Dual role of the adenovirus pVI C terminus as a nuclear localization signal and activator of the viral protease.

Adenain, the protease produced by adenovirus, is regulated by formation of a heterodimer with an 11 aa peptide derived from the C terminus of another adenoviral protein, pVI. Here, the role of the basic motif KRRR, which is conserved in pVI sequences from human adenovirus serotypes, was investigated. It was shown that this motif is less important than the N- or C-terminal regions in the formation of the adenain-peptide heterodimer and in the activity of the subsequent complex. This motif, however, acted as a nuclear localization signal that was capable of targeting heterologous proteins to the nucleus, resulting in a distinctive intranuclear distribution consisting of discrete foci, which is similar to that found for pVI during adenovirus infection.

Two novel muramidases from skin mucosa of rainbow trout (Oncorhynchus mykiss).

Two novel antibacterial muramidases were purified to homogeneity from skin exudates of rainbow trout (Oncorhynchus mykiss). Unusually, one has an acidic isoelectric point and it is the first anionic muramidase to be reported for fish. Its molecular mass is 14,268 Da, as determined by mass spectrometry. The other muramidase is cationic with a mass of 14,252 Da. Partial N-terminal amino acid sequencing and peptide mapping strongly point to it being a c-type lysozyme, the first to be purified and characterised from skin of a salmonid. Its optimum pH ranges from 4.5 to 5.5 and its optimum temperature, at pH 5.0, is 33-49 degrees C, although it still exhibits activity at 5 degrees C. It is strongly bactericidal to the Gram-(+) bacterium Planococcus citreus, with a minimum bactericidal concentration of 100 U ml(-1), but is neither chitinolytic nor haemolytic. These two muramidases probably contribute to epithelial defence of the fish against microbes, either alone or in synergism with antibacterial peptides.

Interaction between hnRNPA1 and IkappaBalpha is required for maximal activation of NF-kappaB-dependent transcription.

Transcriptional activation of NF-kappaB is mediated by signal-induced phosphorylation and degradation of its inhibitor, IkappaBalpha. NF-kappaB activation induces a rapid resynthesis of IkappaBalpha which is responsible for postinduction repression of transcription. Following resynthesis, IkappaBalpha translocates to the nucleus, removes template bound NF-kappaB, and exports NF-kappaB to the cytoplasm in a transcriptionally inactive form. Here we demonstrate that IkappaBalpha interacts directly with another nucleocytoplasmic shuttling protein, hnRNPA1, both in vivo and in vitro. This interaction requires one of the N-terminal RNA binding domains of hnRNPA1 and the C-terminal region of IkappaBalpha. Cells lacking hnRNPA1 are defective in NF-kappaB-dependent transcriptional activation, but the defect in these cells is complemented by ectopic expression of hnRNPA1. hnRNPA1 expression in these cells increased the amount of IkappaBalpha degradation, compared to that of the control cells, in response to activation by Epstein-Barr virus latent membrane protein 1. Thus in addition to regulating mRNA processing and transport, hnRNPA1 also contributes to the control of NF-kappaB-dependent transcription.

Antibacterial proteins in rainbow trout, Oncorhynchus mykiss.

Antibacterial proteins are an important part of the innate immune system for all animals. They have been extensively studied in mammals, amphibians and invertebrates, but have received only scant attention in fish. Their expression and processing, however, provide a way of monitoring defence vigour during development or with seasonal changes in physiology. The aim of the present work was to identify and characterise antibacterial proteins in rainbow trout. In vitro analyses of extracts of the peripheral blood leucocytes, head kidney leucocytes and mucus from adult unstimulated (non-immune) fish showed marked antibacterial activity against Gram positive bacteria. Fractionation by ion exchange chromatography and RP-HPLC of head kidney extracts showed the presence of two forms of lysozyme but no constitutively expressed antimicrobial proteins of < 10 kDa. By contrast, chromatographic analyses of mucus revealed at least four antibacterial proteins. Two are conventional lysozymes, a third is an unusual lysozyme-like protein with a low isoelectric point, and the fourth is a highly hydrophobic, cationic peptide of c. 3 kDa.

Purification and characterization of a cysteine-rich 11.5-kDa antibacterial protein from the granular haemocytes of the shore crab, Carcinus maenas.

Extracts of the granular haemocytes of Carcinus maenas were subjected to ion-exchange chromatography and reverse-phase (RP)-HPLC to investigate the presence of an antibacterial protein of approximately 11 kDa. This protein was isolated, characterized and subjected to partial amino acid sequence analysis. It was found by mass spectrometry to have a molecular mass of 11 534 Da, to be cationic and hydrophobic and active only against marine Gram-positive bacteria. In addition its activity is stable after heating to 100 degrees C and is retained at concentrations as low as 10 microgram.mL-1. It has an unusual amino acid sequence, unlike any known antibacterial peptide described in the literature but bears a consensus disulphide domain signature, indicating that it might be a member of the four-disulphide core proteins. Partial cDNA sequence data has been obtained.

Identification of the enzyme required for activation of the small ubiquitin-like protein SUMO-1.

The ubiquitin-like protein SUMO-1 is conjugated to a variety of proteins including Ran GTPase-activating protein 1 (RanGAP1), IkappaBalpha, and PML. SUMO-1-modified proteins display altered subcellular targeting and/or stability. We have purified the SUMO-1-activating enzyme from human cells and shown that it contains two subunits of 38 and 72 kDa. Isolation of cDNAs for each subunit indicates that they are homologous to ubiquitin-activating enzymes and to the Saccharomyces cerevisiae enzymes responsible for conjugation of Smt3p and Rub-1p. In vitro, recombinant SAE1/SAE2 (SUMO-1-activating enzyme) was capable of catalyzing the ATP-dependent formation of a thioester linkage between SUMO-1 and SAE2. The addition of the SUMO-1-conjugating enzyme Ubch9 resulted in efficient transfer of the thioester-linked SUMO-1 from SAE2 to Ubch9. In the presence of SAE1/SAE2, Ubch9, and ATP, SUMO-1 was efficiently conjugated to the protein substrate IkappaBalpha. As SAE1/SAE2, Ubch9, SUMO-1, and IkappaBalpha are all homogeneous, recombinant proteins, it appears that SUMO-1 conjugation of IkappaBalpha in vitro does not require the equivalent of an E3 ubiquitin protein ligase activity.

Purification and characterization of a proline-rich antibacterial peptide, with sequence similarity to bactenecin-7, from the haemocytes of the shore crab, Carcinus maenas.

Antibacterial peptides are important for non-specific host defence in many animals. They have been extensively characterized from mammals, amphibians, insects and chelicerates but have not so far been found in crustaceans. Here we report the presence of several constitutive antibacterial proteins, active against both gram-positive and gram-negative bacteria, in the haemocytes of the shore crab, Carcinus maenas. These proteins have molecular masses of > 70 kDa, approximately 45 kDa, approximately 14 kDa and 6.5 kDa. The 6.5 kDa peptide has been purified to homogeneity by Sep Pak C18 extraction, gel filtration and reverse-phase HPLC. Partial N-terminal sequence analysis further shows that it is proline rich and shares more than 60% identity in a 28-amino-acid overlap with the mature form of bactenecin 7, an antimicrobial peptide from bovine neutrophils which belongs to the cathelicidin family of mammalian peptide antibiotics.

Chemical mechanisms underlying the vasodilator and platelet anti-aggregating properties of S-nitroso-N-acetyl-DL-penicillamine and S-nitrosoglutathione.

The chemistries of S-nitroso-DL-penicillamine (SNAP) and S-nitrosoglutathione (GSNO) in relation to their ability to relax vascular smooth muscle and prevent platelet aggregation have been investigated. Metal ion catalysis greatly accelerates the decomposition of SNAP, but has little effect on GSNO. Instead, NO release from GSNO is effected either by NO transfer to a free thiol (e.g. cysteine), or by enzymatic cleavage of the glutamyl-cystyl peptide bond. In both cases the resulting nitrosothiol (i.e. S-nitrosocysteine and S-nitrosocystylglycine, respectively) is susceptible to metal ion catalysed NO release. We conclude that transnitrosation or enzymatic cleavage are obligatory steps in the mechanism of NO release from GSNO, whereas SNAP needs only the presence of metal ions to effect this process. The different modes of NO production may go some way towards explaining the different physiological effectiveness of these S-nitrosothiols as vasodilators and inhibitors of platelet aggregation.

Characterization of the adenovirus proteinase: development and use of a specific peptide assay.

An assay system has been developed for the adenovirus endoproteinase which utilizes the synthetic peptide MSGGAFSW, derived from the cleavage site of the adenovirus type 2 (Ad2) protein pVI. MSGGAFSW was shown to be cleaved at the G-A bond when incubated with a source of Ad2 proteinase. Digestions were readily monitored by either fast protein liquid chromatography or thin layer electrophoresis, enabling the rapid production of quantitative data. Comparison of the peptide assay with a previously described [35S]methionine assay system showed it to be faster, cleaner and less prone to extreme conditions of pH and ionic strength. The effect on adenovirus proteinase activity of a number of inhibitors was assessed using both the [35S]methionine and peptide assays. Identical inhibitor profiles were obtained and these suggested that the adenovirus enzyme is a cysteine proteinase. The 23K gene product, thought to be the proteinase, contains cysteine and histidine residues at positions 122 and 54, respectively, that could constitute part of the active site of a cysteine proteinase. These amino acids and their surrounding residues are conserved in all serotypes examined and appear to bear some resemblance to those in the putative active sites of the 3C proteinases in picornaviruses.

Characterization of the adenovirus proteinase: substrate specificity.

Peptides were synthesized based on the cleavage sites in the adenovirus type 2 proteins pVI and pVII. The synthetic peptides were incubated with disrupted, purified adenovirus as a source of proteinase and specific cleavages were monitored by fast protein liquid chromatography and amino acid analysis. Using this approach it was established that all the peptides cleaved were of the form M(L)XGX decreases G or M(L)XGG decreases X. Thus we have shown that the adenoviral proteinase recognizes a specific secondary structure formed by a sequence of at least five amino acids, the main determinants of specificity being two and four residues to the N-terminal side of the bond cleaved. We were able to examine the relevant structural features of the peptide substrates by utilizing the CHEM-X molecular modelling package. Using our consensus sequence we were able to predict the cleavage sites in the viral proteins pVIII, pre-terminal protein (pTP), 11K and IIIa. Octapeptides containing the predicted sites in pVIII and the pTP were synthesized and shown to be cleaved by the proteinase.

Phosphorylation of adenovirus DNA-binding protein.

Evidence is presented here which indicates that the adenovirus DNA-binding protein (DBP) is phosphorylated at a tyrosine residue early in infection. This was suggested by the discovery that a proportion of the label in 32P-labelled DBP was resistant to alkali, and was substantiated by acid hydrolysis of DBP immunoprecipitates and by immunoblotting with a monoclonal antibody against phosphotyrosine. Treatment of [35S]methionine-labelled DBPs with chymotrypsin produced fragments of apparent Mr 45K and 39K whereas digestion of 32P-labelled DBP resulted in fragments of 45K and 26K. Consideration of the distribution of 32P label and its alkali stability in these fragments suggested that chymotrypsin cleaved populations of DBP at different sites depending on their phosphorylation states. The conservation, in all of the seven adenovirus serotypes sequenced, of a tyrosine residue (at amino acid 195 in adenovirus type 2) together with its surrounding residues, suggests that phosphorylation/dephosphorylation at this tyrosine residue may be important in various functions ascribed to the DBP.

The influence of calcium ions on the conformation of fibrinogen fragment D: the use of chemical cross-linking agents.

Fibrinogen fragment D prepared in the presence of calcium ions (fragment D[Ca++]) shows qualitatively similar cross-linking patterns with dimethyl suberimidate, dimethyl adipimidate and tetranitromethane. Fragment D prepared in the presence of EDTA (fragment D[EDTA]) gives a consistently different pattern with these reagents. In the case of fragment D[EDTA] there is much more intermolecular cross-linking suggesting that the loss of the C-terminus of the gamma-chain remnant results in fragment D adopting a more open conformation. Neither the addition of 2M urea nor EDTA to fragment D[Ca++] alters its cross-linking pattern suggesting that the proposed conformational change follows cleavage of a plasmin susceptible bond which is normally protected by the presence of calcium ions.

The preparation of an alkali-soluble collagen from demineralized bone.

Ossein was solubilized by the action of alkali and a resulting high-molecular-weight fraction isolated. The chemical and physical properties of this fraction were studied and compared with those of an acid-soluble collagen prepared from calf skin by conventional techniques. From the results it is concluded that the alkali-soluble protein exhibits only minor differences from acid-soluble collagen, and that these differences can be ascribed for the most part to a decrease in the inter- and intramolecular cross-linking.