Molecular basis for repression of liver X receptor-mediated gene transcription by receptor-interacting protein 140.

Similarities in physiological roles of LXR (liver X receptors) and co-repressor RIP140 (receptor-interacting protein 140) in regulating energy homoeostasis and lipid and glucose metabolism suggest that the effects of LXR could at least partly be mediated by recruitment of the co-repressor RIP140. In the present study, we have elucidated the molecular basis for regulation of LXR transcriptional activity by RIP140. LXR is evenly localized in the nucleus and neither the N-terminal domain nor the LBD (ligand-binding domain) is necessary for nuclear localization. Both LXR subtypes, LXRalpha and LXRbeta, interact with RIP140 and co-localize in diffuse large nuclear domains. Interaction and co-localization are dependent on the LBD of the receptor. The C-terminal domain of RIP140 is sufficient for full repressive effect. None of the C-terminal NR (nuclear receptor)-boxes is required for the co-repressor activity, whereas the NR-box-like motif as well as additional elements in the C-terminal region are required for full repressive function. The C-terminal NR-box-like motif is necessary for interaction with LXRbeta, whereas additional elements are needed for strong interaction with LXRalpha. In conclusion, our results suggest that co-repression of LXR activity by RIP140 involves an atypical binding mode of RIP140 and a repression element in the RIP140 C-terminus.

Deletion of the V1/V2 region does not increase the accessibility of the V3 region of recombinant gp125.

Previous analyses of HIV-1 surface glycoprotein indicate that both the V1/V2 region and the interaction of gp120 with CD4 influence the accessibility of the V3 region on gp120. In this study we investigated the accessibility of the V3 region of HIV-2 recombinant gp125 proteins using V3-specific mAbs (7C8 and 3C4) and analyzed the binding kinetics of soluble CD4 (sCD4) to recombinant HIV-1 gp120 and HIV-2 gp125 proteins by surface plasmon resonance (SPR) analysis. Our results indicated that 7C8 recognized monomers of gp125 and gp125Delta v1v2, (lacking the V1/V2 region) while 3C4 was sensitive to the conformation of gp125, recognizing only oligomers of gp125Delta v1v2. Furthermore, SPR analysis of 7C8 binding to gp125 demonstrated that the deletion of the V1/V2 region did not increase the accessibility of the V3 region in gp125Delta v1v2. Comparative SPR analyses of sCD4 binding HIV recombinant surface glycoproteins revealed a lower affinity of sCD4 to gp125 as compared to gp120. Moreover, the analyses suggest that conformational changes only occur in HIV-1 gp120 upon interaction with CD4. We hypothesize that the V3 region is accessible in HIV-2 gp125 and thus may not require interaction with CD4 to induce conformational reorientation of the V1/V2 region.

Cloning, expression, and purification of HIV-2 gp125: A target for HIV vaccination.

The envelope of the human immunodeficiency virus (HIV) is the main target for neutralizing antibodies. We report the cloning, purification, and characterization of two recombinant forms of the envelope glycoprotein gp125 from a primary HIV-2SBL-6669 isolate. Both constructs were truncated at the N- and C-termini, and in the gp125deltav1v2 construct the variable V1 and V2 loops were deleted. The recombinant glycoproteins were stably expressed in Chinese hamster ovarian cells, producing soluble gp125 and gp125deltav1v2 at molecular weights of 74.2 and 56.9 kDa, respectively, and were purified from cell culture supernatants in a single step using Galanthus nivalis lectin chromatography. Circular dichroism analysis indicated a similar secondary structure for gp125 and gp125deltav1v2, and both proteins were recognized by HIV-2 serum antibodies in surface plasmon resonance assays. The high yield and purity of these constructs makes them suitable for structural and functional analyses, as well as vaccine studies.

Activation functions 1 and 2 of nuclear receptors: molecular strategies for transcriptional activation.

Nuclear receptors (NRs) comprise a family of ligand inducible transcription factors. To achieve transcriptional activation of target genes, DNA-bound NRs directly recruit general transcription factors (GTFs) to the preinitiation complex or bind intermediary factors, so-called coactivators. These coactivators often constitute subunits of larger multiprotein complexes that act at several functional levels, such as chromatin remodeling, enzymatic modification of histone tails, or modulation of the preinitiation complex via interactions with RNA polymerase II and GTFs. The binding of NR to coactivators is often mediated through one of its activation domains. Many NRs have at least two activation domains, the ligand-independent activation function (AF)-1, which resides in the N-terminal domain, and the ligand-dependent AF-2, which is localized in the C-terminal domain. In this review, we summarize and discuss current knowledge regarding the molecular mechanisms of AF-1- and AF-2-mediated gene activation, focusing on AF-1 and AF-2 conformation and coactivator binding.

Spontaneous mutations in the human CMV HLA class I homologue UL18 affect its binding to the inhibitory receptor LIR-1/ILT2/CD85j.

Human cytomegalovirus (HCMV) down-regulates cell surface expression of HLA class I molecules (HLA-I). UL18, an HCMV-encoded HLA-I homologue, has been proposed to protect virus-infected cells against NK cell recognition by engaging the inhibitory receptor leukocyte Ig-like receptor (LIR)-1, which also binds a broad spectrum of HLA-I alleles, including HLA-G1. Because genetic and biological differences exist among HCMV strains, we characterized laboratory (AD169) and clinical (4636, 13B, 109B) strain-derived UL18 proteins. Compared to the known AD169-derived UL18, mutations were found in clinical strain-derived UL18. They were clustered in the alpha3 domain (13B), previously shown to be critical for LIR-1 binding, or in the alpha1 domain (4636). Iotan cytotoxicity assays, pretreatment of LIR-1+ NKL with soluble 4636-UL18 completely abolished LIR-1-dependent protection from NK lysis, conferred by the expression of HLA-G1 on target cells (721.221-HLA-G1+). Similarly, flow cytometry, Biacore and ELISA experiments showed 4636-UL18 and 13B-UL18 to have the strongest binding capacity to LIR-1. Our results suggest the importance of two independent UL18 regions for LIR-1 binding, one localized on the tip of the alpha3 domain, and another composed of two loops that emerge from the alpha1 domain. Strain variations in these domains may result in different UL18-mediated effects on LIR-1+ cells during the course of HCMV infection.

Selection of DNA aptamers against rat liver X receptors.

Liver X receptors alpha and beta (LXRalpha; LXRbeta) are members of the nuclear hormone receptor superfamily of ligand-activated transcription factors. LXRs play an important role in the reverse cholesterol transport and govern the expression of many of the proteins that are indispensable for the regulation of normal cholesterol levels in the body. SELEX, an in vitro selection technology, was used on a single stranded DNA library harboring a 12 randomized nucleotide sequence in order to isolate aptamers showing affinity for LXRalpha. Enzyme-linked assays and surface plasmon resonance measurements showed that the selected aptamers had strong affinities for LXRalpha with apparent dissociation constants, K(d)s, in nanomolar range. All clones carried CG-repeats, indicating a probability for a similar manner of binding to LXRalpha. Very high cross-reactivities were observed when testing the aptamers with LXRbeta (up to 700%) and RXRalpha (up to 50%). If instead we regard the aptamer sequences as selected against LXRbeta, the cross-reactivities decrease considerably, to 17% for LXRalpha and 7% for RXRalpha. Therefore, in the future we are planning to use the obtained aptamers as binders for LXRbeta.

Interaction of transcriptional intermediary factor 2 nuclear receptor box peptides with the coactivator binding site of estrogen receptor alpha.

The activation function 2/ligand-dependent interaction between nuclear receptors and their coregulators is mediated by a short consensus motif, the so-called nuclear receptor (NR) box. Nuclear receptors exhibit distinct preferences for such motifs depending both on the bound ligand and on the NR box sequence. To better understand the structural basis of motif recognition, we characterized the interaction between estrogen receptor alpha and the NR box regions of the p160 coactivator TIF2. We have determined the crystal structures of complexes between the ligand-binding domain of estrogen receptor alpha and 12-mer peptides from the Box B2 and Box B3 regions of TIF2. Surprisingly, the Box B3 module displays an unexpected binding mode that is distinct from the canonical LXXLL interaction observed in other ligand-binding domain/NR box crystal structures. The peptide is shifted along the coactivator binding site in such a way that the interaction motif becomes LXXYL rather than the classical LXXLL. However, analysis of the binding properties of wild type NR box peptides, as well as mutant peptides designed to probe the Box B3 orientation, suggests that the Box B3 peptide primarily adopts the "classical" LXXLL orientation in solution. These results highlight the potential difficulties in interpretation of protein-protein interactions based on co-crystal structures using short peptide motifs.