Ternary complexes and cooperative interplay between NCoA-62/Ski-interacting protein and steroid receptor coactivators in vitamin D receptor-mediated transcription.

The vitamin D receptor (VDR) is a ligand-dependent transcriptional factor that binds to vitamin D-responsive elements as a heterodimer with retinoid X receptor (RXR) to regulate target gene transcription. The steroid receptor coactivator (SRC) proteins are coactivators that interact with the AF-2 domain of VDR to augment 1,25-dihydroxyvitamin D3-dependent transcription. In contrast, NCoA-62/Ski-interacting protein (SKIP) is a distinct, activation function-2-independent coactivator for VDR. The current study examined whether these two distinct classes of coactivators impact functionally on VDR-mediated transcription. Using a ternary complex binding assay, we observed a marked preference for the direct interaction of NCoA-62/SKIP with the VDR-RXR heterodimer as compared with the VDR-VDR homodimer or VDR monomer. The liganded VDR also formed a ternary complex with NCoA-62/SKIP and SRC proteins in vitro. Competition experiments using LXXLL peptides showed that NCoA-62/SKIP and SRC coactivators contact different domains of the VDR-RXR heterodimer. Synergistic interplays were observed between NCoA-62/SKIP and SRC coactivators in VDR-mediated transcriptional assays, and protein interference assays indicated a requirement for both NCoA-62/SKIP and SRCs in VDR- mediated transcription. These studies suggest that the ligand-dependent and simultaneous interaction of NCoA-62/SKIP and SRC coactivators with distinct interaction domains within the VDR-RXR heterodimer results in cooperative interplays between coactivators in VDR-mediated transcription.

Vitamin D receptor and nuclear receptor coactivators: crucial interactions in vitamin D-mediated transcription.

The nuclear actions of 1,25-dihydroxyvitamin D(3) [1alpha,25(OH)(2)D(3)] are mediated by the vitamin D receptor (VDR). Binding of ligand induces conformational changes in the VDR which promote heterodimerization with retinoid X receptor (RXR) and recruitment of a number of nuclear receptor coactivator proteins including the steroid receptor coactivator (SRC) family members, select SMAD proteins, a novel coactivator complex referred to as DRIP, and a variety of other putative factors. We recently described a novel nuclear receptor coactivator termed NCoA-62 that interacts with the VDR to enhance 1alpha,25(OH)(2)D(3)-activated transcription. NCoA-62 is unrelated to the SRC family, the DRIP complex, as well as other nuclear receptor coactivators described thus far. The molecular mechanisms involved in NCoA-62 coactivator function are poorly understood, but protein-protein interactions are likely to play an important role. The purpose of this paper is to briefly review salient features of the coactivators involved in VDR-activated transcription and to focus on our current understanding of NCoA-62 and its interplay with other nuclear receptor coactivator proteins. It is clear from the studies described here that a concerted series of interactions with multiple coactivator proteins are essential for high order transactivation by 1alpha,25(OH)(2)D(3) and the VDR.

Isolation and characterization of a novel coactivator protein, NCoA-62, involved in vitamin D-mediated transcription.

The vitamin D receptor (VDR) forms a heterodimeric complex with retinoid X receptor (RXR) and binds to vitamin D-responsive promoter elements to regulate the transcription of specific genes or gene networks. The precise mechanism of transcriptional regulation by the VDR.RXR heterodimer is not well understood, but it may involve interactions of VDR.RXR with transcriptional coactivator or corepressor proteins. Here, a yeast two-hybrid strategy was used to isolate proteins that selectively interacted with VDR and other nuclear receptors. One cDNA clone designated NCoA-62, encoded a 62, 000-Da protein that is highly related to BX42, a Drosophila melanogaster nuclear protein involved in ecdysone-stimulated gene expression. Yeast two-hybrid studies and in vitro protein-protein interaction assays using glutathione S-transferase fusion proteins demonstrated that NCoA-62 formed a direct protein-protein contact with the ligand binding domain of VDR. Coexpression of NCoA-62 in a vitamin D-responsive transient gene expression system augmented 1, 25-dihydroxyvitamin D3-activated transcription, but it had little or no effect on basal transcription or gal4-VP16-activated transcription. NCoA-62 also interacted with retinoid receptors, and its expression enhanced retinoic acid-, estrogen-, and glucocorticoid-mediated gene expression. These data indicate that NCoA-62 may be classified into an emerging set of transcriptional coactivator proteins that function to facilitate vitamin D- and other nuclear receptor-mediated transcriptional pathways.

A peptidomimetic that specifically inhibits human leukocyte antigen DRB1*0401-restricted T cell proliferation.

The ability of a peptidomimetic (SC-67655) to block the peptide binding site of the rheumatoid arthritis-linked human leukocyte antigen encoded by the DRB1*0401 allele was evaluated. The inhibitor bound to purified DRB1*0401 molecules with an affinity similar to that of the well-characterized peptide ligand HA307-319. Cell binding assays demonstrated that, in contrast to the promiscuous HA307-319 peptide, the peptidomimetic was highly specific for DRB1*0401. The inhibitor also blocked functional T cell responses to peptide antigens but did not block T cell proliferation in response to protein antigens. Furthermore, it did not appear to be taken up by cells. An analog of the peptidomimetic that was conjugated to a signal peptide sequence did inhibit a T cell proliferative response to protein antigen. Thus, the peptidomimetic must be taken up by cells to block the presentation of peptides derived from protein antigens. These findings have implications for the rational development of inhibitors that block the class II peptide binding groove for the treatment of autoimmune diseases.

Negatively charged residues interacting with the p4 pocket confer binding specificity to DRB1*0401.

OBJECTIVE: To identify critical residues involved in the binding of a selective peptide to DRB1*0401. METHODS: The binding of peptides to native or site-directed mutant DR molecules was evaluated using enzyme-linked immunosorbent assay and flow cytometry. RESULTS: Amino acid substitutions at DR and peptide residues, which were predicted to contribute to interactions within the DR p4 pocket, had the greatest effects on the specificity of binding. CONCLUSION: Differences in the peptide-binding repertoires of DR molecules may contribute to associations with autoimmune diseases.