LGL: creating a map of protein function with an algorithm for visualizing very large biological networks

Networks are proving to be central to the study of gene function, protein-protein interaction, and biochemical pathway data. Visualization of networks is important for their study, but visualization tools are often inadequate for working with very large biological networks. Here, we present an algorithm, called large graph layout (LGL), which can be used to dynamically visualize large networks on the order of hundreds of thousands of vertices and millions of edges. LGL applies a force-directed iterative layout guided by a minimal spanning tree of the network in order to generate coordinates for the vertices in two or three dimensions, which are subsequently visualized and interactively navigated with companion programs. We demonstrate the use of LGL in visualizing an extensive protein map summarizing the results of approximately 21 billion sequence comparisons between 145579 proteins from 50 genomes. Proteins are positioned in the map according to sequence homology and gene fusions, with the map ultimately serving as a theoretical framework that integrates inferences about gene function derived from sequence homology, remote homology, gene fusions, and higher-order fusions. We confirm that protein neighbors in the resulting map are functionally related, and that distinct map regions correspond to distinct cellular systems, enabling a computational strategy for discovering proteins' functions on the basis of the proteins' map positions. Using the map produced by LGL, we infer general functions for 23 uncharacterized protein families.     

Computational methods for sequence mapping of large combinatorial libraries and deduced sequence signatures

Here we describe a computational approach for the high-throughput sequence mapping of combinatorial libraries obtained by DNA shuffling. Original algorithms and their software implementation were developed for the automated and reliable analysis of hybridization data of differentially labeled oligonucleotide probes with PCR products spotted on DNA microarrays. This novel approach allows a context-dependent sequence attribution tolerant to fluctuations in experimental conditions and is well adapted to hybridization signals of variable qualities resulting from high-throughput PCR amplification from colonies. In addition, the analysis permits the calculation of sequence signatures that are characteristic of combinatorial library structure, defects, and diversity. The approach is of interest for the characterization and the equalization (library reduction to nonredundant structures) of combinatorial libraries involved in directed evolution and could be extrapolated to high-throughput polymorphism analysis.     

Comparison of sequence profiles. Strategies for structural predictions using sequence information

Distant homologies between proteins are often discovered only after three-dimensional structures of both proteins are solved. The sequence divergence for such proteins can be so large that simple comparison of their sequences fails to identify any similarity. New generation of sensitive alignment tools use averaged sequences of entire homologous families (profiles) to detect such homologies. Several algorithms, including the newest generation of BLAST algorithms and BASIC, an algorithm used in our group to assign fold predictions for proteins from several genomes, are compared to each other on the large set of structurally similar proteins with little sequence similarity. Proteins in the benchmark are classified according to the level of their similarity, which allows us to demonstrate that most of the improvement of the new algorithms is achieved for proteins with strong functional similarities, with almost no progress in recognizing distant fold similarities. It is also shown that details of profile calculation strongly influence its sensitivity in recognizing distant homologies. The most important choice is how to include information from diverging members of the family, avoiding generating false predictions, while accounting for entire sequence divergence within a family. PSI-BLAST takes a conservative approach, deriving a profile from core members of the family, providing a solid improvement without almost any false predictions. BASIC strives for better sensitivity by increasing the weight of divergent family members and paying the price in lower reliability. A new FFAS algorithm introduced here uses a new procedure for profile generation that takes into account all the relations within the family and matches BASIC sensitivity with PSI-BLAST like reliability.     

The integration of genomic and structural information in the development of high affinity plasmepsin inhibitors

The plasmepsins are key enzymes in the life cycle of the Plasmodium parasites responsible for malaria. Since plasmepsin inhibition leads to parasite death, these enzymes have been acknowledged to be important targets for the development of new antimalarial drugs. The development of effective plasmepsin inhibitors, however, is compounded by their genomic diversity which gives rise not to a unique target for drug development but to a family of closely related targets. Successful drugs will have to inhibit not one but several related enzymes with high affinity. Structure-based drug design against heterogeneous targets requires a departure from the classic 'lock-and-key' paradigm that leads to the development of conformationally constrained molecules aimed at a single target. Drug molecules designed along those principles are usually rigid and unable to adapt to target variations arising from naturally occurring genetic polymorphisms or drug-induced resistant mutations. Heterogeneous targets need adaptive drug molecules, characterised by the presence of flexible elements at specific locations that sustain a viable binding affinity against existing or expected polymorphisms. Adaptive ligands have characteristic thermodynamic signatures that distinguish them from their rigid counterparts. This realisation has led to the development of rigorous thermodynamic design guidelines that take advantage of correlations between the structure of lead compounds and the enthalpic and entropic components of the binding affinity. In this paper, we discuss the application of the thermodynamic approach to the development of high affinity (K(i) - pM) plasmepsin inhibitors. In particular, a family of allophenylnorstatine-based compounds is evaluated for their potential to inhibit a wide spectrum of plasmepsins.     

Covalent attachment of peptides for high sensitivity solid-phase sequence analysis

We have developed a method for the high efficiency covalent immobilization of picomole to nanomole quantities of peptides in a form compatible with high sensitivity gas-liquid or solid-phase sequence analysis. Glass fiber filter paper was derivatized with amino-phenyltriethoxysilane and peptides were applied to circular disks cut to 1-cm diameters. Peptides were covalently immobilized on the aminophenyl-glass fiber paper through their terminal alpha-carboxyl groups and amino acid side-chain carboxyl groups by activation with the water-soluble reagent N-ethyl-N'-(3-dimethylaminopropyl)carbodiimide. Disks containing the covalently attached peptide were directly inserted into the cartridge of an automated sequenator for sequence analysis by the Edman degradation. Peptides prepared in this way could be routinely sequenced through to and including the C-terminal amino acid residue, at extraordinarily low backgrounds. The covalent immobilization of peptide fragments allowed far more flexibility in sequencing conditions, including the use of polar extraction solvents to increase the yield of phenylthiohydantoin (PTH)-His and PTH-Arg and the use of alternative Edman-type sequencing reagents with enhanced detectability, such as the chromophoric compound 4- (N,N'-dimethylamino)azobenzene-4'-isothiocyanate. The potential of this high efficiency immobilization method for contributing to the development of sequencing chemistries with enhanced sensitivity is discussed.     

DNAMAT: an efficient graphic matrix sequence homology algorithm and its application to structural analysis

We present a fast algorithm to produce a graphic matrix representationof sequence homology. The algorithm is based on lexicographicalordering of fragments. It preserves most of the options of asimple naive algorithm with a significant increase in speed.This algorithm was the basis for a program, called DNAMAT, thathas been extensively tested during the last three years at theWeizmann Institute of Science and has proven to be very useful.In addition we suggest a way to extend our approach to analysea series of related DNA or RNA sequences, in order to determinecertain common structural features. The analysis is done by‘summing’ a set of dot-matrices to produce an overallmatrix that displays structural elements common to most of thesequences. We give an example of this procedure by analysingtRNA sequences. Received on June 26, 1986; accepted on September 28, 1986     

AutoCSA, an algorithm for high throughput DNA sequence variant detection in cancer genomes

The undertaking of large-scale DNA sequencing screens for somatic variants in human cancers requires accurate and rapid processing of traces for variants. Due to their often aneuploid nature and admixed normal tissue, heterozygous variants found in primary cancers are often subtle and difficult to detect. To address these issues, we have developed a mutation detection algorithm, AutoCSA, specifically optimized for the high throughput screening of cancer samples. Availability: http://www.sanger.ac.uk/genetics/CGP/Software/AutoCSA.     

In vitro selection of an RNA sequence that interacts with high affinity with thymidylate synthase

Previous studies have shown that the repressive effect of thymidylate synthase (TS) mRNA translation is mediated by direct binding of TS itself to two cis-acting elements on its cognate mRNA. To identify the optimal RNA nucleotides that interact with TS, we in vitro synthesized a completely degenerate, linear RNA pool of 25 nt and employed in vitro selection to isolate high affinity RNA ligands that bind human TS protein. After 10 rounds of selection and amplification, a single RNA molecule was selected that bound TS protein with nearly 20-fold greater affinity than native, wild-type TS RNA sequences. Secondary structure analysis of this RNA sequence predicted it to possess a stem–loop structure. Deletion and/or modification of the UGU loop element within the RNA sequence decreased binding to TS by up to 1000-fold. In vivo transfection experiments revealed that the presence of the selected RNA sequence resulted in a significant increase in the expression of a heterologous luciferase reporter construct in human colon cancer H630 and TS-overexpressing HCT-C:His-TS+ cells, but not in HCT-C18 cells expressing a functionally inactive TS. In addition, the presence of this element in H630 cells leads to induced expression of TS protein. An immunoprecipitation method using RT–PCR confirmed a direct interaction between human TS protein and the selected RNA sequence in transfected human cancer H630 cells. This study identified a novel RNA sequence from a degenerate RNA library that specifically interacts with TS.     

To distract or not to distract: an algorithm for airway management in isolated Pierre Robin sequence

Approaches advocated for treatment of airway obstruction among neonates with Pierre Robin sequence include positioning, tongue-lip adhesion, mandibular distraction, and tracheostomy, with no established guidelines regarding which modality is appropriate for a specific patient. This report proposes an algorithm for the management of neonatal upper airway obstruction among patients with isolated Pierre Robin sequence. Data for 21 patients with isolated Pierre Robin sequence who were treated by one surgeon during a 9-year period were reviewed. Eighteen patients presented during the first 1 week of life and three patients presented late, between 12 and 33 months of age. Follow-up periods ranged from 9 to 70 months (median, 33 months). Successful airway management was achieved with positioning alone for 10 patients, with tongue-lip adhesion for seven of nine patients, with tracheostomy for two patients, and with mandibular distraction for three patients. Changes in the maxillary-mandibular discrepancy were significant with natural mandibular growth during the first 1 year of life (p < 0.0001). Oromotor studies performed 3 months or more after tongue-lip adhesion reversal (n = 9) demonstrated no appreciable deficits in tongue function, relative to other children with cleft lips/palates. A multidisciplinary team should evaluate all patients with isolated Pierre Robin sequence, to fully assess the maxillary-mandibular relationship, anatomically define the site of airway obstruction, and identify feeding difficulties. Patients should be evaluated for episodes of desaturation occurring spontaneously, during feeding, or during sleeping. Patients with desaturation should be further evaluated with double endoscopy (nasoendoscopy and bronchoscopy). If the airway obstruction is localized to the tongue base alone and cannot be controlled with positioning, then tongue-lip adhesion is the initial treatment of choice, because such patients demonstrate significant mandibular growth during the first 1 year of life. Mandibular distraction among neonates is reserved for failures of tongue-lip adhesion in which isolated tongue-base airway obstruction is documented. Neither of the patients who experienced failure of tongue-lip adhesion in this series would have been a candidate for distraction with the algorithm presented. Avoiding routine neonatal distraction serves to avoid facial scarring, nerve and tooth bud injury, and potential disturbances of intrinsic mandibular growth. Patients with persistent respiratory difficulties beyond age 9 months require reevaluation for multiple sites of airway obstruction. Mandibular distraction may be one of several modalities required to avoid tracheostomy for such patients.     

Biochemical and structural studies on the high affinity of Hsp70 for ADP

The molecular chaperone 70-kDa heat shock protein (Hsp70) is driven by ATP hydrolysis and ADP-ATP exchange. ADP dissociation from Hsp70 is reportedly slow in the presence of inorganic phosphate (P(i) ). In this study, we investigated the interaction of Hsp70 and its nucleotide-binding domain (NBD) with ADP in detail, by isothermal titration calorimetry measurements and found that Mg(2+) ion dramatically elevates the affinity of Hsp70 for ADP. On the other hand, P(i) increased the affinity in the presence of Mg(2+) ion, but not in its absence. Thus, P(i) enhances the effect of the Mg(2+) ion on the ADP binding. Next, we determined the crystal structures of the ADP-bound NBD with and without Mg(2+) ion. As compared with the Mg(2+) ion-free structure, the ADP- and Mg(2+) ion-bound NBD contains one Mg(2+) ion, which is coordinated with the β-phosphate group of ADP and associates with Asp10, Glu175, and Asp199, through four water molecules. The Mg(2+) ion is also coordinated with one P(i) molecule, which interacts with Lys71, Glu175, and Thr204. In fact, the mutations of Asp10 and Asp199 reduced the affinity of the NBD for ADP, in both the presence and the absence of P(i) . Therefore, the Mg(2+) ion-mediated network, including the P(i) and water molecules, increases the affinity of Hsp70 for ADP, and thus the dissociation of ADP is slow. In ADP-ATP exchange, the slow ADP dissociation might be rate-limiting. However, the nucleotide-exchange factors actually enhance ADP release by disrupting the Mg(2+) ion-mediated network.     

Construction of a high sensitive Escherichia coli alkaline phosphatase reporter system for screening affinity peptides

An enzyme-linker-peptide fusion protein reporter system was constructed for sensitive analysis of affinity of peptide ligands to their receptor. An E. coli alkaline phosphatase (EAP) mutant enzyme with high catalytic activity was selected as the reporter protein. Interaction of affinity peptide and streptavidin was applied as demonstration of the method. Three affinity peptides, strep-tag I (SI), strep-tag II (SII) and streptavidin binding peptide (SBP) were genetically fused to the C-terminal of EAP respectively, with an insertion of a flexible linker peptide in between. The enzyme activity of the EAP fusions showed no obvious change. After expression and purification, the EAP-affinity peptide fusions were applied to the streptavidin modified surface. Binding of the fusions to the surface through interaction of affinity peptides to streptavidin was indicated by color generated from conversion of the substrate by EAP. The relative affinity and specificity of each affinity peptides to the immobilized streptavidin were then evaluated with high sensitivity and broad detection range. This method may be used for effective high-throughput screening of high affinity peptide from the peptide pool.     

Construction of a high sensitive Escherichia coli alkaline phosphatase reporter system for screening affinity peptides

An enzyme-linker-peptide fusion protein reporter system was constructed for sensitive analysis of affinity of peptide ligands to their receptor. An E. coli alkaline phosphatase (EAP) mutant enzyme with high catalytic activity was selected as the reporter protein. Interaction of affinity peptide and streptavidin was applied as demonstration of the method. Three affinity peptides, strep-tag I (SI), strep-tag II (SII) and streptavidin binding peptide (SBP) were genetically fused to the C-terminal of EAP respectively, with an insertion of a flexible linker peptide in between. The enzyme activity of the EAP fusions showed no obvious change. After expression and purification, the EAP-affinity peptide fusions were applied to the streptavidin modified surface. Binding of the fusions to the surface through interaction of affinity peptides to streptavidin was indicated by color generated from conversion of the substrate by EAP. The relative affinity and specificity of each affinity peptides to the immobilized streptavidin were then evaluated with high sensitivity and broad detection range. This method may be used for effective high-throughput screening of high affinity peptide from the peptide pool.     

IGF-II binding on human lymphoid cells: demonstration of a common high affinity receptor for insulin like peptides

We have studied the binding of 125I-GF-II to the IM-9 human lymphoid cell line, and to human placental membranes. All of IGF-II radioligand binding to IM-9 cells, and half of the binding to human placental membranes is to a previously unrecognized common (Type-III) high affinity receptor site for insulin-like peptides, in which IGF-I and IGF-II are equipotent and insulin only slightly less potent. This common receptor represents another mechanism by which insulin, and the somatomedins can exert biological action.     

The iRMSD: a local measure of sequence alignment accuracy using structural information

MOTIVATION: We introduce the iRMSD, a new type of RMSD, independent from any structure superposition and suitable for evaluating sequence alignments of proteins with known structures. RESULTS: We demonstrate that the iRMSD is equivalent to the standard RMSD although much simpler to compute and we also show that it is suitable for comparing sequence alignments and benchmarking multiple sequence alignment methods. We tested the iRMSD score on 6 established multiple sequence alignment packages and found the results to be consistent with those obtained using an established reference alignment collection like Prefab. AVAILABILITY: The iRMSD is part of the T-Coffee package and is distributed as an open source freeware (http://www.tcoffee.org/).     

Photoaffinity labeling identification of thyroid hormone-regulated glucocorticoid-binding peptides in rat liver endoplasmic reticulum: an oligomeric protein with high affinity for 16beta-hydroxylated stanozolol

Steroid-binding proteins unrelated to the classical nuclear receptors have been proposed to play a role in non-genomic actions of the17-alkylated testosterone derivative (17-AA) stanozolol (ST). We have previously reported that male rat liver endoplasmic reticulum contains two steroid-binding sites associated with high molecular mass oligomeric proteins: (1) the ST-binding protein (STBP); and (2) the low-affinity glucocorticoid-binding protein (LAGS). To further explore the role of LAGS on the mechanism of action of ST, we have now studied: (1) the interaction of ST and its hydroxylated metabolites with solubilized LAGS and the cytosolic glucocorticoid receptor (GR); and (2) the effects of hormones on the capability of STBP to bind ST. We found that, unlike 17-methyltestosterone, neither ST nor its hydroxylated metabolites bind to GR. However, the 16β-hydroxylation of ST significantly increases the capability of LAGS to bind ST. Interestingly, 3′-hydroxylation of ST abrogates the capability of LAGS to bind ST. ST (k_i=30 nM) and 16β-hydroxystanozolol (k_i=13 nM) bind with high affinity to LAGS, and are capable of accelerating the rate of dissociation of previously bound dexamethasone from the LAGS. STBP and LAGS are strongly induced by ethinylestradiol. However, unlike STBP, LAGS is regulated by thyroid hormones and growth hormone, which proves that these steroid-binding activities are associated with different binding sites. These findings seem to suggest a novel mechanism for ST whereby membrane-associated glucocorticoid-binding activity is targeted by the 16β-hydroxylated metabolite of ST. ST and its 16β-hydroxylated metabolite modulate glucocorticoid activity in the liver through negative allosteric modulation of LAGS, with the result of this interaction an effective increase in classical GR-signaling by increasing glucocorticoid availability to the cytosolic GR.     

DynaBiS: A hierarchical sampling algorithm to identify flexible binding sites for large ligands and peptides

Knowing the ligand or peptide binding site in proteins is highly important to guide drug discovery, but experimental elucidation of the binding site is difficult. Therefore, various computational approaches have been developed to identify potential binding sites in protein structures. However, protein and ligand flexibility are often neglected in these methods due to efficiency considerations despite the recognition that protein–ligand interactions can be strongly affected by mutual structural adaptations. This is particularly true if the binding site is unknown, as the screening will typically be performed based on an unbound protein structure. Herein we present DynaBiS, a hierarchical sampling algorithm to identify flexible binding sites for a target ligand with explicit consideration of protein and ligand flexibility, inspired by our previously presented flexible docking algorithm DynaDock. DynaBiS applies soft-core potentials between the ligand and the protein, thereby allowing a certain protein–ligand overlap resulting in efficient sampling of conformational adaptation effects. We evaluated DynaBiS and other commonly used binding site identification algorithms against a diverse evaluation set consisting of 26 proteins featuring peptide as well as small ligand binding sites. We show that DynaBiS outperforms the other evaluated methods for the identification of protein binding sites for large and highly flexible ligands such as peptides, both with a holo or apo structure used as input.     

Monohydroxytamoxifen: an antioestrogen with high affinity for the chick oviduct oestrogen receptor

The monohydroxylated derivative of tamoxifen (a non-steroidal triaryl ethylene antioestrogen) shows an apparent affinity (Ki = 0.2 nM) for the chick oviduct oestrogen receptor which is higher than that of oestradiol itself, and ～ 10 times higher than that of tamoxifen. Administered $\text{in}\text{vivo}$ with oestradiol benzoate, it inhibited the increase of tissue growth, progesterone receptor content, ornithine decarboxylase activity (ODC), and ovalbumin and conalbumin synthesis, and also inhibited the oestradiol induced increase of ODC $\text{in}\text{vitro}$. It did not display any oestrogenic effect by itself. We conclude that antioestrogenic action may be exhibited by a molecule with higher affinity binding to the oestrogen receptor than oestradiol itself. Metabolic studies demonstrated that the antioestrogenic action of tamoxifen is not due to its prior conversion to monohydroxytamoxifen.     

Optimized protocols for the isolation of specific protein-binding peptides or peptoids from combinatorial libraries displayed on beads

Many methods have been published by which combinatorial libraries may be screened for compounds capable of manipulating the function(s) of a target protein. One of the simplest approaches is to identify compounds in a library that bind the protein of interest, since these binding events usually occur on functionally important surfaces of the protein. These protein-binding compounds could also be of utility as protein capture agents in the construction of protein-detecting microarrays or related analytical devices and as reagents for the affinity purification of proteins from complex mixtures. In this article, we provide optimized methods for screening libraries of molecules displayed on the beads on which they were synthesized. This is a particularly convenient format for library screening for laboratories with limited budgets and modest robotics capabilities.     

Just-in-time purification: an effective solution for cherry-picking and purifying active compounds from large legacy libraries

Many companies possess a compound collection consisting of purified compounds and of unpurified products from combinatorial libraries. Using commercial and proprietary compounds as examples, this report provides clear examples of the significant impact purification can have on the activity observed for a compound and highlights the need to retest the purified compounds prior to creating structure-activity relationships. Crude mixtures made with commercial compounds led to an increase in the number of false positives in the SXR-GAL4 assay as compared with their pure and purified counterparts. An examination of proprietary compounds in an HIV assay resulted in the purification of 61 active crude synthetic mixtures. Of these 61 compounds, 32 were 5-fold less active and 2 were 5-fold more active after purification. This report details a semiautomated process developed and implemented for cherry-picking, tracking, and selectively purifying compounds found active in high-throughput screening campaigns.