Comparison of mRNA binding by Met-tRNAf binding protein and mRNA-associated proteins

One of the heterogeneous mRNA binding activities in the 0.5 M KCl eluate of rabbit reticulocyte polyribosomes co-purified to apparent homogeneity through phosphocellulose and DEAE-cellulose chromatography and isoelectric focusing with the GTP-dependent Met-tRNAf binding protein. Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis following iodination revealed putative subunits of 51,000 and 39,000 apparent molecular weights. Specificity of mRNA binding by this protein was suggested since the ability of poly(A)-rich mRNA to compete for binding of [3H]poly(A)-rich mRNA exceeded by 10- to 100-fold that of most natural or synthetic RNAs tested, except for the hybrid poly(G) - poly(C) which was almost as effective, and poly(G), which was more effective, at competing for protein-dependent binding. The mRNA binding activity exhibited complete GTP independence and no apparent divalent cation requirement. GDP inhibited Met-tRNAf binding but neither GDP, GMP, nor 7-methylguanosine 5'-monophosphate inhibited mRNA binding by this protein. Similar data were obtained with respect to the ability of natural or synthetic RNAs to compete for binding of [3H]poly(A)-rich mRNA by proteins associated with purified rabbit reticulocyte polyribosomal mRNA-protein particles; while poly(A) was an ineffective competitor, poly(G) was more effective than even mRNA at competing for protein-dependent binding. No significant binding of Met-tRNAf by mRNA-protein particles was detected. Polyacrylamide gel electrophoresis following reduction of mRNA-protein particles revealed apparent co-migration of a major protein with one subunit of the GTP-dependent Met-tRNAf binding protein, but no protein comparable to the 39,000 dalton subunit protein.     

Predicting ligand binding to proteins by affinity fingerprinting

BACKGROUND: There are many ways to represent a molecule's properties, including atomic-connectivity drawings, NMR spectra, and molecular orbital models. Prior methods for predicting the biological activity of compounds have largely depended on these physical representations. Measuring a compound's binding potency against a small reference panel of diverse proteins defines a very different representation of the molecule, which we call an affinity fingerprint. Statistical analysis of such fingerprints provides new insights into aspects of binding interactions that are shared among a wide variety of proteins. These analyses facilitate prediction of the binding properties of these compounds assayed against new proteins. RESULTS: Affinity fingerprints are reported for 122 structurally-diverse compounds using a reference panel of eight proteins that collectively are able to generate unique fingerprints for about 75% of the small organic compounds tested. Application of multivariate regression techniques to this database enables the creation of computational surrogates to represent new proteins that are surprisingly effective at predicting binding potencies. We illustrate this for two enzymes with no previously recognizable similarity to each other or to any of the reference proteins. Fitting of analogous computational surrogates to four other proteins confirms the generality of the method; when applied to a fingerprinted library of 5000 compounds, several sub-micromolar hits were correctly predicted. CONCLUSIONS: An affinity fingerprint database, which provides a rich source of data defining operational similarities among proteins, can be used to test theories of cryptic homology unexpected from current understanding of protein structure. Practical applications to drug design include efficient pre-screening of large numbers of compounds against target proteins using fingerprint similarities, supplemented by a small number of empirical measurements, to select promising compounds for further study.     

AbdB-like Hox proteins stabilize DNA binding by the Meis1 homeodomain proteins

Recent studies show that Hox homeodomain proteins from paralog groups 1 to 10 gain DNA binding specificity and affinity through cooperative binding with the divergent homeodomain protein Pbx1. However, the AbdB-like Hox proteins from paralogs 11, 12, and 13 do not interact with Pbx1a, raising the possibility of different protein partners. The Meis1 homeobox gene has 44% identity to Pbx within the homeodomain and was identified as a common site of viral integration in myeloid leukemias arising in BXH-2 mice. These integrations result in constitutive activation of Meis1. Furthermore, the Hoxa-9 gene is frequently activated by viral integration in the same BXH-2 leukemias, suggesting a biological synergy between these two distinct classes of homeodomain proteins in causing malignant transformation. We now show that the Hoxa-9 protein physically interacts with Meis1 proteins by forming heterodimeric binding complexes on a DNA target containing a Meis1 site (TGACAG) and an AbdB-like Hox site (TTTTACGAC). Hox proteins from the other AbdB-like paralogs, Hoxa-10, Hoxa-11, Hoxd-12, and Hoxb-13, also form DNA binding complexes with Meis1b, while Hox proteins from other paralogs do not appear to interact with Meis1 proteins. DNA binding complexes formed by Meis1 with Hox proteins dissociate much more slowly than DNA complexes with Meis1 alone, suggesting that Hox proteins stabilize the interactions of Meis1 proteins with their DNA targets.     

Weak substrate binding to transport proteins studied by NMR

The weak binding of sugar substrates fails to induce any quantifiable physical changes in the L-fucose-H+ symport protein, FucP, from Escherichia coli, and this protein lacks any strongly binding ligands for competitive binding assays. Access to substrate binding behavior is however possible using NMR methods which rely on substrate immobiliza-tion for detection. Cross-polarization from proton to carbon spins could detect the portion of 13C-labeled substrate associated with 0.2 micromol of the functional transport system overexpressed in the native membranes. The detected substrate was shown to be in the FucP binding site because its signal was diminished by the unlabeled substrates L-fucose and L-galactose but was unaffected by a three- to fivefold molar excess of the non-transportable stereoisomer D-fucose. FucP appeared to bind both anomers of its substrates equally well. An NMR method, designed to measure the rate of substrate exchange, could show that substrate exchanged slowly with the carrier center (>10(-1) s), although its dynamics are not necessarily coupled strongly to this site within the protein. Relaxation measurements support this view that fluctuations in the interaction with substrate would be confined to the binding site in this transport system.     

Interaction of tannin with human salivary proline-rich proteins

Tannins are polyphenolic compounds, widely distributed in plant-based foods, which have harmful effects on animals including humans. Salivary proline-rich proteins (PRPs) may act as a defence against tannins by forming complexes with them and thereby preventing their interaction with other biological compounds and absorption from the intestinal canal. The aim here was to compare the ability of members of the family of human PRPs to form insoluble complexes with tannin and to assess the stability of such complexes under conditions similar to those in the alimentary tract. Basic PRPs (BPRPs), which have no other known biological functions, were very effective in forming insoluble complexes with both condensed tannin and tannic acid. Practically no tannin bound to acidic PRPs (APRPs) and glycosylated PRPs (GPRPs), suggesting that tannin in the diet would not affect their biological activities. There were only small differences in the tannin-precipitating ability of various BPRPs of different sizes or sequences, indicating that, although there is considerable phenotypic variation of PRPs, it is not likely to cause marked individual variation in tannin-binding ability. Tryptic digestion of an APRP led to a marked increase in tannin binding to the resulting proline-rich peptides, supporting observations in other studies that there may be an interaction between the proline-poor N-terminal and the proline-rich C-terminal regions in native APRPs, which inhibits the biological activities of the proteins. Deglycosylation of a GPRP also led to a dramatic increase in tannin-binding ability, showing that the carbohydrate side-chains prevent binding of tannin. Most of the condensed tannin-PRP complexes remained insoluble under conditions similar to those in the stomach and small intestine, supporting the proposal that PRPs act as a defence against tannin.     

DNA binding by cut homeodomain proteins is down-modulated by casein kinase II

Luminal Ag challenge of intestinal segments from sensitized rats results in a rapid (approximately 3 min) secretory response. We previously showed in horseradish peroxidase (HRP)-sensitized rats that the initial phase of transepithelial Ag transport occurred via a transcellular route and was enhanced by sensitization. However, following the hypersensitivity reaction, Ag also crossed between epithelial cells. The aim of this study was to determine the role of mast cells in the altered transepithelial Ag transport. White spotting mast cell-deficient rats and +/+ littermate controls were sensitized to HRP. After 10 to 14 days, jejunal segments were resected, mounted in Ussing chambers, and challenged with HRP on the luminal side. Electron microscopy of jejunum fixed at 2 min showed a similarly enhanced endocytic transport of HRP in sensitized +/+ and Ws/Ws rats compared with naive controls. In sensitized +/+ rats, a secretory response occurred approximately 3 min after challenge, and tissue conductance increased thereafter. Naive +/+ and sensitized Ws/Ws rats did not demonstrate a secretory response to HRP challenge, and conductance remained at baseline levels. The flux of HRP was elevated across tissue from sensitized +/+ rats but not across tissue from naive controls or sensitized Ws/Ws rats. The results indicate that sensitization enhances the initial phase of transepithelial uptake of Ag by transcytosis in a mast cell-independent manner. However, subsequent recruitment of the paracellular pathway for Ag transport in sensitized rats is dependent upon the presence of mast cells and occurs after the activation of such cells.     

Toxins produced by arthropod parasites: salivary gland proteins of human body lice and venom proteins of chelonine wasps

A review is presented of our ongoing research projects on the protein components of the saliva of human body lice and of the non-paralyzing venom of wasps in the subfamily Cheloninae. Sodium dodecyl sulfate-polyacryamide gel electrophoretic analysis of lice salivary gland proteins showed a predominance of high and intermediate mol. wt proteins. Immunoblotting with a low titer polyclonal antiserum to lice salivary proteins indicated that some, but not all, of the predominant high mol. wt salivary gland proteins are injected into the host during feeding. The venom of a Chelonus sp. wasp contains a chitinase, and a 33,000 mol. wt protein with a primary structure composed mostly of a series of 12 tandem repeats of a 14-residue sequence. The N-terminus of this protein and its homologs in a related species of Ascogaster share a conserved adjacent pair of acidic residues. Epitope mapping/immunoprecipitation experiments now in progress will provide information on which linear motifs are on the surface of the protein, and will thereby provide information on the tertiary structure of the protein.     

Secreted poxvirus chemokine binding proteins

Poxviruses encode a variety of immunomodulatory proteins that subvert the cytokine networks of infected hosts. Myxoma virus, a poxvirus pathogen of rabbits, expresses two distinct 35- to 40-kDa secreted glycoproteins that bind a broad spectrum of chemokines. The first of these, designated M-T7, is encoded by the T7 gene and is the first example of what is here referred to as type-I chemokine binding protein (CBP-I). M-T7 was initially discovered as a secreted viral homologue of cellular interferon-gamma receptor but binding studies indicate that purified M-T7 protein also interacts with members of the CXC, CC, and C chemokine families through the conserved heparin-binding domains. The second myxoma protein, M-T1, also called CBP-II, is a member of a larger superfamily of poxvirus proteins that includes related secreted 35-kDa proteins encoded by a wide variety of orthopoxviruses. Deletion analysis of either CBP-I or -II genes within recombinant poxvirus constructs revealed profound alterations in the trafficking of infiltrating leukocytes into virus-infected lesions. It is proposed that the interaction of CBP-I with the conserved heparin-binding domains found on most chemokines represents a novel mechanism for altering multiple chemokine functions in vivo. In summary, CBP-I and CBP-II are the first examples of secreted virus proteins that bind to multiple chemokine family members as part of a strategy to prevent the early phase of inflammatory cell migration into virus-infected tissues.     

RP-HPLC binding domains of proteins

Procedures have been developed to identify the chromatographic binding domains of horse heart cytochrome c (Cyt c) and bovine growth hormone (bGH) during their interaction with reversed-phase sorbent materials. The procedure involves adsorption of the protein solute to the chromatographic sorbent, followed by proteolytic cleavage. Comparison of the proteolytic map obtained for Cyt c and bGH in free solution with the corresponding map obtained when these proteins are adsorbed to the chromatographic sorbent revealed significant differences in the digestion pattern. Following characterization of the peptides generated in both maps, the results indicated that specific regions on the surface of both Cyt c and bGH are inaccessible to tryptic cleavage when adsorbed to the hydrophobic surface of both a C-4 and a C-18 sorbent. Based on the assumption that the region of the protein surface that is in contact with the sorbent remains intact and bound to the sorbent during the digestion step, while the protein surface that is exposed to the solvent is accessible to proteolysis, the regions that were inaccessible to tryptic digestion were found to correspond to hydrophobic domains on the protein surface. These results also suggest that the three-dimensional structures of these proteins remain largely intact upon adsorption to the hydrophobic surface.     

Carcinogenesis of mouse submandibular salivary gland using DMBA implant

Carcinogenesis of 35 ddy male mice submandibular salivary glands were attempted; using implants of a 1 mg (2 mm) prepared pellets of the potent chemical carcinogen 9, 10-dimethyl-1, 2-benzanthracene (DMBA) dry powder without a vehicle or carrier. This method appeared to be easy, fast and effective. Within a period ranging from 7-14 weeks; twenty animals developed epidermoid carcinoma (two of them developed squamous cell carcinoma of covering skin as well), and two animals developed mixed tumors (pleomorphic adenoma like tumor) but neither adenocystic carcinoma nor sarcoma were found, the results were adequately discussed.     

Differential expression of transcripts encoding retinoid binding proteins and retinoic acid receptors during placentation of the mouse

An isocratic reversed-phase high-performance liquid chromatography (RP-HPLC) method for the determination of human growth hormone (hGH) directly in osmotic shock fluids is described. This methodology allows an initial rapid evaluation of the quality and quantity of hGH being secreted in the bacterial periplasmic space right after, or even during fermentation. Considering that RP-HPLC does not identify size isomers, these were determined via a parallel run of the same osmotic shock fluid on high-performance size-exclusion chromatography, coupled with radioimmunoassay, of the eluted fractions. The methodology provides a complete picture, within 24 h from the beginning of the fermentation process, of the recombinant protein being produced with respect to its activity, identity, yield, and hGH-related contaminants. These latter include sulfoxide and desamido derivatives, dimer and high-molecular-mass forms.     

Modulation of P-glycoprotein activity by estramustine is limited by binding to plasma proteins

BACKGROUND: Estramustine previously has been shown to interact with P-glycoprotein and to restore intracellular accumulation of vinblastine and paclitaxel in cells overexpressing this drug transporter. However, the ability of estramustine to potentiate the cytotoxicities of several drugs was less than that expected. To resolve this apparent discordance, the authors examined the effects of serum on the actions of estramustine. METHODS: The cytotoxicities of anticancer drugs with or without estramustine or verapamil toward MCF-7 breast carcinoma cells and a P-glycoprotein-overexpressing subline MCF-7/ADR were determined using the sulforhodamine-binding assay. The extent of intracellular accumulation of [3H]vinblastine and [3H]paclitaxel was determined for each using standard methods, and the binding of radiolabeled drugs to plasma proteins was characterized by equilibrium dialysis. RESULTS: Without serum, the sensitivities of MCF-7/ADR cells to several P-glycoprotein-transported drugs were increased by estramustine and verapamil. Conversely, when the cells were treated with a 10% serum, the cytotoxicities of these drugs were increased by verapamil, but not by estramustine. Without serum, intracellular accumulation of [3H]vinblastine and [3H]paclitaxel by MCF-7/ADR cells was increased markedly by verapamil and estramustine; however, serum suppressed the effects of estramustine much more strongly than those of verapamil. Equilibrium dialysis experiments demonstrated that [3H]estramustine binds to plasma proteins, predominantly albumin, whereas [3H]paclitaxel binds to albumin and alpha 1-acid-glycoprotein, and [3H]vinblastine binds predominantly to alpha 1-acid-glycoprotein. CONCLUSION: Although estramustine can bind to P-glycoprotein, its effectiveness as a reversing agent in vivo likely is limited by binding to plasma proteins.     

Antigenic peptide binding by class I and class II histocompatibility proteins

Enhanced expression of the immediate early gene c-fos has been used as a marker of cellular activation in many different neuronal pathways. We wished to determine the neurochemical content and the connectivity of neurons, in which expression of c-fos is induced. For this purpose, a dual-immunocytochemical staining technique has been developed with avidin-biotin-peroxidase labelling using diaminobenzidine as the chromogen for c-fos protein located in the nucleus, and benzidine dihydrochloride (BDHC) in the presence of sodium nitroprusside to reveal cytoplasmic antigens (neuropeptide or retrograde tracer) in the same section. The blue granular BDHC reaction product in the cytoplasm combined with the homogeneous brown nuclear DAB staining for c-fos protein provides excellent resolution of dual-labelled cells even in tissue sections of 40 microns in thickness. The high sensitivity of the avidin-biotin-peroxidase immunocytochemistry and the stability of the reaction products provide an excellent tool for quantitative analysis of stimulated cells within a neurochemically defined cell group. The BDHC/DAB protocol was developed to identify activated cells in three experimental situations. Firstly, to investigate the phenotype of light-activated cells in the suprachiasmatic nucleus of the hypothalamus, c-fos protein DAB staining was carried out together with BDHC staining for peptide histidine isoleucine (PHI) and vasoactive intestinal peptide (VIP).(ABSTRACT TRUNCATED AT 250 WORDS)     

Redox modulation of selenium binding proteins by cadmium exposures in mice

The mechanism of the antagonistic behaviour of selenium (Se) against cadmium (Cd) toxicity is investigated. This study reports the distribution of Cd at the organ and subcellular level after chronic treatments. The possible role of the selenium binding proteins (SBP) during Cd exposure are also evaluated. Kidney concentrates more Cd than liver following 8 weeks of treatment. Simultaneous administration of Se reduced Cd accumulation in Kidney. This affect did not occur in liver. Among the subcellular fractions, the maximum concentrations of both of the elements were found in the cytosol. The overall uptake of 75Se was enhanced in the cytosol of kidney and liver of the Cd treated animals. These observations support a hypothesis that selenium is complexed with cadmium. The increase in the labeling of SBP as a result of Cd exposures may reflect a change in the conformation of the protein molecule. These proteins (SBP) contain a sequence motif, which may be an active redox centre. Also, Cd significantly reduced the glutathione level, thereby disrupting the thiol/disulfide balance. This in turn may affect the redox status of the proteins leading to a 75Se or 75Se-Cd complex with SBP.     

Inhibition of insulin receptor activation by insulin-like growth factor binding proteins

The insulin-like growth factors (IGFs) are transported by a family of high-affinity binding proteins (IGFBPs) that protect IGFs from degradation, limit their binding to IGF receptors, and modulate IGF actions. The six classical IGFBPs have been believed to have no affinity for insulin. We now demonstrate that IGFBP-7/mac25, a newly identified member of the IGFBP superfamily that binds IGFs specifically with low affinity is a high-affinity insulin binding protein. IGFBP-7 blocks insulin binding to the insulin receptor and thereby inhibiting the earliest steps in insulin action, such as autophosphorylation of the insulin receptor beta subunit and phosphorylation of IRS-1, indicating that IGFBP-7 is a functional insulin-binding protein. The affinity of other IGFBPs for insulin can be enhanced by modifications that disrupt disulfide bonds or remove the conserved COOH terminus. Like IGFBP-7, an NH2-terminal fragment of IGFBP-3 (IGFBP-3((1-87))), also binds insulin with high affinity and blocks insulin action. IGFBPs with enhanced affinity for insulin might contribute to the insulin resistance of pregnancy, type II diabetes mellitus, and other pathological conditions.     

Chemical characterization of the predominant proteins secreted by mouse seminal vesicles

Eosinophilic fasciitis is a disorder characterised by induration of the skin due to chronic inflammation and fibrosis of the subcutaneous septa and muscular fascia. It is different from sclerodermia. This is a clinical entity that presents as swelling, tenderness and stiffness of the extremities associated with peripheral eosinophilia. We described a patient with this disorder with 18 month of evolution who had a bilateral carpal tunnel syndrome. Her disease appeared after steroid treatment. A review of medical literature demonstrated that similar clinical pictures are originated by different causes. Some authors propose to encompass this group of disorder under the designation of "fasciitis-panniculitis syndrome" instead of "eosinophilic fasciitis" although the well-known eosinophilic fasciitis is clearly recognized and demonstrated.     

Protein binding effects of salivary excretion of phenobarbital in dogs

The salivary excretion of phenobarbital was investigated by collecting parotid saliva (Pr) and mandibular-sublingual saliva (MS) separately after intravenous administration in beagle dogs. (1) The alterations in the proportions of saliva secreted by the different glands were produced by salivation stimulants such as citric acid, ascorbic acid, sodium chloride and sodium glutamate. (2) The phenobarbital concentrations in both Pr amd MS were lower than those in plasma. The drug concentrations in MS were significantly lower than in Pr with stimulus of 10% citric acid of 15% sodium chloride (p less than 0.05). There was a significant correlation between phenobarbital concentration in each saliva and plasma specimen ( p less than 0.05). (3) The stimulation with 10% citric acid produced higher saliva /plasma drug concentration ratios (S/P ratios: 0.923 +/- 0.175 for Pr, 0.633 +/- 0.073 for MS) than that with 15% sodium chloride (S/P ratios: 0.597 +/- 0.071 for Pr, 0.509 +/- 0.067 or MS). (4) The S/P ratios were hardly influenced by salivary flow rates, at least under the experimental conditions examined in this study. (5) The increased S/P ratios were observed with higher salivary pH and then the equation of Matin et al. 3) seemed to hold for the average values of salivary pH and S/P ratio. (6) The stimulation with 10% citric acid produced higher protein concentration in saliva and higher S/P ratio than that with 15% sodium chloride following alternate stimulations in the same dog.     

Estrogen receptor activation function 1 works by binding p160 coactivator proteins

Estrogen receptor-alpha contains two transactivation functions, a weak constitutive activation function (AF-1) and a hormone-dependent activation function (AF-2). AF-2 works by recruiting a large coactivator complex, composed of one or more p160s, CREB-binding protein (CBP)/p300, and P/CAF (p300 and CBP-associated factor), via direct contacts with the p160s. We report here that independent AF-1 activity also requires p160 contacts. Unlike AF-2, which binds signature NR boxes in the center of the p160 molecule, AF-1 binds to sequences near the p160 C terminus. We propose that the ability of AF-1 and AF-2 to interact with separate surfaces of the same coactivator is important for the ability of these transactivation functions to synergize.