Identification and molecular characterization of the RNA polymerase-binding motif of infectious bursal disease virus inner capsid protein VP3

Infectious bursal disease virus (IBDV), a member of the Birnaviridae family, is the causative agent of one of the most important infectious poultry diseases. Major aspects of the molecular biology of IBDV, such as assembly and replication, are as yet poorly understood. We have previously shown that encapsidation of the putative virus-encoded RNA-dependent RNA polymerase VP1 is mediated by its interaction with the inner capsid protein VP3. Here, we report the characterization of the VP1-VP3 interaction. RNase A treatment of VP1- and VP3-containing extracts does not affect the formation of VP1-VP3 complexes, indicating that formation of the complex requires the establishment of protein-protein interactions. The use of a set of VP3 deletion mutants allowed the mapping of the VP1 binding motif of VP3 within a highly charged 16-amino-acid stretch on the C terminus of VP3. This region of VP3 is sufficient to confer VP1 binding activity when fused to an unrelated protein. Furthermore, a peptide corresponding to the VP1 binding region of VP3 specifically inhibits the formation of VP1-VP3 complexes. The presence of Trojan peptides containing the VP1 binding motif in IBDV-infected cells specifically reduces infective virus production, thus showing that formation of VP1-VP3 complexes plays a critical role in IBDV replication.     

Identification and characterization of a nuclear interacting partner of anaplastic lymphoma kinase (NIPA)

Anaplastic large-cell lymphoma is a subtype of non-Hodgkin lymphomas characterized by the expression of CD30. More than half of these lymphomas carry a chromosomal translocation t(2;5) leading to expression of the oncogenic tyrosine kinase nucleophosmin-anaplastic lymphoma kinase (NPM-ALK). NPM-ALK is capable of transforming fibroblasts and lymphocytes in vitro and of causing lymphomas in mice. Previously, we and others demonstrated phospholipase C-gamma and phosphatidylinositol 3-kinase as crucial downstream signaling mediators of NPM-ALK-induced oncogenicity. In this study, we used an ALK fusion protein as bait in a yeast two-hybrid screen identifying NIPA (nuclear interacting partner of ALK) as a novel downstream target of NPM-ALK. NIPA encodes a 60-kDa protein that is expressed in a broad range of human tissues and contains a classical nuclear translocation signal in its C terminus, which directs its nuclear localization. NIPA interacts with NPM-ALK and other ALK fusions in a tyrosine kinase-dependent manner and is phosphorylated in NPM-ALK-expressing cells on tyrosine and serine residues with serine 354 as a major phosphorylation site. Overexpression of NIPA in Ba/F3 cells was able to protect from apoptosis induced by IL-3 withdrawal. Mutations of the nuclear translocation signal or the Ser-354 phosphorylation site impaired the antiapoptotic function of NIPA. In NPM-ALK-transformed Ba/F3 cells, apoptosis triggered by wortmannin treatment was enhanced by overexpression of putative dominant-negative NIPA mutants. These results implicate an antiapoptotic role for NIPA in NPM-ALK-mediated signaling events.     

NAB2, a corepressor of EGR-1, inhibits vascular endothelial growth factor-mediated gene induction and angiogenic responses of endothelial cells

In this study we have investigated the role of a specific corepressor of EGR-1, NAB2, to down-regulate vascular endothelial growth factor (VEGF)-induced gene expression in endothelial cells and to inhibit angiogenesis. Firstly, we show a reciprocal regulation of EGR-1 and NAB2 following VEGF treatment. During the initial phase EGR-1 is rapidly induced and NAB2 levels are down-regulated. This is followed by a reduction of EGR-1 and a concomitant increase of NAB2. Secondly, using the tissue factor gene as a readout for VEGF-induced and EGR-1-regulated gene expression we demonstrate that NAB2 can completely block VEGF-induced tissue factor reporter gene activity. Thirdly, by adenovirus-mediated expression we show that NAB2 inhibits up-regulation of tissue factor, VEGF receptor-1, and urokinase plasminogen activator mRNAs even when a combination of VEGF and bFGF is used for induction. In addition, NAB2 overexpression significantly reduced tubule and sprout formation in two different in vitro angiogenesis assays and largely prevented the invasion of cells and formation of vessel-like structures in the murine Matrigel model. These data suggest that NAB2 regulation represents a mechanism to guarantee transient EGR-1 activity following exposure of endothelial cells to VEGF and that NAB2 overexpression could be used to inhibit signals involved in the early phase of angiogenesis.     

Molecular requirements for the regulation of the renal outer medullary K(+) channel ROMK1 by the serum- and glucocorticoid-inducible kinase SGK1

The serum- and glucocorticoid- inducible kinase SGK1 stimulates the renal outer medullary K(+) channel ROMK1 in the presence of the Na(+)/H(+) exchanger regulating factor NHERF2. SGK1/NHERF2 are effective through enhancement of ROMK1 abundance within the cell membrane. The present study aims to define the molecular requirements for the interaction of ROMK1 with SGK1/NHERF2. Pull down assays reveal that SGK1 interacts with NHERF2 through the second PDZ domain of NHERF2. According to chemiluminescence and electrophysiology, deletion of the second PDZ domain of NHERF2 or the putative PDZ binding motif on ROMK1 abrogates the stimulating effect of SGK1 on ROMK1 protein abundance in the plasma membrane and K(+) current.     

Caseinolytic activity of fruit extract from Opuntia ficus-indica on bovine, caprine, and ovine sodium caseinates

The rates and extents of hydrolysis of alpha(S)- and beta-caseins from bovine, caprine, and ovine sodium caseinates produced by an enzymatic extract of the fruit of Opuntia ficus-indica, (L.) Miller were evaluated and compared with those produced by a commercial animal rennet. A mechanistic model based on a pseudo-first-order enzymatic reaction, in the presence of first-order deactivation of the enzyme, was postulated and successfully fitted to the experimental data. The animal rennet exhibited higher enzymatic efficiency than the fruit extract, irrespective of the source (i.e., bovine, caprine, or ovine) and the type (i.e., alpha(S)- or beta-casein) of substrate. The enzymatic efficiency (k(cat)/K(m)) for alpha(S)-casein ranged from 72 to 220 and from 43 to 65 L g(-1) h(-1), and for beta-casein from 242 to 742 and from 55 to 164 L g(-1) h(-1), for the animal rennet and the enzymatic extract of O. ficus-indica, respectively. Finally, it was observed that beta-casein from caprine and ovine caseinates was degraded by O. ficus-indica faster than its alpha(S) counterpart, but the reverse was observed for bovine caseinate.     

Construction of a derivative of Agrobacterium tumefaciens C58 that does not mutate to tetracycline resistance

Agrobacterium tumefaciens C58 mutates to tetracycline resistance at high frequency, complicating the use of many broad-host-range cloning and binary vectors that code for resistance to this antibiotic as the selection marker. Such mutations are associated with a resistant gene unit, tetC58, that is present in the genome of this strain. By deleting the tetC58 locus, we constructed NTL4, a derivative of C58 that no longer mutates to tetracycline resistance. The deletion had no detectable effect on genetic or physiological traits of NTL4 or on the ability of this strain to transform plants.     

Interaction of ochratoxin A with human serum albumin. Binding sites localized by competitive interactions with the native protein and its recombinant fragments

Competitive interactions of ochratoxin A (OTA) and several other acidic compounds were utilized to gain insight into the localization of binding sites and the nature of binding interactions between anionic species and human serum albumin (HSA). Depolarization of OTA fluorescence in the presence of a competing anion was used to quantify ligand-protein interactions. The results obtained were rationalized in terms of OTA displacement from its major binding site. Based on their ability to displace OTA, two distinct groups of the anionic ligands were revealed. The first group contained structurally diverse compounds that shared a common binding site in subdomain IIA (Sudlow Site I). The second group consisted of three non-steroidal anti-inflammatory drugs, which showed much lower affinity to Site I than the OTA dianion. The major site for these drugs was located in domain III. Fluorescence spectroscopy measurements of OTA, warfarin (WAR) and naproxen (NAP) complexes with recombinant proteins corresponding to the domains of HSA (D1-D3) revealed binding to all domains but with different affinities. The binding constants for OTA and WAR decreased in the series D2z.Gt;D3>D1. In contrast, NAP showed the most favorable interaction with D3 and comparable affinities to the two remaining domains. The OTA binding constant for D2, 7.9 x 10(5) M(-1), was smaller than the largest constant for HSA by a factor of approximately 7. The binding constant for OTA with D3, 1.1 x 10(5) M(-1), was very close to that of the secondary binding site for HSA.     

Gliadin T cell epitope selection by tissue transglutaminase in celiac disease. Role of enzyme specificity and pH influence on the transamidation versus deamidation process

Tissue transglutaminase (TG2) can modify proteins by transamidation or deamidation of specific glutamine residues. TG2 has a major role in the pathogenesis of celiac disease as it is both the target of disease-specific autoantibodies and generates deamidated gliadin peptides that are recognized by CD4(+), DQ2-restricted T cells from the celiac lesions. Capillary electrophoresis with fluorescence-labeled gliadin peptides was used to separate and quantify deamidated and transamidated products. In a competition assay, the affinity of TG2 to a set of overlapping gamma-gliadin peptides was measured and compared with their recognition by celiac lesion T cells. Peptides differed considerably in their competition efficiency. Those peptides recognized by intestinal T cell lines showed marked competition indicating them as excellent substrates for TG2. The enzyme fine specificity of TG2 was characterized by synthetic peptide libraries and mass spectrometry. Residues in positions -1, +1, +2, and +3 relative to the targeted glutamine residue influenced the enzyme activity, and proline in position +2 had a particularly positive effect. The characterized sequence specificity of TG2 explained the variation between peptides as TG2 substrates indicating that the enzyme is involved in the selection of gluten T cell epitopes. The enzyme is mainly localized extracellularly in the small intestine where primary amines as substrates for the competing transamidation reaction are present. The deamidation could possibly take place in this compartment as an excess of primary amines did not completely inhibit deamidation of gluten peptides at pH 7.3. However, lowering of the pH decreased the reaction rate of the TG2-catalyzed transamidation, whereas the rate of the deamidation reaction was considerably increased. This suggests that the deamidation of gluten peptides by TG2 more likely takes place in slightly acidic environments.     

Changing the net charge from negative to positive makes ribonuclease Sa cytotoxic

Ribonuclease Sa (pI = 3.5) from Streptomyces aureofaciens and its 3K (D1K, D17K, E41K) (pI = 6.4) and 5K (3K + D25K, E74K) (pI = 10.2) mutants were tested for cytotoxicity. The 5K mutant was cytotoxic to normal and v-ras-transformed NIH3T3 mouse fibroblasts, but RNase Sa and 3K were not. The structure, stability, and activity of the three proteins are comparable, but the net charge at pH 7 increases from -7 for RNase Sa to -1 for 3K and to +3 for 5K. These results suggest that a net positive charge is a key determinant of ribonuclease cytotoxicity. The cytotoxic 5K mutant preferentially attacks v-ras-NIH3T3 fibroblasts, suggesting that mammalian cells expressing the ras-oncogene are potential targets for ribonuclease-based drugs.