Biochemistry and Pharmacology - Research Publications
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ItemA Bioinformatic Strategy for the Detection, Classification and Analysis of Bacterial Autotransporters(PUBLIC LIBRARY SCIENCE, 2012-08-14)Autotransporters are secreted proteins that are assembled into the outer membrane of bacterial cells. The passenger domains of autotransporters are crucial for bacterial pathogenesis, with some remaining attached to the bacterial surface while others are released by proteolysis. An enigma remains as to whether autotransporters should be considered a class of secretion system, or simply a class of substrate with peculiar requirements for their secretion. We sought to establish a sensitive search protocol that could identify and characterize diverse autotransporters from bacterial genome sequence data. The new sequence analysis pipeline identified more than 1500 autotransporter sequences from diverse bacteria, including numerous species of Chlamydiales and Fusobacteria as well as all classes of Proteobacteria. Interrogation of the proteins revealed that there are numerous classes of passenger domains beyond the known proteases, adhesins and esterases. In addition the barrel-domain-a characteristic feature of autotransporters-was found to be composed from seven conserved sequence segments that can be arranged in multiple ways in the tertiary structure of the assembled autotransporter. One of these conserved motifs overlays the targeting information required for autotransporters to reach the outer membrane. Another conserved and diagnostic motif maps to the linker region between the passenger domain and barrel-domain, indicating it as an important feature in the assembly of autotransporters.
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ItemSystems biology: the next frontier for bioinformatics.(Hindawi Limited, 2010)Biochemical systems biology augments more traditional disciplines, such as genomics, biochemistry and molecular biology, by championing (i) mathematical and computational modeling; (ii) the application of traditional engineering practices in the analysis of biochemical systems; and in the past decade increasingly (iii) the use of near-comprehensive data sets derived from 'omics platform technologies, in particular "downstream" technologies relative to genome sequencing, including transcriptomics, proteomics and metabolomics. The future progress in understanding biological principles will increasingly depend on the development of temporal and spatial analytical techniques that will provide high-resolution data for systems analyses. To date, particularly successful were strategies involving (a) quantitative measurements of cellular components at the mRNA, protein and metabolite levels, as well as in vivo metabolic reaction rates, (b) development of mathematical models that integrate biochemical knowledge with the information generated by high-throughput experiments, and (c) applications to microbial organisms. The inevitable role bioinformatics plays in modern systems biology puts mathematical and computational sciences as an equal partner to analytical and experimental biology. Furthermore, mathematical and computational models are expected to become increasingly prevalent representations of our knowledge about specific biochemical systems.
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ItemA Comprehensive Bioinformatics Analysis of the Nudix Superfamily in Arabidopsis thaliana(HINDAWI LTD, 2009)Nudix enzymes are a superfamily with a conserved common reaction mechanism that provides the capacity for the hydrolysis of a broad spectrum of metabolites. We used hidden Markov models based on Nudix sequences from the PFAM and PROSITE databases to identify Nudix hydrolases encoded by the Arabidopsis genome. 25 Nudix hydrolases were identified and classified into 11 individual families by pairwise sequence alignments. Intron phases were strikingly conserved in each family. Phylogenetic analysis showed that all multimember families formed monophyletic clusters. Conserved familial sequence motifs were identified with the MEME motif analysis algorithm. One motif (motif 4) was found in three diverse families. All proteins containing motif 4 demonstrated a degree of preference for substrates containing an ADP moiety. We conclude that HMM model-based genome scanning and MEME motif analysis, respectively, can significantly improve the identification and assignment of function of new members of this mechanistically-diverse protein superfamily.
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ItemExtraction of pure components from overlapped signals in gas chromatography-mass spectrometry (GC-MS)(BMC, 2009)Gas chromatography-mass spectrometry (GC-MS) is a widely used analytical technique for the identification and quantification of trace chemicals in complex mixtures. When complex samples are analyzed by GC-MS it is common to observe co-elution of two or more components, resulting in an overlap of signal peaks observed in the total ion chromatogram. In such situations manual signal analysis is often the most reliable means for the extraction of pure component signals; however, a systematic manual analysis over a number of samples is both tedious and prone to error. In the past 30 years a number of computational approaches were proposed to assist in the process of the extraction of pure signals from co-eluting GC-MS components. This includes empirical methods, comparison with library spectra, eigenvalue analysis, regression and others. However, to date no approach has been recognized as best, nor accepted as standard. This situation hampers general GC-MS capabilities, and in particular has implications for the development of robust, high-throughput GC-MS analytical protocols required in metabolic profiling and biomarker discovery. Here we first discuss the nature of GC-MS data, and then review some of the approaches proposed for the extraction of pure signals from co-eluting components. We summarize and classify different approaches to this problem, and examine why so many approaches proposed in the past have failed to live up to their full promise. Finally, we give some thoughts on the future developments in this field, and suggest that the progress in general computing capabilities attained in the past two decades has opened new horizons for tackling this important problem.
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ItemLeishCyc: a biochemical pathways database for Leishmania major(BMC, 2009-06-05)BACKGROUND: Leishmania spp. are sandfly transmitted protozoan parasites that cause a spectrum of diseases in more than 12 million people worldwide. Much research is now focusing on how these parasites adapt to the distinct nutrient environments they encounter in the digestive tract of the sandfly vector and the phagolysosome compartment of mammalian macrophages. While data mining and annotation of the genomes of three Leishmania species has provided an initial inventory of predicted metabolic components and associated pathways, resources for integrating this information into metabolic networks and incorporating data from transcript, protein, and metabolite profiling studies is currently lacking. The development of a reliable, expertly curated, and widely available model of Leishmania metabolic networks is required to facilitate systems analysis, as well as discovery and prioritization of new drug targets for this important human pathogen. DESCRIPTION: The LeishCyc database was initially built from the genome sequence of Leishmania major (v5.2), based on the annotation published by the Wellcome Trust Sanger Institute. LeishCyc was manually curated to remove errors, correct automated predictions, and add information from the literature. The ongoing curation is based on public sources, literature searches, and our own experimental and bioinformatics studies. In a number of instances we have improved on the original genome annotation, and, in some ambiguous cases, collected relevant information from the literature in order to help clarify gene or protein annotation in the future. All genes in LeishCyc are linked to the corresponding entry in GeneDB (Wellcome Trust Sanger Institute). CONCLUSION: The LeishCyc database describes Leishmania major genes, gene products, metabolites, their relationships and biochemical organization into metabolic pathways. LeishCyc provides a systematic approach to organizing the evolving information about Leishmania biochemical networks and is a tool for analysis, interpretation, and visualization of Leishmania Omics data (transcriptomics, proteomics, metabolomics) in the context of metabolic pathways. LeishCyc is the first such database for the Trypanosomatidae family, which includes a number of other important human parasites. Flexible query/visualization capabilities are provided by the Pathway Tools software and its Web interface. The LeishCyc database is made freely available over the Internet http://www.leishcyc.org.
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ItemProtein secretion and outer membrane assembly in Alphaproteobacteria(OXFORD UNIV PRESS, 2008-11)The assembly of beta-barrel proteins into membranes is a fundamental process that is essential in Gram-negative bacteria, mitochondria and plastids. Our understanding of the mechanism of beta-barrel assembly is progressing from studies carried out in Escherichia coli and Neisseria meningitidis. Comparative sequence analysis suggests that while many components mediating beta-barrel protein assembly are conserved in all groups of bacteria with outer membranes, some components are notably absent. The Alphaproteobacteria in particular seem prone to gene loss and show the presence or absence of specific components mediating the assembly of beta-barrels: some components of the pathway appear to be missing from whole groups of bacteria (e.g. Skp, YfgL and NlpB), other proteins are conserved but are missing characteristic domains (e.g. SurA). This comparative analysis is also revealing important structural signatures that are vague unless multiple members from a protein family are considered as a group (e.g. tetratricopeptide repeat (TPR) motifs in YfiO, beta-propeller signatures in YfgL). Given that the process of the beta-barrel assembly is conserved, analysis of outer membrane biogenesis in Alphaproteobacteria, the bacterial group that gave rise to mitochondria, also promises insight into the assembly of beta-barrel proteins in eukaryotes.
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ItemPyMS: a Python toolkit for processing of gas chromatography-mass spectrometry (GC-MS) data. Application and comparative study of selected tools(BMC, 2012-05-30)BACKGROUND: Gas chromatography-mass spectrometry (GC-MS) is a technique frequently used in targeted and non-targeted measurements of metabolites. Most existing software tools for processing of raw instrument GC-MS data tightly integrate data processing methods with graphical user interface facilitating interactive data processing. While interactive processing remains critically important in GC-MS applications, high-throughput studies increasingly dictate the need for command line tools, suitable for scripting of high-throughput, customized processing pipelines. RESULTS: PyMS comprises a library of functions for processing of instrument GC-MS data developed in Python. PyMS currently provides a complete set of GC-MS processing functions, including reading of standard data formats (ANDI- MS/NetCDF and JCAMP-DX), noise smoothing, baseline correction, peak detection, peak deconvolution, peak integration, and peak alignment by dynamic programming. A novel common ion single quantitation algorithm allows automated, accurate quantitation of GC-MS electron impact (EI) fragmentation spectra when a large number of experiments are being analyzed. PyMS implements parallel processing for by-row and by-column data processing tasks based on Message Passing Interface (MPI), allowing processing to scale on multiple CPUs in distributed computing environments. A set of specifically designed experiments was performed in-house and used to comparatively evaluate the performance of PyMS and three widely used software packages for GC-MS data processing (AMDIS, AnalyzerPro, and XCMS). CONCLUSIONS: PyMS is a novel software package for the processing of raw GC-MS data, particularly suitable for scripting of customized processing pipelines and for data processing in batch mode. PyMS provides limited graphical capabilities and can be used both for routine data processing and interactive/exploratory data analysis. In real-life GC-MS data processing scenarios PyMS performs as well or better than leading software packages. We demonstrate data processing scenarios simple to implement in PyMS, yet difficult to achieve with many conventional GC-MS data processing software. Automated sample processing and quantitation with PyMS can provide substantial time savings compared to more traditional interactive software systems that tightly integrate data processing with the graphical user interface.
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ItemProtein Substrates of a Novel Secretion System Are Numerous in the Bacteroidetes Phylum and Have in Common a Cleavable C-Terminal Secretion Signal, Extensive Post-Translational Modification, and Cell-Surface Attachment(AMER CHEMICAL SOC, 2013-10)The secretion of certain proteins in Porphyromonas gingivalis is dependent on a C-terminal domain (CTD). After secretion, the CTD is cleaved prior to extensive modification of the mature protein, probably with lipopolysaccharide, therefore enabling attachment to the cell surface. In this study, bioinformatic analyses of the CTD demonstrated the presence of three conserved sequence motifs. These motifs were used to construct Hidden Markov Models (HMMs) that predicted 663 CTD-containing proteins in 21 fully sequenced species of the Bacteroidetes phylum, while no CTD-containing proteins were predicted in species outside this phylum. Further HMM searching of Cytophaga hutchinsonii led to a total of 171 predicted CTD proteins in that organism alone. Proteomic analyses of membrane fractions and culture fluid derived from P. gingivalis and four other species containing predicted CTDs (Parabacteroides distasonis, Prevotella intermedia, Tannerella forsythia, and C. hutchinsonii) demonstrated that membrane localization, extensive post-translational modification, and CTD-cleavage were conserved features of the secretion system. The CTD cleavage site of 10 different proteins from 3 different species was determined and found to be similar to the cleavage site previously determined in P. gingivalis, suggesting that homologues of the C-terminal signal peptidase (PG0026) are responsible for the cleavage in these species.
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ItemThe Essentials of Protein Import in the Degenerate Mitochondrion of Entamoeba histolytica(PUBLIC LIBRARY SCIENCE, 2010-03)Several essential biochemical processes are situated in mitochondria. The metabolic transformation of mitochondria in distinct lineages of eukaryotes created proteomes ranging from thousands of proteins to what appear to be a much simpler scenario. In the case of Entamoeba histolytica, tiny mitochondria known as mitosomes have undergone extreme reduction. Only recently a single complete metabolic pathway of sulfate activation has been identified in these organelles. The E. histolytica mitosomes do not produce ATP needed for the sulfate activation pathway and for three molecular chaperones, Cpn60, Cpn10 and mtHsp70. The already characterized ADP/ATP carrier would thus be essential to provide cytosolic ATP for these processes, but how the equilibrium of inorganic phosphate could be maintained was unknown. Finally, how the mitosomal proteins are translocated to the mitosomes had remained unclear. We used a hidden Markov model (HMM) based search of the E. histolytica genome sequence to discover candidate (i) mitosomal phosphate carrier complementing the activity of the ADP/ATP carrier and (ii) membrane-located components of the protein import machinery that includes the outer membrane translocation channel Tom40 and membrane assembly protein Sam50. Using in vitro and in vivo systems we show that E. histolytica contains a minimalist set up of the core import components in order to accommodate a handful of mitosomal proteins. The anaerobic and parasitic lifestyle of E. histolytica has produced one of the simplest known mitochondrial compartments of all eukaryotes. Comparisons with mitochondria of another amoeba, Dictystelium discoideum, emphasize just how dramatic the reduction of the protein import apparatus was after the loss of archetypal mitochondrial functions in the mitosomes of E. histolytica.