Modification of the zonal elution method for detection of transient protein-protein interactions involving ligand exchange.

A new affinity chromatography method was developed by modifying a zonal elution method. The new method targets transient protein-protein interactions, such as those encountered during direct ligand transfer between the ligand transporter and its cognate receptor. A ligand-loaded transport protein is immobilized on the solid support, and a plug containing a putative receptor is flowed through the column. Elution profiles of proteins not interacting with the immobilized transporter can be approximated with a simple Gaussian curve, while the elution profiles of cognate receptors show significant delay and exhibit complex shape. Ligand transfer from the immobilized transporter molecules to the receptors is verified by both UV absorbance measurements and mass spectrometry. The sensitivity of the method is demonstrated using retinoic acid (RA) transfer from various isoforms of cellular RA binding proteins (CRABPs) and RA receptor γ (RARγ). Although these interactions have been hypothesized long ago to proceed via direct mechanism (i.e., via transient docking of the receptor and the transporter), the existing biophysical techniques failed to detect the presence of the transporter-receptor complexes. However, the modified zonal elution method provides unequivocal evidence of direct interaction between RARγ and one of the CRABP isoforms (CRABP II) during the ligand transfer to the receptor.

Characterization of the human tumor suppressors TIG3 and HRASLS2 as phospholipid-metabolizing enzymes.

Tazarotene-induced protein 3 (TIG3) and HRAS-like suppressor family 2 (HRASLS2) exhibit tumor-suppressing activities and belong to the lecithin retinol acyltransferase (LRAT) protein family. Since Ca(2+)-independent N-acyltransferase and H-rev107 (another tumor suppressor), both of which are members of the LRAT family, have been recently reported to possess catalytic activities related to phospholipid metabolism, we examined possible enzyme activities of human TIG3 and HRASLS2 together with human H-rev107. The purified recombinant proteins of TIG3, HRASLS2, and H-rev107 functioned as phospholipase (PL) A(1/2) in a Ca(2+)-independent manner with maximal activities of 0.53, 0.67, and 2.57 micromol/min/mg of protein, respectively. The proteins were active with various phosphatidylcholines (PCs) and phosphatidylethanolamines (PEs), and for most of substrates the PLA(1) activity was much higher than the PLA(2) activity. In addition, HRASLS2 catalyzed N-acylation of PE to form N-acyl-PE and O-acylation of lyso PC to form PC. TIG3 and H-rev107 catalyzed the N-acylation and O-acylation at relatively low rates. Moreover, these three proteins showed different expression profiles in human tissues. These results suggest that the tumor suppressors TIG3, HRASLS2 and H-rev107 are involved in the phospholipid metabolism with different physiological roles.

Expression, purification and biochemical characterization of the N-terminal regions of human TIG3 and HRASLS3 proteins.

Tarzarotene-induced gene 3 (TIG3) and HRAS-like suppressor (HRASLS3) are members of the HREV107 family of class II tumor suppressors, which are down-regulated in various cancer cells. TIG3 and HRASLS3 also exhibit phospholipase activities. Both proteins share a common domain architecture with hydrophilic N-terminal and hydrophobic C-terminal regions. The hydrophobic C-terminal region is important for tumor suppression. However, the function of the hydrophilic N-terminal region remains elusive. To facilitate biochemical characterizations of TIG3 and HRASLS3, we expressed and purified the N-terminal regions of TIG3 and HRASLS3, designated TIG3 (1-134) and HRASLS3 (1-133), in a bacterial system. We found that the N-terminal regions of TIG3 and HRASLS3 have calcium-independent phospholipase A(2) activities. Limited proteolysis revealed that TIG3 (1-132) is a structural domain in the N-terminal region of TIG3. Our data suggest that the hydrophobic C-terminal regions might be crucial for cellular localization, while the hydrophilic N-terminal regions are sufficient for the enzymatic activity of both TIG3 and HRASLS3.

Identification of a membrane receptor for retinol-binding protein functioning in the cellular uptake of retinol.

Retinol-binding protein (RBP) is the transport protein that carries retinol in the circulation from the liver to its target tissues. The existence of a cell-surface receptor on the target cells, which mediates the uptake of retinol from RBP, has been known since 1975. Recently, it was identified as an integral transmembrane protein named STRA6 that is inducible by retinoic acid in certain cancer cells. The receptor was found to be highly specific for RBP, with high affinity, and to be localized in all tissues known to require retinol for their function, particularly the pigment epithelium of the eye.

Rapid identification of DNA-binding proteins by mass spectrometry.

We report a protocol for the rapid identification of DNA-binding proteins. Immobilized DNA probes harboring a specific sequence motif are incubated with cell or nuclear extract. Proteins are analyzed directly off the solid support by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. The determined molecular masses are often sufficient for identification. If not, the proteins are subjected to mass spectrometric peptide mapping followed by database searches. Apart from protein identification, the protocol also yields information on posttranslational modifications. The protocol was validated by the identification of known prokaryotic and eukaryotic DNA-binding proteins, and its use provided evidence that poly(ADP-ribose) polymerase exhibits DNA sequence-specific binding to DNA.

Retinoic acid receptor alpha1 variants, RARalpha1DeltaB and RARalpha1DeltaBC, define a new class of nuclear receptor isoforms.

Retinoic acid (RA) binds and activates retinoid X receptor (RXR)/retinoic acid receptor (RAR) heterodimers, which regulate the transcription of genes that have retinoic acid response elements (RARE). The RAR isotypes (alpha, beta and gamma) are comprised of six regions designated A-F. Two isoforms of RARalpha, 1 and 2, have been identified in humans, which have different A regions generated by differential promoter usage and alternative splicing. We have isolated two new splice variants of RARalpha1 from human B lymphocytes. In one of these variants, exon 2 is juxtaposed to exon 5, resulting in an altered reading frame and a stop codon. This variant, designated RARalpha1DeltaB, does not code for a functional receptor. In the second variant, exon 2 is juxtaposed to exon 6, maintaining the reading frame. This isoform, designated RARalpha1DeltaBC, retains most of the functional domains of RARalpha1, but omits the transactivation domain AF-1 and the DNA-binding domain. Consequently, it does not bind nor transactivate RARE on its own. Nevertheless, RARalpha1DeltaBC interacts with RXRalpha and, as an RXRalpha/RARalpha1DeltaBC heterodimer, transactivates the DR5 RARE upon all-trans-RA binding. The use of RAR- and RXR-specific ligands shows that, whereas transactivation of the DR5 RARE through the RXRalpha/RARalpha1 heterodimer is mediated only by RAR ligands, transactivation through the RXRalpha/RARalpha1DeltaBC heterodimer is mediated by RAR and RXR ligands. Whilst RARalpha1 has a broad tissue distribution, RARalpha1DeltaBC has a more heterogeneous distribution, but with significant expression in myeloid cells. RARalpha1DeltaBC is an infrequent example of a functional nuclear receptor which deletes the DNA-binding domain.

Isolation and characterization of two isoforms of a retinoic acid-binding protein from rabbit epididymal secretions.

Two isoforms of a retinoic acid-binding protein have been purified from rabbit epididymal secretions using a combination of gel filtration and ion-exchange chromatography. The two polypeptides (EP21a and b) present similar molecular mass (21 kDa), under native or denaturing conditions and have very similar amino-acid composition and tryptic peptide maps but differ in net charge. Both isoforms are glycosilated though to a different extent (9.2% and 6.3% of carbohydrate content) and are major components of the epididymal fluid. Binding of retinoic acid to EP21s appears to be specific, since they do not bind retinol, but is non-saturable. EP21s seem to present some similarity to two retinoic acid-binding proteins from rat epididymal secretions (site of biosynthesis, androgen dependence, ligand specificity and association to the spermatozoa) but differ from the rat proteins in amino-acid composition and glycosilation.

Crystal structure of cellular retinoic acid binding protein I shows increased access to the binding cavity due to formation of an intermolecular beta-sheet.

A recombinant form of murine apo-cellular retinoic acid binding protein I (apo-CRABPI) has been purified and crystallized at pH 5.0, and the crystal structure has been refined to an R-factor of 19.6% at a resolution of 2.7 A. CRABPI binds all-trans retinoic acid and some retinoic acid metabolites with nanomolar affinities. Coordinates of the holo form of CRABP were not available during the early stages of the study, and in spite of numerous homologs of known structure, phases were not obtainable through molecular replacement. Instead, an interpretable electron density map was obtained by multiple isomorphous replacement methods after improvement of the heavy-atom parameters with density modified trial phases. Two molecules of apo-CRABPI occupy the P3121 asymmetric unit and are related by pseudo 2-fold rotational symmetry. Unique conformational differences are apparent between the two molecules. In all of the family members studied to date, there is a lack of hydrogen bonds between two of the component beta-strands resulting in a gap in the interstand hydrogen bonding pattern. In the crystallographic dimer described here, a continuous intermolecular beta-sheet is formed by using this gap region. This is possible because of an 8 A outward maximum displacement of the tight turn between the third and fourth beta-strands on one of the molecules. The result is a double beta-barrel containing two apo-CRABPI molecules with a more open, ligand-accessible binding cavity, which has not been observed in other structures of a family of proteins that bind hydrophobic ligands.

Intrinsic tryptophans of CRABPI as probes of structure and folding.

The native state fluorescence and CD spectra of the predominantly beta-sheet cellular retinoic acid-binding protein I (CRABPI) include contributions from its three tryptophan residues and are influenced by the positions of these residues in the three-dimensional structure. Using a combination of spectroscopic approaches and single Trp-mutants of CRABPI, we have deconvoluted these spectra and uncovered several features that have aided in our analysis of the development of structure in the folding pathway of CRABPI. The emission spectrum of native CRABPI is dominated by Trp 7. Trp 109 is fluorescence-silent due to its interaction with the guanidino group of Arg 111. Although the far-UV CD spectrum of CRABPI is largely determined by the protein's secondary structure, aromatic clustering around Trp 87 and the aromatic-charge interaction between Arg 111 and Trp 109 give rise to a characteristic feature in the CD spectrum at 228 nm. The near-UV CD bands of CRABPI arise largely from additive contributions of the three tryptophan residues. Trp 7 and Trp 87 give a negative CD band at 275 nm. The near-UV CD band from Trp 109 is positive and shifted to longer wavelengths (to 302 nm) due to the charge-aromatic interaction between Arg 111 and Trp 109. Our deconvolution of the equilibrium spectra have been used to interpret kinetic folding experiments monitored by stopped-flow fluorescence. These dynamic experiments suggest the early evolution of a well-populated, hydrophobically collapsed intermediate, which undergoes global rearrangement to form the fully folded structure. The results presented here suggest several additional strategies for dissecting the folding pathway of CRABPI.

Ligand specificity and developmental expression of RXR and ecdysone receptor in the migratory locust.

The ecdysone receptor(1), which is a heterodimer of EcR and the retinoic acid receptor (RXR) homolog, Ultraspiracle (USP), has been well studied in the evolutionarily advanced and derived insects, the flies and moths. It is less well characterized in more primitive insect orders such as the Orthoptera, which include the grasshoppers and locusts. Following our previous isolation from Locusta migratoria (Lm) of a shorter RXR isoform (now called LmRXR-S), the isolation of a second, longer isoform (LmRXR-L) that appears to have characteristics of a ligand-modulated nuclear receptor is reported here. Transcripts for both isoforms, as well as LmEcR, were detected in embryos and in females during oocyte maturation. After expression in E. coli, both LmRXR-S and LmRXR-L form heterodimers with recombinant LmEcR in vitro which bind the active ecdysteroid, ponasterone A. Binding was only weakly competed for by ecdysone agonists that are known to be toxic to more advanced insects, suggesting functionally significant divergence in EcR ligand binding domains. In contrast, the DNA binding domain of LmEcR is less divergent and a protein complex, presumably LmEcR/LmRXR, that bound the ecdysone response element, IR-1, was detected in locust nuclear extracts. Because of reports of juvenile hormone (JH III) binding to Drosophila USP and the observed in silico RXR-like ligand-binding site in LmRXR-L, the recombinant proteins were also tested for binding to JH III. Neither LmRXR isoform, alone or in combination with LmEcR, bound JH III at nanomolar concentrations.

Direct electrochemical DNA detection originated from the self-redox signal of sulfonated polyaniline enhanced by graphene oxide in neutral solution.

In this paper, a type of direct DNA impedance detection using the self-redox signal change of sulfonated polyaniline (SPAN) enhanced by graphene oxide (GNO) was reported, here SPAN is a copolymer obtained from aniline and m-aminobenzenesulfonic acid. The resulting nanocomposite was characterized by scanning electron microscopy, transmission electron microscopy, Fourier transform infrared spectroscopy, cyclic voltammetry, and electrochemical impedance spectroscopy. The π-π planar structure of GNO and the carboxyl groups on the surface of GNO ensured it could act as an excellent substrate for adsorption and polymerization of aniline monomer. Because of the existence of GNO, the electrochemical activities of SPAN were enhanced obviously. Because of abundant sulfonic acid groups, the resulting nanocomposite showed obvious self-redox signal even at physiological pH, which is beneficial for biosensing field. DNA probes with amine groups could be covalently attached to the modified electrode surface through the acyl chloride cross-linking reaction of sulfonic groups and amines. When the flexible probe DNA was successfully grafted, the electrode was coated and electron transfer between electrode and buffer was restrained. Thus, the inner impedance value of SPAN (rather than using outer classic EIS probe, [Fe(CN)6](3-/4-)) increased significantly. After hybridization, the rigid helix opened the electron channel, which induced impedance value decreased dramatically. As an initial application of this system, the PML/RARA fusion gene sequence formed from promyelocytic leukemia (PML) and retinoic acid receptor alpha (RARA) was successfully detected.

Fluorescence-based technique for analyzing retinoic acid.

Retinoic acid (RA) is a potent transcriptional activator whose actions are mediated by members of the nuclear hormone receptor family. In addition to playing key roles in embryonic development and in tissue maintenance in the adult, RA is a potent anticarcinogenic agent currently in clinical use for treatment of various cancers. Here, we describe an optical method for measuring the concentrations of RA in biological samples. This method uses cellular retinoic acid-binding protein I (CRABP-I), a protein that binds RA with high affinity and specificity, as a "read-out" for its ligand. Replacing (28)Leu of CRABP-I with a Cys residue allows for covalently attaching an environmentally sensitive fluorescent probe to the protein at a region that undergoes a significant conformational change upon ligand binding. Association of RA with the modified protein thus results in changes in the fluorescence of the probe, enabling reliable measurements of RA concentrations as low as 50 nM. We show that the method can be effectively used to measure RA concentrations in serum and to monitor the biosynthesis and the degradation of RA in cultured mammalian cells.

Dynamic recruitment of axin by Dishevelled protein assemblies.

Dishevelled (Dvl) proteins are cytoplasmic components of the Wnt signalling pathway, which controls numerous cell fate decisions during animal development. During Wnt signalling, Dvl binds to the intracellular domain of the frizzled transmembrane receptors, and also to axin to block its activity, which results in the activation of beta-catenin and, consequently, in a transcriptional switch. We have previously reported that the DIX domain of mammalian Dvl2 allows it to form dynamic protein assemblies. Here, we show that these Dvl2 assemblies recruit axin, and also casein kinase Iepsilon. Using photobleaching experiments of GFP-tagged Dvl2 and axin to study the dynamics of their interaction, we found that the recruitment of axin-GFP by Dvl2 assemblies is accompanied by a striking acceleration of the dynamic properties of axin-GFP. We also show that the interaction between Dvl2 and axin remains highly dynamic even after Wnt-induced relocation to the plasma membrane. We discuss how the recruitment of casein kinase Iepsilon by Dvl2 assemblies might impact on the recruitment of axin to the plasma membrane during Wnt signalling.

Dysregulation of retinoid transporters expression in body fluids of schizophrenia patients.

This study aims to find the biomarkers or associated proteins in body fluids of schizophrenia patients so that we can further understand the etiology of schizophrenia. We applied proteomic technologies combining two-dimensional electrophoresis with Coomassie blue staining and mass spectrometry and identified a procedure for the clinical screening of disease-influenced body fluid proteins in two sets of samples, plasma from 19 schizophrenia patients and cerebrospinal fluid (CSF) from 35 drug-treated schizophrenic patients and 36 healthy controls. The expression of transthyretin (TTR) tetramer increased significantly in plasma of schizophrenic patients after a valid 2 months in-hospital antipsychotic treatment. Conversely, the expression of the TTR tetramer and apolipoprotein E (ApoE) was down-regulated by up to 1.68 and 3.62 times, respectively, in the CSF of schizophrenia patients compared to that of normal controls, which has not been reported previously. Considering that the TTR tetramer and ApoE are both retinoid transporters, retinoid dysfunction might be involved in the pathology of schizophrenia.