1H, 13C and 15N assignment of the GNA1946 outer membrane lipoprotein from Neisseria meningitidis.

GNA1946 (Genome-derived Neisseria Antigen 1946) is a highly conserved exposed outer membrane lipoprotein from Neisseria meningitidis bacteria of 287 amino acid length (31 kDa). Although the structure of NMB1946 has been solved recently by X-Ray crystallography, understanding the behaviour of GNA1946 in aqueuos solution is highly relevant for the discovery of the antigenic determinants of the protein that will possibly lead to a more efficient vaccine development against virulent serogroup B strain of N. meningitidis. Here we report almost complete (1)H, (13)C and (15)N resonance assignments of GNA1946 (residues 10-287) in aqueous buffer solution.

cis-Inhibition of proteasomal degradation by viral repeats: impact of length and amino acid composition.

The Gly-Ala repeat (GAr) of the Epstein-Barr virus nuclear antigen 1 is a cis acting inhibitor of ubiquitin-proteasome proteolysis. We have investigated the capacity of various repeats to inhibit the turnover of the proteasomal substrate IkappaBalpha. Inhibition of TNFalpha-induced degradation was achieved by insertion of octamers containing three alanines or valines, interspersed by no more then three consecutive glycines. The inhibitory activity was abolished by increasing the length of the spacer, by eliminating the spacers, or by substitution of a single hydrophobic residue with a polar or charged residue. A serine containing octamer was inactive but inhibition was partially restored by insertion of three consecutive repeats. These findings suggest a model where inhibition requires the interaction of at least three alanine residues of the GAr in a beta-strand conformation with adjacent hydrophobic binding pockets of a putative receptor.

Molecular cloning, expression, and purification of pig interleukin-5.

Interleukin-5 (IL-5) is thought to be a key cytokine in allergic inflammation. Pig IL-5 was cloned, sequenced, and expressed to enable us to study of the biological role of IL-5 in pigs used in a model for allergen-induced late-phase reactions. These pigs were sensitized to proteins extracted from Ascaris suum, resulting in hypersensitivity to this antigen in both the skin and airways, and a slight blood eosinophilia. Peripheral blood mononuclear cells from antigen-sensitized pigs were isolated and polyclonally stimulated. Total RNA was extracted and reverse transcribed into cDNA. IL-5 primers based on the cow IL-5 cDNA sequence were used to obtain an initial polymerase chain reaction product. 3' rapid amplification of cDNA ends (3'RACE) and 5'RACE procedures were applied to identify the 3' and 5' ends, respectively. The full-length pig IL-5 cDNA is 405 base pairs long. Mature pig IL-5 was expressed in Escherichia coli with a His-tag for purification. The IL-5 protein is 115 amino acids long, has an estimated molecular weight of 14 000 M(r) and forms a biologically active homodimer of 28 000 M(r). Pig IL-5 shows 65% amino acid identity to the human IL-5 sequence and 90, 88, 83, 62, and 61% identity to the cow, sheep, horse, mouse, and rat counterparts.

A minimal glycine-alanine repeat prevents the interaction of ubiquitinated I kappaB alpha with the proteasome: a new mechanism for selective inhibition of proteolysis.

The Epstein-Barr virus nuclear antigen 1 contains a glycine-alanine repeat that inhibits in cis MHC class I-restricted presentation. We report here that insertion of a minimal glycine-alanine repeat motif in different positions of I kappaB alpha protects this NF-kappaB inhibitor from signal-induced degradation dependent on ubiquitin-proteasome, and decreases its basal turnover in vivo resulting in constitutive dominant-negative mutants. The chimeras are phosphorylated and ubiquitinated in response to tumor necrosis factor alpha, but are then released from NF-kappaB and fail to associate with the proteasome. This explains how functionally competent I kappaB alpha is protected from proteasomal disruption and identifies the glycine-alanine repeat as a new regulator of proteolysis.

Random coil conformation of a Gly/Ala-rich insert in IkappaB alpha excludes structural stabilization as the mechanism for protection against proteasomal degradation.

Peptide segments of multiple glycine and alanine residues prevent the proteolytic degradation of ubiquitinated proteins by the proteasome. The structure of a Gly/Ala-rich insert in IkappaB alpha was probed by nuclear magnetic resonance (NMR) spectroscopy, comparing IkappaB alpha samples with and without Gly/Ala-rich insert. Narrow 1H-NMR resonances at chemical shifts indicative of random coil conformations were observed in the difference spectrum. circular dichroism (CD) measurements further confirm that the mechanism of protection against proteolytic degradation is not based on structural transition or stabilization caused by the Gly/Ala-rich segment. In addition, most of the N- and C-terminal residues outside the ankyrin repeats in wild-type IkappaB alpha were found to be flexibly disordered.

Inhibition of ubiquitin/proteasome-dependent protein degradation by the Gly-Ala repeat domain of the Epstein-Barr virus nuclear antigen 1.

The Epstein-Barr virus (EBV) encoded nuclear antigen (EBNA) 1 is expressed in latently infected B lymphocytes that persist for life in healthy virus carriers and is the only viral protein regularly detected in all EBV associated malignancies. The Gly-Ala repeat domain of EBNA1 was shown to inhibit in cis the presentation of major histocompatibility complex (MHC) class I restricted cytotoxic T cell epitopes from EBNA4. It appears that the majority of antigens presented via the MHC I pathway are subject to ATP-dependent ubiquitination and degradation by the proteasome. We have investigated the influence of the repeat on this process by comparing the degradation of EBNA1, EBNA4, and Gly-Ala containing EBNA4 chimeras in a cell-free system. EBNA4 was efficiently degraded in an ATP/ubiquitin/proteasome-dependent fashion whereas EBNA1 was resistant to degradation. Processing of EBNA1 was restored by deletion of the Gly-Ala domain whereas insertion of Gly-Ala repeats of various lengths and in different positions prevented the degradation of EBNA4 without appreciable effect on ubiquitination. Inhibition was also achieved by insertion of a Pro-Ala coding sequence. The results suggest that the repeat may affect MHC I restricted responses by inhibiting antigen processing via the ubiquitin/proteasome pathway. The presence of regularly interspersed Ala residues appears to be important for the effect.