The nine-amino-acid activation domain (9aaTAD) is defined by a short amino acid pattern including two hydrophobic regions (positions p3-4 and p6-7). The KIX domain of mediator transcription CBP interacts with the 9aaTAD domains of transcription factors MLL, E2A, NF-kB, and p53. In this study, we analyzed the 9aaTADs-KIX interactions by nuclear magnetic resonance. The positions of three KIX helixes α1-α2-α3 are influenced by sterically-associated hydrophobic I611, L628, and I660 residues that are exposed to solvent. The positions of two rigid KIX helixes α1 and α2 generate conditions for structural folding in the flexible KIX-L12-G2 regions localized between them. The three KIX I611, L628, and I660 residues interact with two 9aaTAD hydrophobic residues in positions p3 and p4 and together build a hydrophobic core of five residues (5R). Numerous residues in 9aaTAD position p3 and p4 could provide this interaction. Following binding of the 9aaTAD to KIX, the hydrophobic I611, L628, and I660 residues are no longer exposed to solvent and their position changes inside the hydrophobic core together with position of KIX α1-α2-α3 helixes. The new positions of the KIX helixes α1 and α2 allow the KIX-L12-G2 enhanced formation. The second hydrophobic region of the 9aaTAD (positions p6 and p7) provides strong binding with the KIX-L12-G2 region. Similarly, multiple residues in 9aaTAD position p6 and p7 could provide this interaction. In conclusion, both 9aaTAD regions p3, p4 and p6, p7 provide co-operative and highly universal binding to mediator KIX. The hydrophobic core 5R formation allows new positions of the rigid KIX α-helixes and enables the enhanced formation of the KIX-L12-G2 region. This contributes to free energy and is the key for the KIX-9aaTAD binding. Therefore, the 9aaTAD-KIX interactions do not operate under the rigid key-and-lock mechanism what explains the 9aaTAD natural variability.
- MeSH
- aminokyselinové motivy MeSH
- histonlysin-N-methyltransferasa chemie metabolismus MeSH
- interakční proteinové domény a motivy MeSH
- lidé MeSH
- nádorový supresorový protein p53 chemie metabolismus MeSH
- NF-kappa B chemie metabolismus MeSH
- protein vázající CREB chemie metabolismus MeSH
- protoonkogenní protein MLL chemie metabolismus MeSH
- transkripční faktory bHLH chemie metabolismus MeSH
- transkripční faktory chemie metabolismus MeSH
- vazba proteinů MeSH
- vazebná místa MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Gamma-glutamyltransferase (GGT), a marker of liver disease, has been shown to be associated with increased risk of diabetes and relative insulin secretion deficiency. However, the mechanism of hepatic Ggt regulation has not been explored fully. In this study, we made a concerted effort to understand the mechanism by investigating the effects of acetylation of histones H3 and H4, and bindings of histone acetyltransferases, CREB binding protein (CBP) and p300, at the Ggt promoter on the regulation of the expression of Ggt gene in the livers of streptozotocin (STZ)-induced moderate hypoinsulinemia rat model. The rats treated with STZ showed remarkably higher serum GGT level and hepatic Ggt/GGT expression than the untreated control rats. Furthermore, the acetylation of histones H3 and H4, and the binding of CBP not p300 at the Ggt promoter regions were significantly higher in the livers of STZ rats than those of the control rats. These results suggest that an enhanced hepatic expression of Ggt is associated with increased acetylation of histones H3 and H4 and CBP binding at the Ggt promoter in STZ-induced moderate hypoinsulinemic rats.
- MeSH
- acetylace MeSH
- experimentální diabetes mellitus enzymologie genetika MeSH
- gama-glutamyltransferasa biosyntéza genetika MeSH
- histonacetyltransferasy metabolismus MeSH
- histony metabolismus MeSH
- játra enzymologie MeSH
- krysa rodu rattus MeSH
- potkani Wistar MeSH
- promotorové oblasti (genetika) MeSH
- protein p300 asociovaný s E1A metabolismus MeSH
- protein vázající CREB metabolismus MeSH
- zvířata MeSH
- Check Tag
- krysa rodu rattus MeSH
- mužské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
BACKGROUND: Epigenetic modifications including DNA methylation and post-translational modifications of histones are known to regulate gene expression. Antagonistic activities of histone acetyltransferases (HATs) and histone deacetylases (HDACs) mediate transcriptional reprogramming during insect development as shown in Drosophila melanogaster and other insects. Juvenile hormones (JH) play vital roles in the regulation of growth, development, metamorphosis, reproduction and other physiological processes. However, our current understanding of epigenetic regulation of JH action is still limited. Hence, we studied the role of CREB binding protein (CBP, contains HAT domain) and Trichostatin A (TSA, HDAC inhibitor) on JH action. RESULTS: Exposure of Tribolium castaneum cells (TcA cells) to JH or TSA caused an increase in expression of Kr-h1 (a known JH-response gene) and 31 or 698 other genes respectively. Knockdown of the gene coding for CBP caused a decrease in the expression of 456 genes including Kr-h1. Interestingly, the expression of several genes coding for transcription factors, nuclear receptors, P450 and fatty acid synthase family members that are known to mediate JH action were affected by CBP knockdown or TSA treatment. CONCLUSIONS: These data suggest that acetylation and deacetylation mediated by HATs and HDACs play an important role in JH action.
- MeSH
- acetylace MeSH
- dvouvláknová RNA metabolismus MeSH
- epigeneze genetická účinky léků MeSH
- hmyzí proteiny antagonisté a inhibitory genetika metabolismus MeSH
- kyseliny hydroxamové farmakologie MeSH
- protein vázající CREB antagonisté a inhibitory genetika metabolismus MeSH
- RNA interference MeSH
- Tribolium účinky léků růst a vývoj metabolismus MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
Juvenile hormones (JH) and ecdysteroids regulate many biological and metabolic processes. CREB-binding protein (CBP) is a transcriptional co-regulator with histone acetyltransferase (HAT) activity. Therefore, CBP is involved in activation of many transcription factors that regulate expression of genes associated with postembryonic development in insects. However, the function of CBP in JH action in insects is not well understood. Hence, we studied the role of CBP in JH action in the red flour beetle, Tribolium castaneum and the Tribolium cell line. CBP knockdown caused a decrease in JH induction of genes, Kr-h1, 4EBP and G13402 in T. castaneum larvae, adults and TcA cells whereas, Trichostatin A [TSA, a histone deacetylase (HDAC) inhibitor] induced the expression of these JH-response genes. Western blot analysis with specific antibodies revealed the requirement of CBP for the acetylation of H3K18 and H3K27 in both T. castaneum and TcA cells. Chromatin immunoprecipitation (Chip) assays showed the importance of CBP-mediated acetylation of H3K27 for JH induction of Kr-h1, 4EBP, and G13402 in TcA cells. These data suggest that CBP plays an important role in JH action in the model insect, T.castaneum.
- MeSH
- acetylace MeSH
- genový knockout MeSH
- histony metabolismus MeSH
- hmyzí proteiny genetika metabolismus MeSH
- juvenilní hormony farmakologie MeSH
- protein vázající CREB genetika metabolismus MeSH
- Tribolium genetika růst a vývoj metabolismus MeSH
- vývojová regulace genové exprese účinky léků MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- Research Support, N.I.H., Extramural MeSH
- Research Support, U.S. Gov't, Non-P.H.S. MeSH
BACKGROUND: Juvenile hormones (JH) and ecdysteroids control postembryonic development in insects. They serve as valuable targets for pest management. Hence, understanding the molecular mechanisms of their action is of crucial importance. CREB-binding protein (CBP) is a universal transcriptional co-regulator. It controls the expression of several genes including those from hormone signaling pathways through co-activation of many transcription factors. However, the role of CBP during postembryonic development in insects is not well understood. Therefore, we have studied the role of CBP in postembryonic development in Tribolium, a model coleopteran insect. RESULTS: CBP is ubiquitously expressed in the red flour beetle, Tribolium castaneum. RNA interference (RNAi) mediated knockdown of CBP resulted in a decrease in JH induction of Kr-h1 gene expression in Tribolium larvae and led to a block in their development. Moreover, the injection of CBP double-stranded RNA (dsRNA) showed lethal phenotypes within 8 days of injection. RNA-seq and subsequent differential gene expression analysis identified CBP target genes in Tribolium. Knockdown of CBP caused a decrease in the expression of 1306 genes coding for transcription factors and other proteins associated with growth and development. Depletion of CBP impaired the expression of several JH response genes (e.g., Kr-h1, Hairy, early trypsin) and ecdysone response genes (EcR, E74, E75, and broad complex). Further, GO enrichment analyses of the downregulated genes showed enrichment in different functions including developmental processes, pigmentation, anatomical structure development, regulation of biological and cellular processes, etc. CONCLUSION: These data suggest diverse but crucial roles for CBP during postembryonic development in the coleopteran model insect, Tribolium. It can serve as a target for RNAi mediated pest management of this stored product pest.
- MeSH
- exprese genu MeSH
- hmyzí proteiny antagonisté a inhibitory genetika metabolismus fyziologie MeSH
- juvenilní hormony farmakologie MeSH
- larva genetika metabolismus MeSH
- protein vázající CREB antagonisté a inhibitory genetika metabolismus fyziologie MeSH
- RNA interference MeSH
- Tribolium genetika růst a vývoj metabolismus MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- Research Support, N.I.H., Extramural MeSH
- Research Support, U.S. Gov't, Non-P.H.S. MeSH
A number of transactivation domains for transcription factors including p53, E2A/HEB, MLL, cMyb, CREB, FOXO3, Gcn4, Oaf1 and Pdr1 have been reported to interact with the KIX domain of general transcriptional mediators CBP, p300 or MED15. Most of those factors belong to the already established Nine amino acid Transactivation Domain (9aaTAD) family. By using available structural data, we found binding analogy for the 9aaTAD in the MLL-KIX and also E2A/HEB-KIX complexes. We recognized two distinct TAD formations in the KIX complex. In the E2A/HEB-KIX complex, the leucine position is determined by the prolonged helical structure including the 9aaTAD and the leucine (long-helical TAD). However in the MLL-KIX complex, the equal position of 9aaTAD and proximal leucine is achieved differently by leucine-turn-helix structural architecture. Furthermore, the FOXO3-KIX complex shares structural analogy with the E2A-KIX complex in respect of both 9aaTAD and proximal leucine. Next, from (i) sequence alignment of the identified 9aaTADs in p53, E2A/HEB and MLL proteins and (ii) the resolved structure of the MLL-KIX and E2A/HEB-KIX complexes, we generated a plausible structural model for p53 that could be used also for other members of the 9aaTAD family. The position of 9aaTADs in Oaf1-, Pdr1- and Gcn4-MED15 KIX complexes and 9aaTAD composition are in good agreement with E2A, MLL, FOXO3 and p53. Analyses of structural data in this study define fundamental structural requirements and shed more light on the ambiguous 9aaTAD domain.
- MeSH
- aminokyselinové motivy MeSH
- databáze proteinů MeSH
- forkhead transkripční faktory chemie metabolismus MeSH
- interakční proteinové domény a motivy * MeSH
- molekulární konformace * MeSH
- molekulární modely * MeSH
- nádorový supresorový protein p53 chemie metabolismus MeSH
- protein vázající CREB chemie metabolismus MeSH
- protoonkogenní protein MLL chemie MeSH
- sekvence aminokyselin MeSH
- transkripční faktory chemie metabolismus MeSH
- vazba proteinů MeSH
- vazebná místa MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Prolactin-releasing peptide (PrRP)-induced secretion of prolactin is not currently considered a primary function of PrRP, but the development of late-onset obesity in both PrRP and PrRP receptor knock-out mice indicates the unique anorexigenic properties of PrRP. In our recent study, we showed comparable potencies of peptides PrRP31 and PrRP20 in binding, intracellular signaling and prolactin release in pituitary RC-4B/C cells, and anorexigenic effect after central administration in fasted mice. In the present study, eight analogs of PrRP20 with C-terminal Phe amide modified with a bulky side chain or a halogenated aromatic ring revealed high binding potency, activation of mitogen-activated protein kinase/extracellular-regulated kinase (MAPK/ERK1/2) and cAMP response element-binding protein (CREB) and prolactin release in RC-4B/C cells. In particular, [PheNO(2)(31)]PrRP20, [1-Nal(31)]PrRP20, [2-Nal(31)]PrRP20 and [Tyr(31)]PrRP20 showed not only in vitro effects comparable or higher than those of PrRP20, but also a very significant and long-lasting anorexigenic effect after central administration in fasted mice. The design of potent and long-lasting PrRP analogs with selective anorexigenic properties promises to contribute to the study of food intake disorders.
- MeSH
- anorektika aplikace a dávkování farmakologie MeSH
- fenylalanin analogy a deriváty metabolismus MeSH
- fosforylace MeSH
- hormon uvolňující prolaktin aplikace a dávkování analogy a deriváty farmakologie MeSH
- krysa rodu rattus MeSH
- mitogenem aktivované proteinkinasy metabolismus MeSH
- myši inbrední C57BL MeSH
- myši MeSH
- nádorové buněčné linie MeSH
- omezení příjmu potravy fyziologie MeSH
- přijímání potravy účinky léků fyziologie MeSH
- prolaktin metabolismus MeSH
- protein vázající CREB metabolismus MeSH
- receptory neuropeptidů metabolismus MeSH
- receptory spřažené s G-proteiny metabolismus MeSH
- signální transdukce MeSH
- vazba proteinů MeSH
- vztahy mezi strukturou a aktivitou MeSH
- western blotting MeSH
- zvířata MeSH
- Check Tag
- krysa rodu rattus MeSH
- mužské pohlaví MeSH
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
