Prediction of cis/trans isomerization in proteins using PSI-BLAST profiles and secondary structure information

Background The majority of peptide bonds in proteins are found to occur in the trans conformation. However, for proline residues, a considerable fraction of Prolyl peptide bonds adopt the cis form. Proline cis/trans isomerization is known to play a critical role in protein folding, splicing, cell signaling and transmembrane active transport. Accurate prediction of proline cis/trans isomerization in proteins would have many important applications towards the understanding of protein structure and function. Results In this paper, we propose a new approach to predict the proline cis/trans isomerization in proteins using support vector machine (SVM). The preliminary results indicated that using Radial Basis Function (RBF) kernels could lead to better prediction performance than that of polynomial and linear kernel functions. We used single sequence information of different local window sizes, amino acid compositions of different local sequences, multiple sequence alignment obtained from PSI-BLAST and the secondary structure information predicted by PSIPRED. We explored these different sequence encoding schemes in order to investigate their effects on the prediction performance. The training and testing of this approach was performed on a newly enlarged dataset of 2424 non-homologous proteins determined by X-Ray diffraction method using 5-fold cross-validation. Selecting the window size 11 provided the best performance for determining the proline cis/trans isomerization based on the single amino acid sequence. It was found that using multiple sequence alignments in the form of PSI-BLAST profiles could significantly improve the prediction performance, the prediction accuracy increased from 62.8% with single sequence to 69.8% and Matthews Correlation Coefficient (MCC) improved from 0.26 with single local sequence to 0.40. Furthermore, if coupled with the predicted secondary structure information by PSIPRED, our method yielded a prediction accuracy of 71.5% and MCC of 0.43, 9% and 0.17 higher than the accuracy achieved based on the singe sequence information, respectively. Conclusion A new method has been developed to predict the proline cis/trans isomerization in proteins based on support vector machine, which used the single amino acid sequence with different local window sizes, the amino acid compositions of local sequence flanking centered proline residues, the position-specific scoring matrices (PSSMs) extracted by PSI-BLAST and the predicted secondary structures generated by PSIPRED. The successful application of SVM approach in this study reinforced that SVM is a powerful tool in predicting proline cis/trans isomerization in proteins and biological sequence analysis.

Predicting residue-wise contact orders in proteins by support vector regression

Background The residue-wise contact order (RWCO) describes the sequence separations between the residues of interest and its contacting residues in a protein sequence. It is a new kind of one-dimensional protein structure that represents the extent of long-range contacts and is considered as a generalization of contact order. Together with secondary structure, accessible surface area, the B factor, and contact number, RWCO provides comprehensive and indispensable important information to reconstructing the protein three-dimensional structure from a set of one-dimensional structural properties. Accurately predicting RWCO values could have many important applications in protein three-dimensional structure prediction and protein folding rate prediction, and give deep insights into protein sequence-structure relationships. Results We developed a novel approach to predict residue-wise contact order values in proteins based on support vector regression (SVR), starting from primary amino acid sequences. We explored seven different sequence encoding schemes to examine their effects on the prediction performance, including local sequence in the form of PSI-BLAST profiles, local sequence plus amino acid composition, local sequence plus molecular weight, local sequence plus secondary structure predicted by PSIPRED, local sequence plus molecular weight and amino acid composition, local sequence plus molecular weight and predicted secondary structure, and local sequence plus molecular weight, amino acid composition and predicted secondary structure. When using local sequences with multiple sequence alignments in the form of PSI-BLAST profiles, we could predict the RWCO distribution with a Pearson correlation coefficient (CC) between the predicted and observed RWCO values of 0.55, and root mean square error (RMSE) of 0.82, based on a well-defined dataset with 680 protein sequences. Moreover, by incorporating global features such as molecular weight and amino acid composition we could further improve the prediction performance with the CC to 0.57 and an RMSE of 0.79. In addition, combining the predicted secondary structure by PSIPRED was found to significantly improve the prediction performance and could yield the best prediction accuracy with a CC of 0.60 and RMSE of 0.78, which provided at least comparable performance compared with the other existing methods. Conclusion The SVR method shows a prediction performance competitive with or at least comparable to the previously developed linear regression-based methods for predicting RWCO values. In contrast to support vector classification (SVC), SVR is very good at estimating the raw value profiles of the samples. The successful application of the SVR approach in this study reinforces the fact that support vector regression is a powerful tool in extracting the protein sequence-structure relationship and in estimating the protein structural profiles from amino acid sequences.

p53-independent NOXA induction overcomes apoptotic resistance of malignant melanomas

Once melanoma metastasizes, no effective treatment modalities prolong survival in most patients. This notorious refractoriness to therapy challenges investigators to identify agents that overcome melanoma resistance to apoptosis. Whereas many survival pathways contribute to the death-defying phenotype in melanoma, a defect in apoptotic machinery previously highlighted inactivation of Apaf-1, an apoptosome component engaged after mitochondrial damage. During studies involving Notch signaling in melanoma, we observed a gamma-secretase tripeptide inhibitor (GSI; z-Leu-Leu-Nle-CHO), selected from a group of compounds originally used in Alzheimer's disease, induced apoptosis in nine of nine melanoma lines. GSI only induced G2-M growth arrest (but not killing) in five of five normal melanocyte cultures tested. Effective killing of melanoma cells by GSI involved new protein synthesis and a mitochondrial-based pathway mediated by up-regulation of BH3-only members (Bim and NOXA). p53 activation was not necessary for up-regulation of NOXA in melanoma cells. Blocking GSI-induced NOXA using an antisense (but not control) oligonucleotide significantly reduced the apoptotic response. GSI also killed melanoma cell lines with low Apaf-1 levels. We conclude that GSI is highly effective in killing melanoma cells while sparing normal melanocytes. Direct enhancement of BH3-only proteins executes an apoptotic program overcoming resistance of this lethal tumor. Identification of a p53-independent apoptotic pathway in melanoma cells, including cells with low Apaf-1, bypasses an impediment to current cytotoxic therapy and provides new targets for future therapeutic trials involving chemoresistant tumors.

Combined mitochondrial and nuclear DNA sequences resolve the interrelations of the major Australasian marsupial radiations

Australasian marsupials include three major radiations, the insectivorous/carnivorous Dasyuromorphia, the omnivorous bandicoots (Peramelemorphia), and the largely herbivorous diprotodontians. Morphologists have generally considered the bandicoots and diprotodontians to be closely related, most prominently because they are both syndactylous (with the 2nd and 3rd pedal digits being fused). Molecular studies have been unable to confirm or reject this Syndactyla hypothesis. Here we present new mitochondrial (mt) genomes from a spiny bandicoot (Echymipera rufescens) and two dasyurids, a fat-tailed dunnart (Sminthopsis crassicaudata) and a northern quoll (Dasyurus hallucatus). By comparing trees derived from pairwise base-frequency differences between taxa with standard (absolute, uncorrected) distance trees, we infer that composition bias among mt protein-coding and RNA sequences is sufficient to mislead tree reconstruction. This can explain incongruence between trees obtained from mt and nuclear data sets. However, after excluding major sources of compositional heterogeneity, both the “reduced-bias” mt and nuclear data sets clearly favor a bandicoot plus dasyuromorphian association, as well as a grouping of kangaroos and possums (Phalangeriformes) among diprotodontians. Notably, alternatives to these groupings could only be confidently rejected by combining the mt and nuclear data. Elsewhere on the tree, Dromiciops appears to be sister to the monophyletic Australasian marsupials, whereas the placement of the marsupial mole (Notoryctes) remains problematic. More generally, we contend that it is desirable to combine mt genome and nuclear sequences for inferring vertebrate phylogeny, but as separately modeled process partitions. This strategy depends on detecting and excluding (or accounting for) major sources of nonhistorical signal, such as from compositional nonstationarity.

Dating of divergences within the Rattus genus phylogeny using whole mitochondrial genomes

The timing and order of divergences within the genus Rattus have, to date, been quite speculative. In order to address these important issues we sequenced six new whole mitochondrial genomes from wild-caught specimens from four species, Rattus exulans, Rattus praetor, Rattus rattus and Rattus tanezumi. The only rat whole mitochondrial genomes available previously were all from Rattus norvegicus specimens. Our phylogenetic and dating analyses place the deepest divergence within Rattus at ∼3.5 million years ago (Mya). This divergence separates the New Guinean endemic R. praetor lineage from the Asian lineages. Within the Asian/Island Southeast Asian clade R. norvegicus diverged earliest at ∼2.9 Mya. R. exulans and the ancestor of the sister species R. rattus and R. tanezumi subsequently diverged at ∼2.2 Mya, with R. rattus and R. tanezumi separating as recently as ∼0.4 Mya. Our results give both a better resolved species divergence order and diversification dates within Rattus than previous studies.

Branch-length estimation bias misleads molecular dating for a vertebrate mitochondrial phylogeny

Despite recent methodological advances in inferring the time-scale of biological evolution from molecular data, the fundamental question of whether our substitution models are sufficiently well specified to accurately estimate branch-lengths has received little attention. I examine this implicit assumption of all molecular dating methods, on a vertebrate mitochondrial protein-coding dataset. Comparison with analyses in which the data are RY-coded (AG → R; CT → Y) suggests that even rates-across-sites maximum likelihood greatly under-compensates for multiple substitutions among the standard (ACGT) NT-coded data, which has been subject to greater phylogenetic signal erosion. Accordingly, the fossil record indicates that branch-lengths inferred from the NT-coded data translate into divergence time overestimates when calibrated from deeper in the tree. Intriguingly, RY-coding led to the opposite result. The underlying NT and RY substitution model misspecifications likely relate respectively to “hidden” rate heterogeneity and changes in substitution processes across the tree, for which I provide simulated examples. Given the magnitude of the inferred molecular dating errors, branch-length estimation biases may partly explain current conflicts with some palaeontological dating estimates.

Tinamous and moa flock together : mitochondrial genome sequence analysis reveals independent losses of flight among ratites

Ratites are large, flightless birds and include the ostrich, rheas, kiwi, emu, and cassowaries, along with extinct members, such as moa and elephant birds. Previous phylogenetic analyses of complete mitochondrial genome sequences have reinforced the traditional belief that ratites are monophyletic and tinamous are their sister group. However, in these studies ratite monophyly was enforced in the analyses that modeled rate heterogeneity among variable sites. Relaxing this topological constraint results in strong support for the tinamous (which fly) nesting within ratites. Furthermore, upon reducing base compositional bias and partitioning models of sequence evolution among protein codon positions and RNA structures, the tinamou–moa clade grouped with kiwi, emu, and cassowaries to the exclusion of the successively more divergent rheas and ostrich. These relationships are consistent with recent results from a large nuclear data set, whereas our strongly supported finding of a tinamou–moa grouping further resolves palaeognath phylogeny. We infer flight to have been lost among ratites multiple times in temporally close association with the Cretaceous–Tertiary extinction event. This circumvents requirements for transient microcontinents and island chains to explain discordance between ratite phylogeny and patterns of continental breakup. Ostriches may have dispersed to Africa from Eurasia, putting in question the status of ratites as an iconic Gondwanan relict taxon. [Base composition; flightless; Gondwana; mitochondrial genome; Palaeognathae; phylogeny; ratites.]

Bird evolution: testing the Metaves clade with six new mitochondrial genomes

Background Evolutionary biologists are often misled by convergence of morphology and this has been common in the study of bird evolution. However, the use of molecular data sets have their own problems and phylogenies based on short DNA sequences have the potential to mislead us too. The relationships among clades and timing of the evolution of modern birds (Neoaves) has not yet been well resolved. Evidence of convergence of morphology remain controversial. With six new bird mitochondrial genomes (hummingbird, swift, kagu, rail, flamingo and grebe) we test the proposed Metaves/Coronaves division within Neoaves and the parallel radiations in this primary avian clade. Results Our mitochondrial trees did not return the Metaves clade that had been proposed based on one nuclear intron sequence. We suggest that the high number of indels within the seventh intron of the β-fibrinogen gene at this phylogenetic level, which left a dataset with not a single site across the alignment shared by all taxa, resulted in artifacts during analysis. With respect to the overall avian tree, we find the flamingo and grebe are sister taxa and basal to the shorebirds (Charadriiformes). Using a novel site-stripping technique for noise-reduction we found this relationship to be stable. The hummingbird/swift clade is outside the large and very diverse group of raptors, shore and sea birds. Unexpectedly the kagu is not closely related to the rail in our analysis, but because neither the kagu nor the rail have close affinity to any taxa within this dataset of 41 birds, their placement is not yet resolved. Conclusion Our phylogenetic hypothesis based on 41 avian mitochondrial genomes (13,229 bp) rejects monophyly of seven Metaves species and we therefore conclude that the members of Metaves do not share a common evolutionary history within the Neoaves.

Two SusD-like proteins encoded within a polysaccharide utilisation locus of an uncultured ruminant bacteroidetes bind strongly to cellulose

We demonstrate that two characteristic Sus-like proteins encoded within a Polysaccharide Utilisation Locus (PUL) bind strongly to cellulosic substrates and interact with plant primary cell walls. This shows associations between uncultured Bacteroidetes-affiliated lineages and cellulose in the rumen, and thus presents new PUL-derived targets to pursue regarding plant biomass degradation.

Isolation and characterisation of novel microsatellite and mitochondrial DNA markers for the Eastern Water Dragon (Physignathus lesueurii)

Habitat fragmentation as a result of urbanisation is a growing problem for native lizard species. The Eastern Water Dragon (Physignathus lesueurii) is a social arboreal agamid lizard, native to Australia. This species represents an ideal model species to investigate the effect of urbanisation because of their prominent abundance in the urban landscape. Here we describe the isolation and characterisation of a novel set of 74 di-, tri-, and tetramicrosatellites from which 18 were selected and optimised into two multiplexes. The 18 microsatellites generated a total 148 alleles across the two populations. The number of alleles per locus varied from 2 to 18 alleles and measures of Ho and He varied from 0.395 to 0.877 and from 0.441 to 0.880, respectively. We also present primers for four novel mitochondrial DNA (mtDNA) markers. The combined length of the four mtDNA marker pairs was 2,528 bp which included 15 nucleotides changes. In comparison to threatened species, which are generally characterised by small population sizes, the Eastern Water Dragon represents an ideal model species to investigate the effect of urbanisation on their behavioural ecology and connectivity patterns among populations.