Cellular retinol-binding protein I is essential for vitamin A homeostasis.

The gene encoding cellular retinol (ROL, vitA)-binding protein type I (CRBPI) has been inactivated. Mutant mice fed a vitA-enriched diet are healthy and fertile. They do not present any of the congenital abnormalities related to retinoic acid (RA) deficiency, indicating that CRBPI is not indispensable for RA synthesis. However, CRBPI deficiency results in an approximately 50% reduction of retinyl ester (RE) accumulation in hepatic stellate cells. This reduction is due to a decreased synthesis and a 6-fold faster turnover, which are not related to changes in the levels of RE metabolizing enzymes, but probably reflect an impaired delivery of ROL to lecithin:retinol acyltransferase. CRBPI-null mice fed a vitA-deficient diet for 5 months fully exhaust their RE stores. Thus, CRBPI is indispensable for efficient RE synthesis and storage, and its absence results in a waste of ROL that is asymptomatic in vitA-sufficient animals, but leads to a severe syndrome of vitA deficiency in animals fed a vitA-deficient diet.

Versatile copurification procedure for rapid isolation of homogeneous RAR-RXR heterodimers.

RAR-RXR heterodimeric complexes (RARalphaDeltaAB-RXRalphaDeltaAB) bound to cognate DR5 DNA response elements were purified to apparent structural and functional homogeneity using a novel versatile immobilized metal affinity chromatography (IMAC) copurification procedure. Dynamic light scattering studies indicated that the complexes were more than 85% monodisperse. By electron microscopy the negatively stained RAR-RXR-DNA complexes appeared homogeneous and corresponded to a dimeric arrangement of the molecules. Using heterodimers purified according to this procedure we demonstrate ligand binding of RXR in the context of the RAR-RXR heterodimer-DNA complex. The present copurification procedure is rapid and has yielded high quality heterodimer-DNA complexes suitable for both structural and biochemical studies.

Surface-enhanced Raman scattering and fluorescence spectroscopy reveal molecular interactions of all-trans retinoic acid and RAR gamma ligand-binding domain.

Surface-enhanced Raman scattering and fluorescence were used to investigate the interactions of all-trans retinoic acid with the gamma-type retinoic acid receptor. Raman data revealed a significant attenuation in intensity of the bands originating from the retinoic acid polyenic chain upon receptor binding, with the spectrum being dominantly that of the beta-ionone ring. Fluorescence measurements supported the hydrophobic character of the ligand binding. These novel spectroscopic results are fully consistent with the published X-ray crystallographic data and suggest that these techniques may be valuable additional tools to characterize the interactions of agonists and antagonists with residues in the ligand-binding pockets of retinoid receptor homo- and heterodimers.

Purification of the human RARgamma ligand-binding domain and crystallization of its complex with all-trans retinoic acid.

A 28-kDa fragment (residues 178-423) of the human retinoic acid receptor gamma, hRARgamma D3E, encompassing the ligand-binding domain (LBD) was overproduced in Escherichia coli and purified as a monomer to more than 95% purity and homogeneity. The Kd for all-trans retinoic acid binding was 0.6 +/- 0.1 nM. Crystals of the LBD complexed with all-trans retinoic acid were grown at pH 7 from sodium acetate in the presence of detergents using the vapor diffusion method. They diffract to 2.0 A using a synchrotron radiation (lambda=0.91 A) and belong to the tetragonal space group P4(1)2(1)2 with unit cell parameters a=b=60.6 A and c=155.3 A, one monomer per asymmetric unit, a solvent content of ca. 33%, and a Vm value of approximately 2 A3/dalton.

Purification, functional characterization, and crystallization of the ligand binding domain of the retinoid X receptor.

The ligand binding domain (LBD) of the human retinoid X receptor alpha (hRXR alpha) was overproduced in Escherichia coli and purified to more than 95% purity and functional homogeneity. Circular dichroism spectra of the purified RXR alpha LBD indicated that the protein was composed predominantly of alpha-helical structures and coils. Crystals were grown from ammonium citrate using the vapor diffusion method against a reservoir containing 100 mM Pipes (pH 7.0) and 1.5 M ammonium citrate. They belong to the hexagonal space group P6(3)22 with unit cell parameters a = b = 110.8 A and c = 109.9 A, alpha = beta = 90 degrees, gamma = 120 degrees, and they diffract X rays to a resolution limit of 2.5 A using synchrotron radiation. The asymmetric unit of the crystals contains one molecule with a solvent content of approximately 55% and a Vm value of 3.6 A3/dalton.

New Zealand white rabbits immunized with RNA-complexed total histones develop an autoimmune-like response.

The antibody response of rabbits immunized with a total histone mixture containing randomly coiled H1/H5, H2A, H2B, H3 and H4 devoid of DNA was investigated in direct and competitive ELISA. The antisera were tested with isolated histones and chromatin and with a series of overlapping synthetic peptides covering the entire sequences of the four core histones and two peptides of H1. It was found that the New Zealand (NZ) white rabbits immunized with the total histone (TH) mixture complexed with RNA produced IgG antibodies reacting with histones and with a number of histone peptides but not with chromatin. The antisera also contained IgG antibodies which bound components that correspond to common target antigens in autoimmune diseases such as native dsDNA, peptides of Sm-D antigen, ubiquitin, branched peptides of ubiquitinated H2A and poly(ADP-ribose). By competition experiments, it was shown that these antibodies corresponded to non-crossreacting antibody populations. New Zealand rabbits immunized with TH in the absence of RNA or random outbred rabbits immunized with the RNA-complexed histone fraction produced antibodies reacting with histone, chromatin and very few histone peptides, while no activity with non-related antigens was observed. The pattern of reactivity of antisera raised in NZ rabbits with RNA-complexed TH was found to be very similar to that observed in sera of patients with systemic lupus erythematosus while, in contrast, the antibody response was very different in NZ or outbred rabbits immunized with various native nuclear particles and with individual histones. Altered nucleosome particles rather than native nucleosomes may represent the antigenic stimulus giving rise to autoantibodies.

Reactivity of autoantibodies in systemic lupus erythematosus with synthetic core histone peptides.

The specificity of autoantibodies present in the serum of 151 patients with systemic lupus erythematosus (SLE) was investigated by ELISA using as antigen individual histones as well as 17 different core histone synthetic peptides. Many of the sera reacted with four terminal peptides (residues 1-21 and 130-135 of H3, 1-29 of H4 and 1-25 of H2B) while fewer reacted with internal peptides (residues 65-85 of H2A and 40-55 of H3). Of the 151 SLE sera, 88% reacted with one or more of the six core histone peptides whereas only 57% reacted with one or more of the complete core histone molecules. Antibodies to mononucleosomes from chicken erythrocytes were also prepared in rabbits. The rabbit antisera were tested by ELISA using as antigen chromatin subunits, native and denatured DNA, individual histones and 23 natural and synthetic peptides of histones. The antinucleosome antibodies were found to recognize the same peptide fragments as those recognized by the SLE sera.