Discovery and hypothesis generation through bioinformatics

A report on the 4th European Conference on Computational Biology and the 6th Spanish Annual Meeting on Bioinformatics, Madrid, Spain, 28 September-1 October 2005.     

Unique SARS-CoV protein nsp1: bioinformatics, biochemistry and potential effects on virulence.

Viruses have evolved a myriad of strategies for promoting viral replication, survival and spread. Sequence analysis of the Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV) genome predicts several proteins that are unique to SARS-CoV. The search to understand the high virulence of SARS-CoV compared with related coronaviruses, which cause lesser respiratory illnesses, has recently focused on the unique nsp1 protein of SARS-CoV and suggests evolution of a possible new virulence mechanism in coronaviruses. The SARS-CoV nsp1 protein increases cellular RNA degradation and thus might facilitate SARS-CoV replication or block immune responses.     

Analysis of protein-protein interactions by mutagenesis: direct versus indirect effects

Site-directed mutagenesis, including double-mutant cycles, is used routinely for studying protein-protein interactions. We now present a case analysis of chymotrypsin inhibitor 2 (CI2) and subtilisin BPN' using (i) a residue in CI2 that is known to interact directly with subtilisin (Tyr42) and (ii) two CI2 residues that do not have direct contacts with subtilisin (Arg46 and Arg48). We find that there are similar changes in binding energy on mutation of these two sets of residues. It can thus be difficult to interpret mutagenesis data in the absence of structural information.     

Unraveling microalgal molecular interactions using evolutionary and structural bioinformatics

Microalgae are unicellular microorganisms indispensible for environmental stability and life on earth, because they produce approximately half of the atmospheric oxygen, with simultaneously feeding on the harmful greenhouse gas carbon dioxide. Using gene fusion analysis, a series of five fusion/fission events was identified, that provided the basis for critical insights to their evolutionary history. Moreover, the three-dimensional structures of both the fused and the component proteins were predicted, allowing us to envisage putative protein–protein interactions that are invaluable for the efficient usage, handling and exploitation of microalgae. Collectively, our proposed approach on the five fusion/fission alga protein events contributes towards the expansion of the microalgae knowledgebase, bridging protein evolution of the ancient microalgal species and the rapidly evolving, modern, bioinformatics field.     

Analysis of Escherichia coli RecA interactions with LexA, lambda CI, and UmuD by site-directed mutagenesis of recA

An early event in the induction of the SOS system of Escherichia coli is RecA-mediated cleavage of the LexA repressor. RecA acts indirectly as a coprotease to stimulate repressor self-cleavage, presumably by forming a complex with LexA. How complex formation leads to cleavage is not known. As an approach to this question, it would be desirable to identify the protein-protein interaction sites on each protein. It was previously proposed that LexA and other cleavable substrates, such as phage lambda CI repressor and E. coli UmuD, bind to a cleft located between two RecA monomers in the crystal structure. To test this model, and to map the interface between RecA and its substrates, we carried out alanine-scanning mutagenesis of RecA. Twenty double mutations were made, and cells carrying them were characterized for RecA-dependent repair functions and for coprotease activity towards LexA, lambda CI, and UmuD. One mutation in the cleft region had partial defects in cleavage of CI and (as expected from previous data) of UmuD. Two mutations in the cleft region conferred constitutive cleavage towards CI but not towards LexA or UmuD. By contrast, no mutations in the cleft region or elsewhere in RecA were found to specifically impair the cleavage of LexA. Our data are consistent with binding of CI and UmuD to the cleft between two RecA monomers but do not provide support for the model in which LexA binds in this cleft.     

Analysis of the psychrotolerant property of hormone-sensitive lipase through site-directed mutagenesis

Mammalian hormone-sensitive lipase (HSL) has given its name to a family of primarily prokaryotic proteins which are structurally related to type B carboxylesterases. In many of these alpha/beta hydrolases, a conserved HG-dipeptide flanks the catalytic pocket. In HSL this dipeptide is followed by two additional glycine residues. Through site-directed mutagenesis, we have investigated the importance of this motif for enzyme activity. Since the presence of multiple glycine residues in a critical region could contribute to cold adaptation by providing local flexibility, we studied the effect of mutating these residues on the psychrotolerant property of HSL. Any double mutation rendered the enzyme completely inactive, without any major effect on the enzyme stability. The partially active single mutants retained the same proportion of activity at reduced temperatures as the wild-type enzyme. These results do not support a role for the HGGG motif in catalysis at low temperatures, but provide further validation of the current three-dimensional model of HSL. Rat HSL was found to be relatively more active than human HSL at low temperatures. This difference was, however, not due to the 12 amino acids which are present in the regulatory module of the rat enzyme but absent in human HSL.     

Dissecting the human spliceosome through bioinformatics and proteomics approaches

The precise excision of introns from mRNAs is executed by the spliceosome, a cellular machinery composed by five small nuclear RNAs and hundreds of proteins. In the last few years, several groups have used proteomics and computational biology tools to characterize the components of the human spliceosome. These reports have identified basically all known splicing factors and several new proteins. The composition of the human spliceosome confirms the link between splicing and other steps in gene expression. Here we comment on these reports and discuss the perspectives for the coming years.     

Binding interactions of kistrin with platelet glycoprotein IIb-IIIa: Analysis by site-directed mutagenesis

The binding interactions between platelet fibrinogen receptor, glycoprotein (GP) IIb-IIIa, and kistrin, a snake venom disintegrin protein that contains the adhesion site recognition sequence Arg-Gly-Asp (RGD) and potently inhibits platelet aggregation, have been investigated by site-directed mutagenesis of a synthetic kistrin gene. Kistrin was expressed as a fusion protein in Escherichia coli under control of the alkaline phosphatase promoter. This construction included the stII signal sequence to direct secretion to the periplasmic space and one synthetic (Z) domain of Staphylococcal protein A to allow affinity purification using IgG Sepharose. Kistrin was cleaved from the Z-domain by site-specific proteolysis using a mutant subtilisin BPN' and purified by reverse-phase HPLC. This approach facilitated the rapid purification of a set of 43 alanine replacement mutants whose relative affinity for GP IIb-IIIa was measured by competition with immobilized kistrin and by inhibition of platelet aggregation in human platelet-rich plasma. Alanine replacements at R49, G50, and D51 led to weaker inhibitors of platelet aggregation by 90-fold, 2-fold, and >200-fold, respectively. The conservative D51E mutant was still >100-fold less potent whereas R49K had a minor effect (1.8-fold), implying the critical nature of the aspartate for high affinity binding. However, mutations outside of the RGD region led to proteins indistinguishable from kistrin, suggesting no substantial secondary binding interactions. Furthermore, reduced kistrin is not active. We therefore propose that a favorable conformation of the RGD region alone is responsible for the high affinity binding of kistrin to GP IIb-IIIa. © 1993 Wiley-Liss, Inc.     

Biocatalysis, biodegradation and bioinformatics

Biocatalysis, biodegradation and bioinformatics are prominent scientific fields in industrial microbiology and biotechnology. This paper describes developments in these fields with a focus on the role of David T Gibson as a researcher and mentor. He has pioneered studies on the mechanisms by which aerobic microorganisms transform aromatic hydrocarbons. In addition, his research has served as a model for further investigations into bacterial atrazine and dichloromethane catabolism described here. Microbial catabolism research requires information on organic chemistry, microorganisms, metabolic pathways, catabolic genes, and enzymes. These information needs are now being met more comprehensively by development of the University of Minnesota Biocatalysis/Biodegradation Database. http://dragon.labmed.umn.edu/∼lynda/index.html The database is built on the ideas championed by David Gibson that a knowledge of microbial catabolic reactions should be organized in a mechanistic fashion and in a systematic format. Received 27 March 1997/ Accepted in revised form 05 June 1997     

生物芯片、生物传感器和生物信息学

近年来,在生物技术和医学研究领域涌现出了许多新技术平台,其中就包括生物芯片技术和生物传感器技术。生物芯片和生物传感器的构建都必须以生物信息学为基础,而两种技术平台应用所得出的数据和结果又反过来大大丰富和充实了生物信息学本身。本分析概述了生物芯片和生物传感器两种技术平台以及生物信息学,对三之间的相互关系进行了讨论。     

Deriving folds of macromolecular complexes through electron cryomicroscopy and bioinformatics approaches

Intermediate-resolution (7-9A) structures of large macromolecular complexes can be obtained by electron cryomicroscopy. This structural information, combined with bioinformatics data for the individual protein components or domains, can lead to a fold model for the entire complex. Such approaches have been demonstrated with the 6.8 A structure of the rice dwarf virus to derive models for the major capsid shell proteins.     

Redox metabolism in malaria: from genes,through biochemistry and pathology,to drugs

Abstract

The role of Saccharomyces cerevisiae flavohemoglobin (Yhb1) is controversial and far from understood. This study compares the effects of nitrosative and oxidative challenge on the yeast mutant lacking the YHB1 gene. Growth of the mutant was impaired by nitrosoglutathione and peroxynitrite, whereas increased sensitivity to reactive oxygen species was not observed. Increased levels of intracellular NO^? after incubation with NO^? donors were found in the mutants cells as compared to the wild-type cells. Deletion of the YHB1 gene was found to augment the reduction of Fe³⁺ by yeast cells which suggests that flavohemoglobin participates in regulation of the activity of plasma membrane ferric reductase(s).     

Allosteric modulation of caspase 3 through mutagenesis

A mutation in the allosteric site of the caspase 3 dimer interface of Val266 to histidine abolishes activity of the enzyme, and models predict that the mutation mimics the action of small molecule allosteric inhibitors by preventing formation of the active site. Mutations were coupled to His266 at two sites in the interface, E124A and Y197C. We present results from X-ray crystallography, enzymatic activity and molecular dynamics simulations for seven proteins, consisting of single, double and triple mutants. The results demonstrate that considering allosteric inhibition of caspase 3 as a shift between discrete 'off-state' or 'on-state' conformations is insufficient. Although His266 is accommodated in the interface, the structural defects are propagated to the active site through a helix on the protein surface. A more comprehensive view of allosteric regulation of caspase 3 requires the representation of an ensemble of inactive states and shows that subtle structural changes lead to the population of the inactive ensemble.     

Generation of nonvernal-obligate, faster-cycling Noccaea caerulescens lines through fast neutron mutagenesis

Noccaea caerulescens (formerly Thlaspi caerulescens) is a widely studied metal hyperaccumulator. However, molecular genetic studies are challenging in this species because of its vernal-obligate biennial life cycle of 7-9months. Here, we describe the development of genetically stable, faster cycling lines of N. caerulescens which are nonvernal-obligate. A total of 5500 M(0) seeds from Saint Laurent Le Minier (France) were subjected to fast neutron mutagenesis. Following vernalization of young plants, 79% of plants survived to maturity. In all, 80,000 M(2) lines were screened for flowering in the absence of vernalization. Floral initials were observed in 35 lines, with nine flowering in <12wk. Two lines (A2 and A7) were selfed to the M(4) generation. Floral initials were observed 66 and 87d after sowing (DAS) in A2 and A7, respectively. Silicle development occurred for all A2 and for most A7 at 92 and 123 DAS, respectively. Floral or silicle development was not observed in wild-type (WT) plants. Leaf zinc (Zn) concentration was similar in WT, A2 and A7 lines. These lines should facilitate future genetic studies of this remarkable species. Seed is publicly available through the European Arabidopsis Stock Centre (NASC).