eukaryotic cells are coated with at least one of the perilipin (Plin) family of proteins. They all regulate key intracellular lipases but do so to significantly different extents. Where more than one Plin is expressed in a cell, they associate with LDs in a hierarchical manner. In vivo, this means that lipid flux control in a particular cell or tissue type is heavily influenced by the specific Plins present on its LDs. Despite their early discovery, exactly how Plins target LDs and why they displace each other in a “hierarchical” manner remains unclear. They all share an amino-terminal 11-mer repeat (11mr) amphipathic region suggested to be involved in LD targeting. Here, we show that, in vivo, this domain functions as a primary highly reversible LD targeting motif in Plin1–3, and, in vitro, we document reversible and competitive binding between a wild-type purified Plin1 11mr peptide and a mutant with reduced binding affinity to both “naked” and phospholipid-coated oil–water interfaces. We also present data suggesting that a second carboxy-terminal 4-helix bundle domain stabilizes LD binding in Plin1 more effectively than in Plin2, whereas it weakens binding in Plin3. These findings suggest that dual amphipathic helical regions mediate LD targeting and underpin the hierarchical binding of Plin1–3 to LDs.

scholarly journals Acyclic nucleoside phosphonates with adenine nucleobase inhibit Trypanosoma brucei adenine phosphoribosyltransferase in vitro

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Eva Doleželová ◽  
Tomáš Klejch ◽  
Petr Špaček ◽  
Martina Slapničková ◽  
Luke Guddat ◽  
...  
Keyword(s):  
Trypanosoma Brucei ◽  
De Novo ◽  
Major Effect ◽  
Nucleotide Analogs ◽  
Rnai Silencing ◽  
Acyclic Nucleoside Phosphonates ◽  
A Cell ◽  
Actual Target ◽  
Promising Source

AbstractAll medically important unicellular protozoans cannot synthesize purines de novo and they entirely rely on the purine salvage pathway (PSP) for their nucleotide generation. Therefore, purine derivatives have been considered as a promising source of anti-parasitic compounds since they can act as inhibitors of the PSP enzymes or as toxic products upon their activation inside of the cell. Here, we characterized a Trypanosoma brucei enzyme involved in the salvage of adenine, the adenine phosphoribosyl transferase (APRT). We showed that its two isoforms (APRT1 and APRT2) localize partly in the cytosol and partly in the glycosomes of the bloodstream form (BSF) of the parasite. RNAi silencing of both APRT enzymes showed no major effect on the growth of BSF parasites unless grown in artificial medium with adenine as sole purine source. To add into the portfolio of inhibitors for various PSP enzymes, we designed three types of acyclic nucleotide analogs as potential APRT inhibitors. Out of fifteen inhibitors, four compounds inhibited the activity of the recombinant APRT1 with Ki in single µM values. The ANP phosphoramidate membrane-permeable prodrugs showed pronounced anti-trypanosomal activity in a cell-based assay, despite the fact that APRT enzymes are dispensable for T. brucei growth in vitro. While this suggests that the tested ANP prodrugs exert their toxicity by other means in T. brucei, the newly designed inhibitors can be further improved and explored to identify their actual target(s).

The influence of hemolymph-binding protein on juvenile hormone stability and distribution in Manduca sexta fat body and imaginal discs in vitro

1975 ◽  
Vol 3 (3) ◽  
pp. 167-184 ◽  
Author(s):  
Bruce Hammock ◽  
Joachim Nowock ◽  
Walter Gooddman ◽  
Vassiliki Stamoudis ◽  
Lawrence I. Gilbert
Keyword(s):  
Juvenile Hormone ◽  
Manduca Sexta ◽  
Fat Body ◽  
Binding Protein ◽  
Imaginal Discs

Cuticle Deposition in Imaginal Disks: Effects of Juvenile Hormone and Fat Body in vitro

Science ◽  
1972 ◽  
Vol 177 (4047) ◽  
pp. 441-442 ◽  
Author(s):  
H. Oberlander ◽  
C. Tomblin
Keyword(s):  
Juvenile Hormone ◽  
Fat Body ◽  
Imaginal Disks

scholarly journals Juvenile hormone regulation of microRNAs is mediated by E75 in the Dengue vector mosquito Aedes aegypti

2021 ◽  
Vol 118 (29) ◽  
pp. e2102851118
Author(s):  
Emre Aksoy ◽  
Alexander S. Raikhel
Keyword(s):  
Aedes Aegypti ◽  
Juvenile Hormone ◽  
Fat Body ◽  
Mirna Gene ◽  
Luciferase Assay ◽  
Small Rna Sequencing ◽  
Hormone Regulation ◽  
Gene Target ◽  
Body Tissues

MicroRNAs (miRNAs) are small noncoding RNAs that play critical roles in controlling posttranscriptional gene regulation and have a profound effect on mosquito reproduction and metabolism. Juvenile hormone (JH) is critical for achieving reproductive competence in the main vector of human arboviral diseases, Aedes aegypti. We report a JH-mediated mechanism governing miRNA expression. Using a transcription factor screen with multiple primary miRNA (pri-miRNA) promoters, we identified that the Ecdysone-induced protein E75 (E75) isoform (E75-RD) induced miRNA gene promoter activity. E75 binding sites were determined in miRNA promoters by means of cell transfection assay. E75-RD was found to be up-regulated by JH, as shown by the JH application and RNA interference (RNAi) of the JH receptor Methoprene-tolerant (Met). Small RNA sequencing from RNAi of Met and E75 displayed an overlapping miRNA cohort, suggesting E75 to be an intermediate component within the JH hierarchical network controlling miRNAs. Further experiments confirmed that E75-RD positively regulates several miRNAs including miR-2940. Reducing miR-2940 resulted in the arrest of follicle development and number of eggs laid. Performing miRNA target predictions and RT-qPCR from antagomir Ant-2940-3p–treated fat body tissues identified the mRNA target Clumsy (AAEL002518). The molecular interaction between this gene target and miR-2940 was confirmed using an in vitro dual luciferase assay in Drosophila S2 cells and in Ae. aegypti Aag2 cell lines. Finally, we performed a phenotypic rescue experiment to demonstrate that miR-2940/Clumsy is responsible for the disruption in egg development. Collectively, these results established the role of JH-mediated E75-RD in regulation of miRNA gene expression during the mosquito reproductive cycle.

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