Profiling serine protease substrate specificity with solution phase fluorogenic peptide microarrays

A novel microarray-based proteolytic profiling assay enabled the rapid determination of protease substrate specificities with minimal sample and enzyme usage. A 722-member library of fluorogenic protease substrates of the general format Ac-Ala-X-X-(Arg/Lys)-coumarin was synthesized and microarrayed, along with fluorescent calibration standards, in glycerol nanodroplets on microscope slides. The arrays were then activated by deposition of an aerosolized enzyme solution, followed by incubation and fluorometric scanning. The specificities of human blood serine proteases (human thrombin, factor Xa, plasmin, and urokinase plasminogen activator) were examined. The arrays provided complete maps of protease specificity for all of the substrates tested and allowed for detection of cooperative interactions between substrate subsites. The arrays were further utilized to explore the conservation of thrombin specificity across species by comparing the proteolytic fingerprints of human, bovine, and salmon thrombin. These enzymes share nearly identical specificity profiles despite approximately 390 million years of divergent evolution. Fluorogenic substrate microarrays provide a rapid way to determine protease substrate specificity information that can be used for the design of selective inhibitors and substrates, the study of evolutionary divergence, and potentially, for diagnostic applications.     

Conservation of the microstructure of genome segments in Brassica napus and its diploid relatives

The cultivated Brassica species are the group of crops most closely related to Arabidopsis thaliana (Arabidopsis). They represent models for the application in crops of genomic information gained in Arabidopsis and provide an opportunity for the investigation of polyploid genome formation and evolution. The scientific literature contains contradictory evidence for the dynamics of the evolution of polyploid genomes. We aimed at overcoming the inherent complexity of Brassica genomes and clarify the effects of polyploidy on the evolution of genome microstructure in specific segments of the genome. To do this, we have constructed bacterial artificial chromosome (BAC) libraries from genomic DNA of B. rapa subspecies trilocularis (JBr) and B. napus var Tapidor (JBnB) to supplement an existing BAC library from B. oleracea. These allowed us to analyse both recent polyploidization (under 10,000 years in B. napus) and more ancient polyploidization events (ca. 20 Myr for B. rapa and B. oleracea relative to Arabidopsis), with an analysis of the events occurring on an intermediate time scale (over the ca. 4 Myr since the divergence of the B. rapa and B. oleracea lineages). Using the Arabidopsis genome sequence and clones from the JBr library, we have analysed aspects of gene conservation and microsynteny between six regions of the genome of B. rapa with the homoeologous regions of the genomes of B. oleracea and Arabidopsis. Extensive divergence of gene content was observed between the B. rapa paralogous segments and their homoeologous segments within the genome of Arabidopsis. A pattern of interspersed gene loss was identified that is similar, but not identical, to that observed in B. oleracea. The conserved genes show highly conserved collinearity with their orthologues across genomes, but a small number of species-specific rearrangements were identified. Thus the evolution of genome microstructure is an ongoing process. Brassica napus is a recently formed polyploid resulting from the hybridization of B. rapa (containing the Brassica A genome) and B. oleracea (containing the Brassica C genome). Using clones from the JBnB library, we have analysed the microstructure of the corresponding segments of the B. napus genome. The results show that there has been little or no change to the microstructure of the analysed segments of the Brassica A and C genomes as a consequence of the hybridization event forming natural B. napus. The observations indicate that, upon polyploid formation, these segments of the genome did not undergo a burst of evolution discernible at the scale of microstructure.     

Binding interaction of SARS coronavirus 3CL(pro) protease with vacuolar-H+ ATPase G1 subunit

The pathogenesis of severe acute respiratory syndrome coronavirus (SARS-CoV) is an important issue for treatment and prevention of SARS. Recently, SARS-CoV 3CL(pro) protease has been implied to be possible relevance to SARS-CoV pathogenesis. In this study, we intended to identify potential 3CL(pro)-interacting cellular protein(s) using the phage-displayed human lung cDNA library. The vacuolar-H+ ATPase (V-ATPase) G1 subunit that contained a 3CL(pro) cleavage site-like motif was identified as a 3CL(pro)-interacting protein, as confirmed using the co-immunoprecipitation assay and the relative affinity assay. In addition, our result also demonstrated the cleavage of the V-ATPase G1 fusion protein and the immunoprecipitation of cellular V-ATPase G1 by the 3CL(pro). Moreover, loading cells with SNARF-1 pH-sensitive dye showed that the intracellular pH in 3CL(pro)-expressing cells was significantly lower as compared to mock cells.     

A method for the preparation of normalized cDNA libraries enriched with full-length sequences

We developed a new method for the preparation of normalized cDNA libraries enriched with full-length sequences. It is based on the properties of the recently characterized duplex-specific nuclease from the hepatopancreas of the Kamchatka crab. The duplex-specific nuclease is thermostable, effectively cleaves double-stranded DNA, and is inactive toward single-stranded DNA (Shagin et al., Genome Res., 2002, vol. 12, pp. 1935–1942). Our method enables the normalization of cDNA samples enriched with full-length sequences without use of laborious and ineffective stages of physical separation. The efficiency of the method was demonstrated in model experiments using cDNA samples from several human tissues.__________Translated from Bioorganicheskaya Khimiya, Vol. 31, No. 2, 2005, pp. 186–194.Original Russian Text Copyright © 2005 by Zhulidov, Bogdanova, Shcheglov, Shagina, Wagner, Khazpekov, Kozhemyako, Lukyanov, Shagin.     

Identification of the<Emphasis Type="Italic">bphC</Emphasis> gene for<Emphasis Type="Italic">meta</Emphasis>-cleavage of aromatic pollutants from a metagenomic library derived from lake waters

Useful genes can be screened from various environments by construction of metagenomic DNA libraries. In this study, water samples were collected from several lakes in mid Korea, and analyzed by T-RFLP to examine diversities of the microbial communities. The crude DNAs were extracted by the SDS-based freezing-thawing method, and then further purified using an UltraClean^TM kit (MoBio, USA). The metagenomic libraries were constructed with the DNAs partially digested withEcoR I,BamH I, andSac II inEscherichia coli DH 10B using the pBACe3.6 vector. About 44.0 Mb of metagenomic libraries were obtained with average inserts 13∼15 kb in size. ThebphC genes responsible for degradation of aromatic hydrocarbons viameta-cleavage were identified from the metagenomic libraries by colony hybridization using thebphC specific sequence as a probe. The 2,3-dihydroxybiphenyl (2,3-DHBP) dioxygenase gene (bphC), capable of degradation of 2,3-DHBP, was cloned and its nucleotide sequences analyzed. The genes consisted of 966 and 897 base pairs with an ATG initiation codon and a TGA termination codon. The activity of the 2,3-DHBP dioxygenase was highly expressed to 2,3-DHBP and showed a broad substrate range to 2,3-DHBP, catechol, 3-methylcatechol and 4-methylcatechol. These results indicated that thebphC gene identified from the metagenomes derived from lake water might be useful in the development of a potent strain for degradation of aromatic pollutants.     

Fluorescence correlation spectroscopy as a method for assessment of interactions between phage displaying antibodies and soluble antigen

Phage display is widely used for expression of combinatorial libraries, not least for protein engineering purposes. Precise selection at the single molecule level will provide an improved tool for generating proteins with complex and distinct properties from large molecular libraries. To establish such an improved selection system, we here report the detection of specific interactions between phage with displayed antibody fragments and fluorescently labeled soluble antigen based on Fluorescence Correlation Spectroscopy (FCS). Our novel strategy comprises the use of two separate fluorochromes for detection of the phage-antigen complex, either with labeled antiphage antibody or using a labeled antigen. As a model system, we studied a human monoclonal antibody to the hepatitis-C virus (HCV) envelope protein E2 and its cognate antigen (rE2 or rE1/E2). We could thus assess the specific interactions and determine the fraction of specific versus background phage (26% specific phage). Aggregation of these particular antigens made it difficult to reliably utilize the full potential of cross-correlation studies using the two labels simultaneously. However, with true monomeric proteins, this will certainly be possible, offering a great advantage in a safer and highly specific detection system.     

A mass-differentiated library strategy for identification of sugar nucleotidyltransferase activities from cell lysates

Sugar nucleotidyltransferases, or nucleotide sugar pyrophosphorylases, are ubiquitous enzymes whose activities have been correlated to disease states and pathogen virulence. Here we report a rapid “one-pot” method to identify a range of sugar nucleotidyltransferase activities of purified proteins or in cell lysates using a mass-differentiated carbohydrate library designed for mass spectrometry-based analysis.     

De novo isolation of antibodies with pH-dependent binding properties

pH-dependent antibodies are engineered to release their target at a slightly acidic pH, a property making them suitable for clinical as well as biotechnological applications. Such antibodies were previously obtained by histidine scanning of pre-existing antibodies, a labor-intensive strategy resulting in antibodies that displayed residual binding to their target at pH 6.0. We report here the de novo isolation of pH-dependent antibodies selected by phage display from libraries enriched in histidines. Strongly pH-dependent clones with various affinity profiles against CXCL10 were isolated by this method. Our best candidate has nanomolar affinity for CXCL10 at pH 7.2, but no residual binding was detected at pH 6.0. We therefore propose that this new process is an efficient strategy to generate pH-dependent antibodies.     

StreamingTrim 1.0: a Java software for dynamic trimming of 16S rRNA sequence data from metagenetic studies

Next‐generation sequencing technologies are extensively used in the field of molecular microbial ecology to describe taxonomic composition and to infer functionality of microbial communities. In particular, the so‐called barcode or metagenetic applications that are based on PCR amplicon library sequencing are very popular at present. One of the problems, related to the utilization of the data of these libraries, is the analysis of reads quality and removal (trimming) of low‐quality segments, while retaining sufficient information for subsequent analyses (e.g. taxonomic assignment). Here, we present StreamingTrim, a DNA reads trimming software, written in Java, with which researchers are able to analyse the quality of DNA sequences in fastq files and to search for low‐quality zones in a very conservative way. This software has been developed with the aim to provide a tool capable of trimming amplicon library data, retaining as much as taxonomic information as possible. This software is equipped with a graphical user interface for a user‐friendly usage. Moreover, from a computational point of view, StreamingTrim reads and analyses sequences one by one from an input fastq file, without keeping anything in memory, permitting to run the computation on a normal desktop PC or even a laptop. Trimmed sequences are saved in an output file, and a statistics summary is displayed that contains the mean and standard deviation of the length and quality of the whole sequence file. Compiled software, a manual and example data sets are available under the BSD‐2‐Clause License at the GitHub repository at https://github.com/GiBacci/StreamingTrim/ .     

Refining similarity scoring to enable decoy‐free validation in spectral library searching

Spectral library searching is a maturing approach for peptide identification from MS/MS, offering an alternative to traditional sequence database searching. Spectral library searching relies on direct spectrum‐to‐spectrum matching between the query data and the spectral library, which affords better discrimination of true and false matches, leading to improved sensitivity. However, due to the inherent diversity of the peak location and intensity profiles of real spectra, the resulting similarity score distributions often take on unpredictable shapes. This makes it difficult to model the scores of the false matches accurately, necessitating the use of decoy searching to sample the score distribution of the false matches. Here, we refined the similarity scoring in spectral library searching to enable the validation of spectral search results without the use of decoys. We rank‐transformed the peak intensities to standardize all spectra, making it possible to fit a parametric distribution to the scores of the nontop‐scoring spectral matches. The statistical significance of the top‐scoring match can then be estimated in a rigorous manner according to Extreme Value Theory. The overall result is a more robust and interpretable measure of the quality of the spectral match, which can be obtained without decoys. We tested this refined similarity scoring function on real datasets and demonstrated its effectiveness. This approach reduces search time, increases sensitivity, and extends spectral library searching to situations where decoy spectra cannot be readily generated, such as in searching unidentified and nonpeptide spectral libraries.