Millisecond to microsecond time scale dynamics of the retinoid X and retinoic acid receptor DNA-binding domains and dimeric complex formation.

The all-trans retinoic acid and 9-cis retinoic acid receptors (RAR and RXR, respectively) belong to a family of ligand inducible transcription factors, which exert their effect via binding to hormone response elements. Both are members of the class II sub-family of nuclear receptors, which bind DNA as dimers, on tandem repeats of a hexamer motif separated by a variable spacer. The variability in spacer length and the head-to-tail organization of the hormone response elements result in different protein-protein interactions in each of the complexes. We show that the zinc-coordinating loop regions of RXR and RAR DNA-binding domains exhibit dynamics on the millisecond to microsecond time scale. The highly dynamic second zinc finger of RXR constitutes the primary protein-protein interface in many nuclear receptor assemblies on DNA. Dynamics is also observed in the first and second zinc fingers of RAR, which are implicated in dimeric interactions with RXR on response elements with spacers of 5 base pairs and 1 base pair, respectively. The striking correspondence between the regions that exhibit conformational exchange and the dimer interfaces of the proteins complexed with DNA suggests a functional role for the dynamics. The observed flexibility may allow the proteins to adapt to various partners and with different orientations upon assembly on DNA. Furthermore, the more extensive dynamics observed for RXR may reflect the greater ability of this protein to modulate its interaction surface since it participates in a wide variety of receptor complexes.

A dual mechanism mediates repression of NF-kappaB activity by glucocorticoids.

Repression of nuclear factor (NF)-kappaB-dependent gene expression is one of the key characteristics by which glucocorticoids exert their antiinflammatory and immunosuppressive effects. In vitro studies have shown protein-protein interactions between NF-kappaB and the glucocorticoid receptor, possibly explaining their mutual repression of transcriptional activity. Furthermore, glucocorticoid-induced transcription of IkappaBalpha was presented as a mechanism in mediation of immunosuppression by glucocorticoids. At present, the relative contribution of each mechanism has not been investigated. We show that dexamethasone induced IkappaBalpha gene transcription in human pulmonary epithelial A549 cells. However, this enhanced IkappaBalpha synthesis did not cause repression of NF-kappaB DNA-binding activity. In addition, dexamethasone was still able to inhibit the expression of NF-kappaB target genes (cyclooxygenase-2, intercellular adhesion molecule-1) in the absence of protein synthesis. Furthermore, we show that the antihormone RU486 did not induce IkappaBalpha expression. However, RU486 was still able to induce, albeit less efficiently, both glucocorticoid- and progesterone receptor-mediated repression of endogenous NF-kappaB target gene expression in A549 cells and the breast cancer cell line T47D, respectively. Taken together, these results indicate that induced IkappaBalpha expression accounts for only part of the repression of NF-kappaB activity by glucocorticoids and progestins. In addition, protein-protein interactions between NF-kappaB and the glucocorticoid or progesterone receptor, resulting in repression of NF-kappaB activity, seem also to be involved. We therefore conclude that NF-kappaB activity is repressed via a dual mechanism involving both protein-protein interactions and induction of IkappaBalpha.

Distinct domains of the RelA NF-kappaB subunit are required for negative cross-talk and direct interaction with the glucocorticoid receptor.

The RelA subunit of NF-kappaB and the glucocorticoid receptor mutually repress each others transcriptional activity, thus providing a mechanism for immunosuppression. Deletion analysis of the glucocorticoid receptor has shown that the DNA binding domain and the ligand binding domain are essential components for repression. Here, we show by deletions and point mutations that both the Rel homology domain and the transactivation domains of RelA are required for repression of the transcriptional activity of the glucocorticoid receptor in intact cells. However, only the Rel homology domain of RelA was found to associate with the glucocorticoid receptor in vitro. RelA mutants, not able to repress glucocorticoid receptor activity, but still able to dimerize, behaved as transdominant inhibitors of the repressive activity of wild type RelA. Furthermore, we show that the 13 S E1A protein is able to interfere with the transrepressive activity of RelA. We propose that negative cross-talk between the glucocorticoid receptor and RelA is due to direct interaction via the Rel homology domain of RelA and the DNA binding domain of the glucocorticoid receptor in combination with interference by the transactivation domains of RelA with the transcriptional activity of the glucocorticoid receptor.

Activation function 1 of retinoic acid receptor beta 2 is an acidic activator resembling VP16.

The mechanisms underlying transcriptional activation are not very well understood, and knowledge is based on experiments with a small number of mostly viral activators. We have investigated the mechanism underlying transactivation by the activation domain present in the N-terminal part of retinoic acid receptor (RAR) beta 2 (AF-1). We show that RAR beta 2 phosphorylation is not crucial for its activity although it may modulate AF-1 activity. Sequential mutation of the negatively charged residues (Asp) resulted in a stepwise decrease in activity, while mutation of all aspartic acid residues resulted in complete loss of activity. Comparison of the critical region for activation with other activators revealed moderate homology with the viral activator VP16. The hydrophobic amino acids surrounding the negatively charged residues reported to be critical for activation by VP16 are all conserved in AF-1. The hydrophobic residues are required for AF-1, since mutation of these residues resulted in a decrease in activity. Furthermore, the activity of this activator, VP16 and TA1 of RelA, is squelched by overexpression of an AF-1-containing expression construct, indicating that AF-1 is an acidic activator. Squelching experiments further indicate that AF-1 and AF-2 function by different mechanisms. Comparison of activation functions present in the AB region of other members of the steroid/thyroid hormone receptor family: RAR alpha 2, RAR gamma 2, and GR suggested that also these receptors contain an acidic activation domain. The mechanism underlying activation by AF-1 is discussed.

Gene mapping and expression of two immunodominant Epstein-Barr virus capsid proteins.

The genomic localization of two immunodominant genes encoding two proteins of the Epstein-Barr virus capsid antigen (VCA) complex, VCA-p18 and VCA-p40, has been identified. For that purpose, lambda gt11-based cDNA libraries were constructed from HH514.c16 cells induced for virus production. The libraries were screened with a monoclonal antibody, EBV.OT41A, directed against VCA-p40 or with affinity-purified human antibodies against VCA-p18. Sequencing of the inserts of positive plaques showed that VCA-p18 and VCA-p40 are encoded within open reading frames (ORFs) BFRF3 and BdRF1, respectively. Peptide scanning analysis of the predicted protein of ORF BdRF1 resulted in defining the epitope of monoclonal antibody EBV.OT41A at the C-terminal region. The dominant VCA-p18 reactivity of human sera can be completely inhibited by preadsorption with Escherichia coli-expressed BFRF3-beta-galactosidase. Serum of a rabbit immunized with BFRF3-beta galactosidase reacts with a VCA-specific protein of 18 kDa. In addition, BFRF3-beta-galactosidase affinity-purified antibodies react with VCA-p18 of virus-producing cells (HH514.c16). Complete inhibition of viral DNA polymerase activity by phosphonoacetic acid is associated with the absence of RNAs and protein products of both ORFs, indicating that VCA-p18 and VCA-p40 are true late antigens.