Functional studies on the ligand binding domain of Ultraspiracle from Drosophila melanogaster

Functional studies on the ligand-binding domain of Ultraspiracle from Drosophila melanogaster. Biol. Chem., 385,[21][22][23][24][25][26][27][28][29][30]. Abstract: The functional insect ecdysteroid receptor is comprised of the ecdysone receptor (EcR) and Ultraspiracle (USP). The ligand-binding domain (LBD) of USP was fused to the GAL4 DNA-binding domain (GAL4-DBD) and char-acterized by analyzing the effect of site-directed mutations in the LBD. Normal and mutant proteins were tested for ligand and DNA binding, dimerization, and their ability to induce gene expression. The presence of helix 12 proved to be essential for DNA binding and was necessary to confer efficient ecdysteroid binding to the hetero-dimer with the EcR (LBD), but did not influence dimerization. The antagonistic position of helix 12 is indispensible for interaction between the fusion protein and DNA, whereas hormone binding to the EcR (LBD) was only partially reduced if fixation of helix 12 was disturbed. The mutation of amino acids, which presumably bind to a fatty acid evoked a profound negative influence on transactivation ability, although enhanced transactivation potency and ligand binding to the ecdysteroid receptor was impaired to varying degrees by mutation of these residues. Mutations of one fatty acid-binding residue within the ligand-binding pocket, I323, however, evoked enhanced transactivation. The results confirmed that the LBD of Ultraspiracle modifies ecdysteroid receptor function through intermolecular interactions and demonstrated that the ligandbinding pocket of USP modifies the DNA-binding and transactivation abilities of the fusion protein. Keywords: dimerization; DNA binding; ecdysteroid; nuclear receptor; transactivation; two-hybrid assay. Article: Introduction The ligand-binding domain (LBD) of nuclear receptors participates in several functions: ligand binding, dimerization, hormonal regulation of transactivation and inter-action with comodulators. Among insects, two nuclear receptors, the ecdysone receptor (EcR) and Ultraspiracle (USP), an ortholog of the vertebrate RXR, form the functional ecdysteroid receptor. The interaction of 20-hydroxy ecdysone with this heterodimer sets off the transcription-al changes associated with insect larval and metamorphic development. Previous studies have conclusively demonstrated that ecdysteroids bind only to EcR, and there is less certainty about what ligand, if any, interacts with the LBD of USP. Other studies have shown that USP physically interacts with juvenile hormone III (JHIII) and can be induced by JHIII with specific promoters The combination of LBDs including the C-terminal part of the hinge region of EcR and USP show the same ligand-binding properties as reported for full-length receptors of Drosophila melanogaster (Grebe et al., unpublished

IL36 is a critical upstream amplifier of neutrophilic lung inflammation in mice

IL-36, which belongs to the IL-1 superfamily, is increasingly linked to neutrophilic inflammation. Here, we combined in vivo and in vitro approaches using primary mouse and human cells, as well as, acute and chronic mouse models of lung inflammation to provide mechanistic insight into the intercellular signaling pathways and mechanisms through which IL-36 promotes lung inflammation. IL-36 receptor deficient mice exposed to cigarette smoke or cigarette smoke and H1N1 influenza virus had attenuated lung inflammation compared with wild-type controls. We identified neutrophils as a source of IL-36 and show that IL-36 is a key upstream amplifier of lung inflammation by promoting activation of neutrophils, macrophages and fibroblasts through cooperation with GM-CSF and the viral mimic poly(I:C). Our data implicate IL-36, independent of other IL-1 family members, as a key upstream amplifier of neutrophilic lung inflammation, providing a rationale for targeting IL-36 to improve treatment of a variety of neutrophilic lung diseases