SNOSite: exploiting maximal dependence decomposition to identify cysteine S-nitrosylation with substrate site specificity

S-nitrosylation, the covalent attachment of a nitric oxide to (NO) the sulfur atom of cysteine, is a selective and reversible protein post-translational modification (PTM) that regulates protein activity, localization, and stability. Despite its implication in the regulation of protein functions and cell signaling, the substrate specificity of cysteine S-nitrosylation remains unknown. Based on a total of 586 experimentally identified S-nitrosylation sites from SNAP/L-cysteine-stimulated mouse endothelial cells, this work presents an informatics investigation on S-nitrosylation sites including structural factors such as the flanking amino acids composition, the accessible surface area (ASA) and physicochemical properties, i.e. positive charge and side chain interaction parameter. Due to the difficulty to obtain the conserved motifs by conventional motif analysis, maximal dependence decomposition (MDD) has been applied to obtain statistically significant conserved motifs. Support vector machine (SVM) is applied to generate predictive model for each MDD-clustered motif. According to five-fold cross-validation, the MDD-clustered SVMs could achieve an accuracy of 0.902, and provides a promising performance in an independent test set. The effectiveness of the model was demonstrated on the correct identification of previously reported S-nitrosylation sites of Bos taurus dimethylarginine dimethylaminohydrolase 1 (DDAH1) and human hemoglobin subunit beta (HBB). Finally, the MDD-clustered model was adopted to construct an effective web-based tool, named SNOSite (http://csb.cse.yzu.edu.tw/SNOSite/), for identifying S-nitrosylation sites on the uncharacterized protein sequences.     

TM-MOTIF: an alignment viewer to annotate predicted transmembrane helices and conserved motifs in aligned set of sequences

Multiple sequence alignments become biologically meaningful only if conserved and functionally important residues and secondary structural elements preserved can be identified at equivalent positions. This is particularly important for transmembrane proteins like G-protein coupled receptors (GPCRs) with seven transmembrane helices. TM-MOTIF is a software package and an effective alignment viewer to identify and display conserved motifs and amino acid substitutions (AAS) at each position of the aligned set of homologous sequences of GPCRs. The key feature of the package is to display the predicted membrane topology for seven transmembrane helices in seven colours (VIBGYOR colouring scheme) and to map the identified motifs on its respective helices /loop regions. It is an interactive package which provides options to the user to submit query or pre-aligned set of GPCR sequences to align with a reference sequence, like rhodopsin, whose structure has been solved experimentally. It also provides the possibility to identify the nearest homologue from the available inbuilt GPCR or Olfactory Receptor cluster dataset whose association is already known for its receptor type. AVAILABILITY: The database is available for free at mini@ncbs.res.in.     

CREDO: a web-based tool for computational detection of conserved sequence motifs in noncoding sequences

SUMMARY: CREDO is a user-friendly, web-based tool that integrates the analysis and results of different algorithms widely used for the computational detection of conserved sequence motifs in noncoding sequences. It enables easy comparison of the individual results. CREDO offers intuitive interfaces for easy and rapid configuration of the applied algorithms and convenient views on the results in graphical and tabular formats. AVAILABILITY: http://mips.gsf.de/proj/regulomips/credo.htm.     

Phylomark, a tool to identify conserved phylogenetic markers from whole-genome alignments

The sequencing and analysis of multiple housekeeping genes has been routinely used to phylogenetically compare closely related bacterial isolates. Recent studies using whole-genome alignment (WGA) and phylogenetics from >100 Escherichia coli genomes has demonstrated that tree topologies from WGA and multilocus sequence typing (MLST) markers differ significantly. A nonrepresentative phylogeny can lead to incorrect conclusions regarding important evolutionary relationships. In this study, the Phylomark algorithm was developed to identify a minimal number of useful phylogenetic markers that recapitulate the WGA phylogeny. To test the algorithm, we used a set of diverse draft and complete E. coli genomes. The algorithm identified more than 100,000 potential markers of different fragment lengths (500 to 900 nucleotides). Three molecular markers were ultimately chosen to determine the phylogeny based on a low Robinson-Foulds (RF) distance compared to the WGA phylogeny. A phylogenetic analysis demonstrated that a more representative phylogeny was inferred for a concatenation of these markers compared to all other MLST schemes for E. coli. As a functional test of the algorithm, the three markers (genomic guided E. coli markers, or GIG-EM) were amplified and sequenced from a set of environmental E. coli strains (ECOR collection) and informatically extracted from a set of 78 diarrheagenic E. coli strains (DECA collection). In the instances of the 40-genome test set and the DECA collection, the GIG-EM system outperformed other E. coli MLST systems in terms of recapitulating the WGA phylogeny. This algorithm can be employed to determine the minimal marker set for any organism that has sufficient genome sequencing.     

Use of thioredoxin as a reporter to identify a subset of Escherichia coli signal sequences that promote signal recognition particle-dependent translocation

We have previously reported that the DsbA signal sequence promotes efficient, cotranslational translocation of the cytoplasmic protein thioredoxin-1 via the bacterial signal recognition particle (SRP) pathway. However, two commonly used signal sequences, those of PhoA and MalE, which promote export by a posttranslational mechanism, do not export thioredoxin. We proposed that this difference in efficiency of export was due to the rapid folding of thioredoxin in the cytoplasm; cotranslational export by the DsbA signal sequence avoids the problem of cytoplasmic folding (C. F. Schierle, M. Berkmen, D. Huber, C. Kumamoto, D. Boyd, and J. Beckwith, J. Bacteriol. 185:5706-5713, 2003). Here, we use thioredoxin as a reporter to distinguish SRP-dependent from non-SRP-dependent cleavable signal sequences. We screened signal sequences exhibiting a range of hydrophobicity values based on a method that estimates hydrophobicity. Successive iterations of screening and refining the method defined a threshold hydrophobicity required for SRP recognition. While all of the SRP-dependent signal sequences identified were above this threshold, there were also a few signal sequences above the threshold that did not utilize the SRP pathway. These results suggest that a simple measure of the hydrophobicity of a signal sequence is an important but not a sufficient indicator for SRP recognition. In addition, by fusing a number of both classes of signal sequences to DsbA, we found that DsbA utilizes an SRP-dependent signal sequence to achieve efficient export to the periplasm. Our results suggest that those proteins found to be exported by SRP-dependent signal sequences may require this mode of export because of their tendency to fold rapidly in the cytoplasm.     

Recognition of distantly related protein sequences using conserved motifs and neural networks.

A sensitive technique for protein sequence motif recognition based on neural networks has been developed. It involves three major steps. (1) At each appropriate alignment position of a set of N matched sequences, a set of N aligned oligopeptides is specified with preselected window length. N neural nets are subsequently and successively trained on N-1 amino acid spans after eliminating each ith oligopeptide. A test for recognition of each of the ith spans is performed. The average neural net recognition over N such trials is used as a measure of conservation for the particular windowed region of the multiple alignment. This process is repeated for all possible spans of given length in the multiple alignment. (2) The M most conserved regions are regarded as motifs and the oligopeptides within each are used to train intensively M individual neural networks. (3) The M networks are then applied in a search for related primary structures in a databank of known protein sequences. The oligopeptide spans in the database sequence with strongest neural net output for each of the M networks are saved and then scored according to the output signals and the proper combination that follows the expected N- to C-terminal sequence order. The motifs from the database with highest similarity scores can then be used to retrain the M neural nets, which can be subsequently utilized for further searches in the databank, thus providing even greater sensitivity to recognize distant familial proteins. This technique was successfully applied to the integrase, DNA-polymerase and immunoglobulin families.     

An approach to delineate primers for a group of poorly conserved sequences incorporating the common motif region

Glutathione synthetase (gshB) has previously been reported to confer tolerance to acidic soil condition in Rhizobium species. Cloning the gene coding for this enzyme necessitates the designing of proper primer sets which in turn depends on the identification of high quality sequence similarity in multiple global alignments. In this experiment, a group of homologous gene sequences related to gshB gene (accession no: gi-86355669:327589-328536) of Rhizobium etli CFN 42, were extracted from NCBI nucleotide sequence databases using BLASTN and were analyzed for designing degenerate primers. However, the T-coffee multiple global alignment results did not show any block of conserved region for the above sequence set to design the primers. Therefore, we attempted to identify the location of common motif region based on multiple local alignments employing the MEME algorithm supported with MAST and Primer3. The results revealed some common motif regions that enabled us to design the primer sets for related gshB gene sequences. The result will be validated in wet lab.     

A novel conserved nuclear localization signal is recognized by a group of yeast importins

Nucleo-cytoplasmic transport of proteins is mostly mediated by specific interaction between transport receptors of the importin beta family and signal sequences present in their cargo. While several signal sequences, in particular the classical nuclear localization signal (NLS) recognized by the heterodimeric importin alpha/beta complex are well known, the signals recognized by other importin beta-like transport receptors remain to be characterized in detail. Here we present the systematic analysis of the nuclear import of Saccharomyces cerevisiae Asr1p, a nonessential alcohol-responsive Ring/PHD finger protein that shuttles between nucleus and cytoplasm but accumulates in the nucleus upon alcohol stress. Nuclear import of Asr1p is constitutive and mediated by its C-terminal domain. A short sequence comprising residues 243-280 is sufficient and necessary for active targeting to the nucleus. Moreover, the nuclear import signal is conserved from yeast to mammals. In vitro, the nuclear localization signal of Asr1p directly interacts with the importins Kap114p, Kap95p, Pse1p, Kap123p, or Kap104p, interactions that are sensitive to the presence of RanGTP. In vivo, these importins cooperate in nuclear import. Interestingly, the same importins mediate nuclear transport of histone H2A. Based on mutational analysis and sequence comparison with a region mediating nuclear import of histone H2A, we identified a novel type of NLS with the consensus sequence R/KxxL(x)(n)V/YxxV/IxK/RxxxK/R that is recognized by five yeast importins and connects them into a highly efficient network for nuclear import of proteins.     

Exploiting the triple response of Arabidopsis to identify ethylene-related mutants.

Alterations in the response of dark-grown seedlings to ethylene (the "triple response") were used to isolate a collection of ethylene-related mutants in Arabidopsis thaliana. Mutants displaying a constitutive response (eto1) were found to produce at least 40 times more ethylene than the wild type. The morphological defects in etiolated eto1-1 seedlings reverted to wild type under conditions in which ethylene biosynthesis or ethylene action were inhibited. Mutants that failed to display the apical hook in the absence of ethylene (his1) exhibited reduced ethylene production. In the presence of exogenous ethylene, hypocotyl and root of etiolated his1-1 seedlings were inhibited in elongation but no apical hook was observed. Mutants that were insensitive to ethylene (ein1 and ein2) produced increased amounts of ethylene, displayed hormone insensitivity in both hypocotyl and root responses, and showed an apical hook. Each of the "triple response" mutants has an effect on the shape of the seedling and on the production of the hormone. These mutants should prove to be useful tools for dissecting the mode of ethylene action in plants.     

Recovering motifs from biased genomes: application of signal correction

A significant problem in biological motif analysis arises when the background symbol distribution is biased (e.g. high/low GC content in the case of DNA sequences). This can lead to overestimation of the amount of information encoded in a motif. A motif can be depicted as a signal using information theory (IT). We apply two concepts from IT, distortion and patterned interference (a type of noise), to model genomic and codon bias respectively. This modeling approach allows us to correct a raw signal to recover signals that are weakened by compositional bias. The corrected signal is more likely to be discriminated from a biased background by a macromolecule. We apply this correction technique to recover ribosome-binding site (RBS) signals from available sequenced and annotated prokaryotic genomes having diverse compositional biases. We observed that linear correction was sufficient for recovering signals even at the extremes of these biases. Further comparative genomics studies were made possible upon correction of these signals. We find that the average Euclidian distance between RBS signal frequency matrices of different genomes can be significantly reduced by using the correction technique. Within this reduced average distance, we can find examples of class-specific RBS signals. Our results have implications for motif-based prediction, particularly with regards to the estimation of reliable inter-genomic model parameters.     

Regulation of Dlx gene expression in the zebrafish pharyngeal arches: from conserved enhancer sequences to conserved activity

The Dlx genes play an important role in the development of the pharyngeal arches and the structures derived from these tissues, including the craniofacial skeleton. They are typically expressed in a nested pattern along the proximo‐distal axis of the branchial arches in mice. This pattern is known as the “Dlx code” and has been proposed to be responsible for an early regional patterning of branchial arches in mammals. A number of cis‐ regulatory elements (CREs) have been identified within the Dlx loci, which target reporter expression to the developing pharyngeal arches of the mouse. Most of these CREs are located in the intergenic regions between the two genes constituting a Dlx bigene cluster. Therefore, the regionalized dlx expression in the branchial arches could be the result of the shared activities of these regulatory regions. Here we analyze previously published and new results showing these CREs are highly conserved in both their sequence and their activity in the pharyngeal arches of two distantly related vertebrates, the zebrafish and the mouse. A better understanding of Dlx gene regulation of the Dlx genes and of the genetic cascades in which they are involved can lead to clues explaining variations in morphology of the craniofacial regions of vertebrates.     

PROMOT: a FORTRAN program to scan protein sequences against a library of known motifs

Information about the three-dimensional structure or functionof a newly determined protein sequence can be obtained if theprotein is found to contain a characterized motif or patternof residues. Recently a database (PROSITE) has been establishedthat contains 337 known motifs encoded as a list of allowedresidue types at specific positions along the sequence. PROMOTis a FORTRAN computer program that takes a protein sequenceand examines if it contains any of the motifs in PROSITE. Theprogram also extends the definitions of patterns beyond thoseused in PROSITE to provide a simple, yet flexible, method toscan either a PROSITE or a user-defined pattern against a proteinsequence database. Received on October 17, 1990; accepted on November 15, 1990     

Utilization of oriented peptide libraries to identify substrate motifs selected by ATM

The ataxia telangiectasia mutated (ATM) gene encodes a serine/threonine protein kinase that plays a critical role in genomic surveillance and development. Here, we use a peptide library approach to define the in vitro substrate specificity of ATM kinase activity. The peptide library analysis identified an optimal sequence with a central core motif of LSQE that is preferentially phosphorylated by ATM. The contributions of the amino acids surrounding serine in the LSQE motif were assessed by utilizing specific peptide libraries or individual peptide substrates. All amino acids comprising the LSQE sequence were critical for maximum peptide substrate suitability for ATM. The DNA-dependent protein kinase (DNA-PK), a Ser/Thr kinase related to ATM and important in DNA repair, was compared with ATM in terms of peptide substrate selectivity. DNA-PK was found to be unique in its preference of neighboring amino acids to the phosphorylated serine. Peptide library analyses defined a preferred amino acid motif for ATM that permits clear distinctions between ATM and DNA-PK kinase activity. Data base searches using the library-derived ATM sequence identified previously characterized substrates of ATM, as well as novel candidate substrate targets that may function downstream in ATM-directed signaling pathways.