Disease relevance of usp

• We describe here the expression of the CF1/USP protein in an E. coli T7 RNA polymerase-driven expression system [1].

High impact information on usp

• DHR38 also heterodimerizes with USP, and this complex responds to a distinct class of ecdysteroids in a manner that is independent of EcR [2].
• This response is unusual in that it does not involve direct binding of ecdysteroids to either DHR38 or USP [2].
• We now demonstrate that ultraspiracle (usp), a Drosophila RXR homolog, can substitute for RXR in stimulating the DNA binding of receptors for retinoic acid, T3, vitamin D, and peroxisome proliferator activators [3].
• Replacement of USP with retinoid X receptor produces heterodimers with distinct pharmacological and functional properties [4].
• Here we report that native EcR and USP are co-localized on ecdysone-responsive loci of polytene chromosomes [4].

Biological context of usp

• Terminal filament morphogenesis is severely disrupted in homozygous usp clones [5].
• We have identified the regions of Drosophila EcR and USP responsible for transcriptional activation of a semisynthetic Eip71CD promoter in Kc cells [6].
• Mutagenesis reveals that AF2 function depends on EcR helix 12, but not on the cognate USP region [6].
• Among other phenotypes, loss of functional USP in Drosophila eye development results in an accelerated morphogenetic furrow, although loss of ecdysone arrests the furrow [7].
• A chimeric transgene (d/cusp), produced by replacing the ligand-binding domain (LBD) of Drosophila USP with the equivalent domain from another Diptera, Chironomus tentans, was tested for its ability to rescue Drosophila usp mutants from early larval lethality [8].

Anatomical context of usp

• Cell-autonomous requirement of the USP/EcR-B ecdysone receptor for mushroom body neuronal remodeling in Drosophila [9].
• In cells destined to form the basal stalks and anterior oviduct, USP colocalizes with what appears to be the EcR-B2 isoform [5].
• The imaginal discs in usp mutants begin to evert but do not elongate or differentiate, the larval midgut and salivary glands fail to undergo programmed cell death and the adult midgut fails to form [10].
• In gel mobility shift assays using fat body nuclear extracts or nuclear receptors synthesized in vitro, a 27-bp sequence harboring the EcRE bound both the Drosophila ecdysone receptor and the Drosophila retinoid-X homologue, Ultraspiracle, in a cooperative manner [11].
• We identified the ecdysteroid receptor components in this cell line, using monoclonal antibodies against the Drosophila ecdysteroid receptor (DmEcR) and ultraspiracle (DmUSP) proteins [12].

Associations of usp with chemical compounds

• In vitro experiments with cultured wing discs showed that BRC-Z1 expression and early metamorphic development are rendered steroid-independent in the usp mutant clones [13].
• Like, USP, it activates transcription in Drosophila Schneider S2 cells in a 20-hydroxyecdysone-dependent manner, via its interaction with the endogenous EcR protein [14].
• EcRB1 is normally induced with a form of USP that is missing its DNA-binding domain (DBD), although potentiation by juvenile hormone (JH) III is reduced [15].
• Substitution of E19, the most characteristic amino acid for estrogen receptor-like P-boxes, with alanine showed that the mutation did not appreciably alter the affinity of the wild-type Usp DNA-binding domain (UspDBD(WT)) for a probe containing natural ecdysone response element (hsp27(wt)) [16].
• Toward this end, we have expressed the EcR DNA-binding domain and the Usp DNA-binding domain as proteins with an affinity tag consisting of six histidine residues (6xHis-EcRDBD and 6xHis-UspDBD, respectively) using the expression vector pQE-30 [17].

Physical interactions of usp

• These results are consistent with a model in which these latter processes are induced by a signal arising during the middle of the last larval stage but suppressed by the unliganded EcR/USP complex [13].
• In fluorescence-based binding assays, USP protein binds JH III and JH III acid with specificity, adopting for each ligand a different final conformational state [18].
• Although members of the NGFI-B family are thought to function exclusively as monomers, we show that DHR38 and BHR38 in fact interact strongly with USP and that this interaction is evolutionarily conserved [19].
• We have determined the optimal binding site of partially purified bacterially produced CF1/USP by an in vitro selection procedure and also have characterized its binding to the follicular-specific chorion s15 promoter [20].

Regulatory relationships of usp

• JH III treatment of Saccharomyces cerevisiae expressing a LexA-USP fusion protein stabilizes an oligomeric association containing this protein, as detected by formation of a protein-DNA complex, and induces USP-dependent transcription in a reporter assay [18].
• Seven-up inhibits ultraspiracle-based signaling pathways in vitro and in vivo [21].

Other interactions of usp

• Surprisingly, most identified primary EcR/USP targets are dispensable for MB neuronal remodeling [9].
• In vivo footprinting experiments using ligation-mediated PCR and wild-type or ecdysteroid-deficient larvae show that occupancy of the Fbp1 EcR/USP binding site and adjacent region is dependent on a high concentration of ecdysteroids [22].
• Electrophoretic mobility shift assays indicated that CcEcR binds specifically to the Drosophila hsp27 ecdysone response element as a heterodimer with Drosophila USP, the product of the ultraspiracle gene [23].
• CF2 is a member of the C2H2 family of zinc finger proteins, whereas CF1 is a member of the family of steroid hormone receptors [24].
• Binding affinity of the Drosophila melanogaster CF1/USP protein to the chorion s15 promoter [1].

Analytical, diagnostic and therapeutic context of usp

• AF2 function was studied using a two-hybrid assay in Kc cells, based on the observation that potent hormone-dependent activation by the EcR/USP ligand-binding domain heterodimer requires the participation of both partners [6].
• We used in vivo, cell culture, and biochemical approaches to analyze the functions of two usp alleles, usp(3) and usp(4), which encode stable proteins with defective DNA-binding domains [25].
• Molecular cloning and expression analysis of ultraspiracle (USP) from the rice stem borer Chilo suppressalis [26].
• Biological activities have been determined in Drosophila melanogaster B(II) cell-based bioassay for ecdysteroid agonists and in an in vitro radioligand-displacement assay using bacterially-expressed D. melanogaster EcR/USP receptor proteins [27].
• Using an antibody raised against DmUSP, Western blot analysis of proteins from epidermis and other tissues revealed five immunoreactive bands, corresponding to different phosphorylated forms of a unique polypeptide, as shown by lambda-phosphatase treatment [28].

References