XLF and APLF bind Ku80 at two remote sites to ensure DNA repair by non-homologous end joining.

The Ku70-Ku80 (Ku) heterodimer binds rapidly and tightly to the ends of DNA double-strand breaks and recruits factors of the non-homologous end-joining (NHEJ) repair pathway through molecular interactions that remain unclear. We have determined crystal structures of the Ku-binding motifs (KBM) of the NHEJ proteins APLF (A-KBM) and XLF (X-KBM) bound to a Ku-DNA complex. The two KBM motifs bind remote sites of the Ku80 α/β domain.

[Evidence of antihypertensive effect of a land snail (Helix aspersa) by-product hydrolysate - Identification of involved peptides].

The antihypertensive potential of a land snail by-product hydrolysate, obtained after an agri-food processing of the raw material, was studied in vitro and in vivo. First, the ACE inhibitory activity of hydrolysates obtained before and after an ultrafiltration step with a 10kDa membrane cutoff was assayed. The IC50 obtained were of 98.

PAXX Is an Accessory c-NHEJ Factor that Associates with Ku70 and Has Overlapping Functions with XLF.

In mammalian cells, classical non-homologous end joining (c-NHEJ) is critical for DNA double-strand break repair induced by ionizing radiation and during V(D)J recombination in developing B and T lymphocytes. Recently, PAXX was identified as a c-NHEJ core component. We report here that PAXX-deficient cells exhibit a cellular phenotype uncharacteristic of a deficiency in c-NHEJ core components.

C-terminal region of bacterial Ku controls DNA bridging, DNA threading and recruitment of DNA ligase D for double strand breaks repair.

Non-homologous end joining is a ligation process repairing DNA double strand breaks in eukaryotes and many prokaryotes. The ring structured eukaryotic Ku binds DNA ends and recruits other factors which can access DNA ends through the threading of Ku inward the DNA, making this protein a key ingredient for the scaffolding of the NHEJ machinery. However, this threading ability seems unevenly conserved among bacterial Ku.

Structural characterization of filaments formed by human Xrcc4-Cernunnos/XLF complex involved in nonhomologous DNA end-joining.

Cernunnos/XLF is a core protein of the nonhomologous DNA end-joining (NHEJ) pathway that processes the majority of DNA double-strand breaks in mammals. Cernunnos stimulates the final ligation step catalyzed by the complex between DNA ligase IV and Xrcc4 (X4). Here we present the crystal structure of the X4(1-157)-Cernunnos(1-224) complex at 5.

Conformational exchange is critical for the productivity of an oxidative folding intermediate with buried free cysteines.

Much has been learned about the folding of proteins from comparative studies of the folding of proteins that are related in sequence and structure. Observation of the effects of mutations helps account for sequence-specific properties and large variations in folding rates observed in homologous proteins, which are not explained by structure-derived descriptions. The folding kinetics of variants of a β-stranded protein, toxin α from Naja nigricollis, depends on the length of their loop lk1.

Delineation of the Xrcc4-interacting region in the globular head domain of cernunnos/XLF.

In mammals, the majority of DNA double-strand breaks are processed by the nonhomologous end-joining (NHEJ) pathway, composed of seven factors: Ku70, Ku80, DNA-PKcs, Artemis, Xrcc4 (X4), DNA-ligase IV (L4), and Cernunnos/XLF. Cernunnos is part of the ligation complex, constituted by X4 and L4. To improve our knowledge on the structure and function of Cernunnos, we performed a systematic mutagenesis study on positions selected from an analysis of the recent three-dimensional structures of this factor.

Conserved structural determinants in three-fingered protein domains.

The three-dimensional structures of some components of snake venoms forming so-called 'three-fingered protein' domains (TFPDs) are similar to those of the ectodomains of activin, bone morphogenetic protein and transforming growth factor-beta receptors, and to a variety of proteins encoded by the Ly6 and Plaur genes. The analysis of sequences of diverse snake toxins, various ectodomains of the receptors that bind activin and other cytokines, and numerous gene products encoded by the Ly6 and Plaur families of genes has revealed that they differ considerably from each other. The sequences of TFPDs may consist of up to six disulfide bonds, three of which have the same highly conserved topology.

Increasing the humoral immunogenic properties of the HIV-1 Tat protein using a ligand-stabilizing strategy.

Tat is regarded as an attractive target for the development of an AIDS vaccine. However, works suggest that Tat is a poorly immunogenic protein and therefore we attempted to increase its immunogenic potency. As we observed that Tat is highly sensitive to enzymatic degradation in vitro we tried to make it less susceptible to proteolysis using ligands.

Dual expression system suitable for high-throughput fluorescence-based screening and production of soluble proteins.

Many studies that aim to characterize the proteome structurally or functionally require the production of pure protein in a high-throughput format. We have developed a fast and flexible integrated system for cloning, protein expression in Escherichia coli, solubility screening and purification that can be completely automated in a 96-well microplate format. We used recombination cloning in custom-designed vectors including (i) a (His)(6) tag-encoding sequence, (ii) a variable solubilizing partner gene, (iii) the DNA sequence corresponding to the TEV protease cleavage site, (iv) the gene (or DNA fragment) of interest, (v) a suppressible amber stop codon, and (vi) an S.

HIV-1 Tat raises an adjuvant-free humoral immune response controlled by its core region and its ability to form cysteine-mediated oligomers.

Proteins are poor immunogens that require an adjuvant to raise an immune response. Here we show that the human immunodeficiency virus, type 1 Tat protein possesses an autoadjuvant property, and we have identified the determinants and the molecular events that are associated with this unusual property. Using a series of chemically synthesized Tat101 derivatives, we show that the core region controls the autoadjuvant phenomenon independently of the B-cell recognition and T-cell stimulation that are associated with epitopes respectively located on the N-terminal region and the cysteine-rich region.

Toxicity-based selection of Escherichia coli mutants for functional recombinant protein production: application to an antibody fragment.

We propose a novel approach to the selection of Escherichia coli bacterial strains improved for the production of recombinant functional proteins. This approach is based on aggregation-induced toxicity of recombinant proteins. We show that selection of clones displaying a reduced toxicity is an efficient means of isolating bacteria producing recombinant protein with reduced aggregation in favour of correct folding.

Relative spatial position of a snake neurotoxin and the reduced disulfide bond alpha (Cys192-Cys193) at the alpha gamma interface of the nicotinic acetylcholine receptor.

We determined the distances separating five functionally important residues (Gln(10), Lys(27), Trp(29), Arg(33), and Lys(47)) of a three-fingered snake neurotoxin from the reduced disulfide bond alpha(Cys(192)-Cys(193)) located at the alphagamma interface of the Torpedo nicotinic acetylcholine receptor. Each toxin position was substituted individually for a cysteine, which was then linked to a maleimido moiety through three different spacers, varying in length from 10 to 22 A. We estimated the coupling efficiency between the 15 toxin derivatives and the reduced cystine alpha(192-193) by gel densitometry of Coomassie Blue-stained gels.

Recombinant Staphylococcus strains as live vectors for the induction of neutralizing anti-diphtheria toxin antisera.

We have investigated whether the nonpathogenic gram-positive bacteria Staphylococcus xylosus and S. carnosus can display a whole domain of a toxic protein on their surface and if such vectors are suitable for immunization of BALB/c mice. The nucleotide sequence encoding the receptor-binding domain (DTR; amino acids 382 to 535) of diphtheria toxin (DT) was inserted into plasmids pSE'mp18ABPXM and pSPPmABPXM, which were designed to display heterologous proteins on S.

Increasing immunogenicity of antigens fused to Ig-binding proteins by cell surface targeting.

Fusion of antigenic proteins to Ig-binding proteins such as protein A from Staphylococcus aureus and its derived ZZ fragment is known to increase immunogenicity of the fused Ag in vivo. To shed light on the origin of this effect, we used snake toxins as Ags and observed that 1) fusion of toxins to ZZ enhanced their presentation to a toxin-specific T cell hybridoma (T1B2), using A20 B lymphoma cells, splenocytes, or peritoneal exudate cells as APCs; 2) this enhancement further increased when the number of fused Ig-binding domains varied from two with ZZ to five with protein A; and 3) the phenomenon vanished when the fusion protein was preincubated with an excess of free ZZ or when P388D1 monocytes cells were used as APCs. Therefore, ZZ-fused toxins are likely to be targeted to surface Igs of APCs by their ZZ moiety.

Transformation of a non-enzymatic toxin into a toxoid by genetic engineering.

Curaremimetic toxins are typical non-enzymatic toxins that bind to their target [the nicotinic acetylcholine receptor (AChR)] through multiple residues. Nevertheless, we show that the concomitant substitutions of only three of the ten functionally important residues of such a toxin sufficed to cause an affinity decrease of the toxin for AChR that is higher than four orders of magnitude. Despite these triple mutations, the overall conformation of the mutated protein remains similar to that of a related recombinant toxin, as judged from both circular dichroism analysis and investigation of antigenicity, using monoclonal and polyclonal antibodies.

Towards a recombinant vaccine against diphtheria toxin.

Two recombinant fragments of diphtheria toxin (DT) were fused to an engineered tandem repeat of the immunoglobulin (Ig) binding domain of protein A, called ZZ. These fragments are (i) the receptor binding domain (DTR), which comprises amino acids 382 to 535 of DT, and (ii) a linear peptide (DT(168-220)) which comprises residues 168 to 220 of the loop between fragment A and fragment B of DT. The fusion proteins were produced in Escherichia coli and purified by affinity chromatography.

Picosecond to hour time scale dynamics of a "three finger" toxin: correlation with its toxic and antigenic properties.

Toxin alpha from Naja nigricollis (61 amino acids, four disulfide bridges) belongs to the "three finger" fold family, which contains snake toxins with various biological activities and nontoxic proteins from different origins. In this paper, we report an extensive 1H and 15N NMR study of the dynamics of toxin alpha in solution. 15N relaxation, 1H off-resonance ROESY, and H-D exchange experiments allowed us to probe picosecond to hour motions in the protein.

Only snake curaremimetic toxins with a fifth disulfide bond have high affinity for the neuronal alpha7 nicotinic receptor.

Long chain and short chain curaremimetic toxins from snakes possess 66-74 residues with five disulfide bonds and 60-62 residues with four disulfide bonds, respectively. Despite their structural differences all of these toxins bind with high affinity to the peripheral nicotinic acetylcholine receptors (AChR). Binding experiments have now revealed that long chain toxins only, like the neuronal kappa-bungarotoxin, have a high affinity for a chimeric form of the neuronal alpha7 receptor, with Kd values ranging from about 1 to 12 nM.

High-level production and isotope labeling of snake neurotoxins, disulfide-rich proteins.

The aim of this work was to produce and to label snake neurotoxins, disulfide-rich proteins. A mutant of a snake toxin, erabutoxin a, was used as a model. Its N-terminal part was fused to ZZ, a synthetic IgG-binding domain of protein A (B.

Mimicry between receptors and antibodies. Identification of snake toxin determinants recognized by the acetylcholine receptor and an acetylcholine receptor-mimicking monoclonal antibody.

In several instances, a monoclonal antibody raised against a receptor ligand has been claimed to mimic the ligand receptor. Thus, a specific monoclonal antibody (Malpha2-3) raised against a short-chain toxin from snake was proposed to mimic the nicotinic acetylcholine receptor (AChR) (). Further confirming this mimicry, we show that (i) like AChR, Malpha2-3 elicits anti-AChR antibodies, which in turn elicit anti-toxin antibodies; and (ii) the region 106-122 of the alpha-chain of AChR shares 66% primary structure identity with complementarity-determining regions of Malpha2-3.

Recombinant antibody-alkaline phosphatase conjugates for diagnosis of human IgGs: application to anti-HBsAg detection.

We have designed an expression vector permitting the production in the periplasm of Escherichia coli of a fusion protein comprising a dimer of bacterial alkaline phosphatase (PhoA) and two Fab or scFv fragments of a monoclonal antibody directed against human IgG. Each hybrid protein expressed both high specificity for the antigen and full PhoA activity. We show that crude periplasmic extracts containing these conjugates can be used as such in enzyme immunoassays for the detection of human IgG, as exemplified in the case of anti-hepatitis B immunoglobulin.

Genetic engineering of snake toxins. The functional site of Erabutoxin a, as delineated by site-directed mutagenesis, includes variant residues.

Using site-directed mutagenesis, we previously identified some residues that probably belong to the site by which Erabutoxin a (Ea), a sea snake toxin, recognizes the nicotinic acetylcholine receptor (AcChoR) (Pillet, L., Trémeau, O., Ducancel, F.

On the site by which alpha-dendrotoxin binds to voltage-dependent potassium channels: site-directed mutagenesis reveals that the lysine triplet 28-30 is not essential for binding.

We constructed a synthetic gene encoding the published amino acid sequence of DTx from Dendroaspis angusticeps, a ligand of voltage-dependent postassium channels that facilitates neurotransmitter release. We expressed it in Escherichia coli as a fusion protein secreted in the culture medium. The recombinant DTx was generated in vitro by chemical treatment and recovered as two isoforms.