Natively unstructured regions in proteins identified from contact predictions

MOTIVATION: Natively unstructured (also dubbed intrinsically disordered) regions in proteins lack a defined 3D structure under physiological conditions and often adopt regular structures under particular conditions. Proteins with such regions are overly abundant in eukaryotes, they may increase functional complexity of organisms and they usually evade structure determination in the unbound form. Low propensity for the formation of internal residue contacts has been previously used to predict natively unstructured regions. RESULTS: We combined PROFcon predictions for protein-specific contacts with a generic pairwise potential to predict unstructured regions. This novel method, Ucon, outperformed the best available methods in predicting proteins with long unstructured regions. Furthermore, Ucon correctly identified cases missed by other methods. By computing the difference between predictions based on specific contacts (approach introduced here) and those based on generic potentials (realized in other methods), we might identify unstructured regions that are involved in protein-protein binding. We discussed one example to illustrate this ambitious aim. Overall, Ucon added quality and an orthogonal aspect that may help in the experimental study of unstructured regions in network hubs. AVAILABILITY: http://www.predictprotein.org/submit_ucon.html. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.     

Variability of the 16S-23S rRNA gene internal transcribed spacer in Pseudomonas avellanae strains

The 16S-23S rRNA gene internal transcribed spacer region (ITS1) from 34 strains of Pseudomonas avellanae and some strains of Pseudomonas syringae pathovars was amplified and assessed by restriction fragment length polymorphism (RFLP) using 10 restriction enzymes. In addition, the ITS1 region of four representative P. avellanae strains was sequenced and compared by the neighbour-joining algorithm with that of P. syringae pathovars. Two main groups of P. avellanae strains were observed that did not correlate with their origin. The ITS1 region sequencing revealed a high similarity with the P. syringae complex. One group of P. avellanae strains showed high similarity to P. s. pv. actinidiae and P. s. pv. tomato; another group showed similarity with P. s. pv. tabaci and P. s. pv. glycinea. Two strains clustered with P. s. pv. pisi. The difficulties to unambiguously classify the strains associated with hazelnut decline in Greece and Italy are discussed.     

Repetitive DNA, molecular cytogenetics and genome organization in the King scallop (Pecten maximus)

We studied the structure, organization and relationship of repetitive DNA sequences in the genome of the scallop, Pecten maximus, a bivalve that is important both commercially and in marine ecology. Recombinant DNA libraries were constructed after partial digestion of genomic DNA from scallop with PstI and ApaI restriction enzymes. Clones containing repetitive DNA were selected by hybridisation to labelled DNA from scallop, oyster and mussel; colonies showing strong hybridisation only to scallop were selected for analysis and sequencing. Six non-homologous tandemly repeated sequences were identified in the sequences, and Southern hybridisation with all repeat families to genomic DNA digests showed characteristic ladders of hybridised bands. Three families had monomer lengths around 40 bp while three had repeats characteristic of the length wrapping around one (170 bp), or two (326 bp) nucleosomes. In situ hybridisation to interphase nuclei showed each family had characteristic numbers of clusters indicating contrasting arrangements. Two of the repeats had unusual repetitions of bases within their sequence, which may relate to the nature of microsatellites reported in bivalves. The study of these rapidly evolving sequences is valuable to understand an important source of genomic diversity, has the potential to provide useful markers for population studies and gives a route to identify mechanisms of DNA sequence evolution.     

High sensitivity of human centrin 2 toward radiolytical oxidation: C-terminal tyrosinyl residue as the main target

Centrins are calcium-binding proteins that play a significant role in the maintenance of the centrosomal organization, mainly in the continuity between centrosome and microtubular network. Recent data showed that centrosome duplication abnormalities, like overduplication for example, could be due to hydrogen peroxide, suggesting an important impact of oxidative stress. To challenge this hypothesis, we performed one-electron oxidation experiments with human centrin 2, starting from azide radicals. Our results first revealed several intermolecular cross-links generating dimers, tetramers, hexamers, and higher molecular mass species. Dimers result from covalent bond linking the C-terminal tyrosines of each monomer. Second, the methionyl residue at position 19 was oxidized on the monomeric centrin. Further, electron microscopy experiments on centrin 2 showed a preexisting hexameric organization that was stabilized by covalent bonds as a result of irradiation. Overall, these results show that centrin 2 is highly sensitive to ionizing radiation, which could have important consequences on its biological functions.     

BibA: a novel immunogenic bacterial adhesin contributing to group B Streptococcus survival in human blood

By the analysis of the recently sequenced genomes of Group B Streptococcus (GBS) we have identified a novel immunogenic adhesin with anti-phagocytic activity, named BibA. The bibA gene is present in 100% of the 24 GBS strains analysed. BibA-specific IgG were found in human sera from normal healthy donors. The putative protein product is a polypeptide of 630 amino acids containing a helix-rich N-terminal domain, a proline-rich region and a canonical LPXTG cell wall-anchoring domain. BibA is expressed on the surface of several GBS strains, but is also recovered in GBS culture supernatants. BibA specifically binds to human C4-binding protein, a regulator of the classic complement pathway. Deletion of the bibA gene severely reduced the capacity of GBS to survive in human blood and to resist opsonophagocytic killing by human neutrophils. In addition, BibA expression increased the virulence of GBS in a mouse infection model. The role of BibA in GBS adhesion was demonstrated by the impaired ability of a bibA knockout mutant strain to adhere to both human cervical and lung epithelial cells. Furthermore, we calculated that recombinant BibA bound to human epithelial cells of distinct origin with an affinity constant of approximately 10(-8) M for cervical epithelial cells. Hence BibA is a novel multifunctional protein involved in both resistance to phagocytic killing and adhesion to host cells. The identification of this potential new virulence factor represents an important step in the development of strategies to combat GBS-associated infections.     

CcpA mutants with differential activities in Bacillus subtilis

CcpA is the master regulator for carbon catabolite regulation in Bacillus subtilis and regulates more than 300 genes by repression or activation. To revealthe effects of different functional domains of CcpA on various regulatory modes, we compared the activities of CcpA point mutants in activation (alsS, ackA) and repression (xynP, gntR). CcpA variants mutated at residues in the HPrSerP-binding region without allosteric functions are inactive. On the other hand, CcpA variants mutated at residues that change their conformation upon HPrSerP or CrhP binding regulate only ackA. Another set of mutants with alterations in the corepressor-binding region show glucose-independent regulation of xynP. The data presented here demonstrate the involvement of HPrSerP and/or CrhP in activation of ackA and alsS by CcpA. Furthermore, these data also indicate that activation and repression mediated by CcpA may utilize different conformational changes of the protein.     

Surfactant disaturated-phosphatidylcholine kinetics in acute respiratory distress syndrome by stable isotopes and a two compartment model

Background

In patients with acute respiratory distress syndrome (ARDS), it is well known that only part of the lungs is aerated and surfactant function is impaired, but the extent of lung damage and changes in surfactant turnover remain unclear. The objective of the study was to evaluate surfactant disaturated-phosphatidylcholine turnover in patients with ARDS using stable isotopes.

Methods

We studied 12 patients with ARDS and 7 subjects with normal lungs. After the tracheal instillation of a trace dose of ¹³C-dipalmitoyl-phosphatidylcholine, we measured the ¹³C enrichment over time of palmitate residues of disaturated-phosphatidylcholine isolated from tracheal aspirates. Data were interpreted using a model with two compartments, alveoli and lung tissue, and kinetic parameters were derived assuming that, in controls, alveolar macrophages may degrade between 5 and 50% of disaturated-phosphatidylcholine, the rest being lost from tissue. In ARDS we assumed that 5–100% of disaturated-phosphatidylcholine is degraded in the alveolar space, due to release of hydrolytic enzymes. Some of the kinetic parameters were uniquely determined, while others were identified as lower and upper bounds.

Results

In ARDS, the alveolar pool of disaturated-phosphatidylcholine was significantly lower than in controls (0.16 ± 0.04 vs. 1.31 ± 0.40 mg/kg, p < 0.05). Fluxes between tissue and alveoli and de novo synthesis of disaturated-phosphatidylcholine were also significantly lower, while mean resident time in lung tissue was significantly higher in ARDS than in controls. Recycling was 16.2 ± 3.5 in ARDS and 31.9 ± 7.3 in controls (p = 0.08).

Conclusion

In ARDS the alveolar pool of surfactant is reduced and disaturated-phosphatidylcholine turnover is altered.     

Assessment of algorithms for high throughput detection of genomic copy number variation in oligonucleotide microarray data

Background

Genomic deletions and duplications are important in the pathogenesis of diseases, such as cancer and mental retardation, and have recently been shown to occur frequently in unaffected individuals as polymorphisms. Affymetrix GeneChip whole genome sampling analysis (WGSA) combined with 100 K single nucleotide polymorphism (SNP) genotyping arrays is one of several microarray-based approaches that are now being used to detect such structural genomic changes. The popularity of this technology and its associated open source data format have resulted in the development of an increasing number of software packages for the analysis of copy number changes using these SNP arrays.

Results

We evaluated four publicly available software packages for high throughput copy number analysis using synthetic and empirical 100 K SNP array data sets, the latter obtained from 107 mental retardation (MR) patients and their unaffected parents and siblings. We evaluated the software with regards to overall suitability for high-throughput 100 K SNP array data analysis, as well as effectiveness of normalization, scaling with various reference sets and feature extraction, as well as true and false positive rates of genomic copy number variant (CNV) detection.

Conclusion

We observed considerable variation among the numbers and types of candidate CNVs detected by different analysis approaches, and found that multiple programs were needed to find all real aberrations in our test set. The frequency of false positive deletions was substantial, but could be greatly reduced by using the SNP genotype information to confirm loss of heterozygosity.