Fast and accurate mutation detection in whole genome sequences of multiple isogenic samples with IsoMut.

BACKGROUND: Detection of somatic mutations is one of the main goals of next generation DNA sequencing. A wide range of experimental systems are available for the study of spontaneous or environmentally induced mutagenic processes. However, most of the routinely used mutation calling algorithms are not optimised for the simultaneous analysis of multiple samples, or for non-human experimental model systems with no reliable databases of common genetic variations. Most standard tools either require numerous in-house post filtering steps with scarce documentation or take an unpractically long time to run. To overcome these problems, we designed the streamlined IsoMut tool which can be readily adapted to experimental scenarios where the goal is the identification of experimentally induced mutations in multiple isogenic samples. METHODS: Using 30 isogenic samples, reliable cohorts of validated mutations were created for testing purposes. Optimal values of the filtering parameters of IsoMut were determined in a thorough and strict optimization procedure based on these test sets. RESULTS: We show that IsoMut, when tuned correctly, decreases the false positive rate compared to conventional tools in a 30 sample experimental setup; and detects not only single nucleotide variations, but short insertions and deletions as well. IsoMut can also be run more than a hundred times faster than the most precise state of art tool, due its straightforward and easily understandable filtering algorithm. CONCLUSIONS: IsoMut has already been successfully applied in multiple recent studies to find unique, treatment induced mutations in sets of isogenic samples with very low false positive rates. These types of studies provide an important contribution to determining the mutagenic effect of environmental agents or genetic defects, and IsoMut turned out to be an invaluable tool in the analysis of such data.

Predicting protein conformation by statistical methods.

The unique folded structure makes a polypeptide a functional protein. The number of known sequences is about a hundred times larger than the number of known structures and the gap is increasing rapidly. The primary goal of all structure prediction methods is to obtain structure-related information on proteins, whose structures have not been determined experimentally. Besides this goal, the development of accurate prediction methods helps to reveal principles of protein folding. Here we present a brief survey of protein structure predictions based on statistical analyses of known sequence and structure data. We discuss the background of these methods and attempt to elucidate principles, which govern structure formation of soluble and membrane proteins.

The HMMTOP transmembrane topology prediction server.

UNLABELLED: The HMMTOP transmembrane topology prediction server predicts both the localization of helical transmembrane segments and the topology of transmembrane proteins. Recently, several improvements have been introduced to the original method. Now, the user is allowed to submit additional information about segment localization to enhance the prediction power. This option improves the prediction accuracy as well as helps the interpretation of experimental results, i.e. in epitope insertion experiments. AVAILABILITY: HMMTOP 2.0 is freely available to non-commercial users at http://www.enzim.hu/hmmtop. Source code is also available upon request to academic users.

Prion protein: evolution caught en route.

The prion protein displays a unique structural ambiguity in that it can adopt multiple stable conformations under physiological conditions. In our view, this puzzling feature resulted from a sudden environmental change in evolution when the prion, previously an integral membrane protein, got expelled into the extracellular space. Analysis of known vertebrate prions unveils a primordial transmembrane protein encrypted in their sequence, underlying this relocalization hypothesis. Apparently, the time elapsed since this event was insufficient to create a "minimally frustrated" sequence in the new milieu, probably due to the functional constraints set by the importance of the very flexibility that was created in the relocalization. This scenario may explain why, in a structural sense, the prion protein is still en route toward becoming a foldable globular protein.

Topology of membrane proteins.

Integral membrane proteins play important roles in living cells. Due to difficulties of experimental techniques, theoretical approaches, i.e., topology prediction methods, are important for structure determination of this class of proteins. Here we show a detailed comparison of transmembrane topology prediction methods. According to this comparison, we conclude that the topology of integral membrane proteins is determined by the maximum divergence of the amino acid composition of sequence segments. These segments are located in different areas of the cell, which can be characterized by different physicochemical properties. The results of these prediction methods compared to the X-ray diffraction data of several transmembrane proteins will also be discussed.

Characterization of the amino-terminal regions in the human multidrug resistance protein (MRP1).

The human multidrug resistance protein (MRP1) contributes to drug resistance in cancer cells. In addition to an MDR1-like core, MRP1 contains an N-terminal membrane-bound (TMD(0)) region and a cytoplasmic linker (L(0)), both characteristic of several members of the MRP family. In order to study the role of the TMD(0) and L(0) regions, we constructed various truncated and mutated MRP1, and chimeric MRP1-MDR1 molecules, which were expressed in insect (Sf9) and polarized mammalian (MDCKII) cells. The function of the various proteins was examined in isolated membrane vesicles by measuring the transport of leukotriene C(4) and other glutathione conjugates, and by vanadate-dependent nucleotide occlusion. Cellular localization, and glutathione-conjugate and drug transport, were also studied in MDCKII cells. We found that chimeric proteins consisting of N-terminal fragments of MRP1 fused to the N terminus of MDR1 preserved the transport, nucleotide occlusion and apical membrane routing of wild-type MDR1. As shown before, MRP1 without TMD(0)L(0) (Delta MRP1), was non-functional and localized intracellularly, so we investigated the coexpression of Delta MRP1 with the isolated L(0) region. Coexpression yielded a functional MRP1 molecule in Sf9 cells and routing to the lateral membrane in MDCKII cells. Interestingly, the L(0) peptide was found to be associated with membranes in Sf9 cells and could only be solubilized by urea or detergent. A 10-amino-acid deletion in a predicted amphipathic region of L(0) abolished its attachment to the membrane and eliminated MRP1 transport function, but did not affect membrane routing. Taken together, these experiments suggest that the L(0) region forms a distinct domain within MRP1, which interacts with hydrophobic membrane regions and with the core region of MRP1.

Functional multidrug resistance protein (MRP1) lacking the N-terminal transmembrane domain.

The human multidrug resistance protein (MRP1) causes drug resistance by extruding drugs from tumor cells. In addition to an MDR-like core, MRP1 contains an N-terminal membrane-bound region (TMD0) connected to the core by a cytoplasmic linker (L0). We have studied truncated MRP1 versions containing either the MDR-like core alone or the core plus linker L0, produced in the baculovirus-insect (Sf9) cell system. Their function was examined in isolated membrane vesicles. Full-length MRP1 showed ATP-dependent, vanadate-sensitive accumulation of leukotriene C4 and N-ethylmaleimide glutathione. In addition, leukotriene C4-stimulated, vanadate-dependent nucleotide occlusion was detected. The MDR-like core was virtually inactive. Co-expression of the core with the N-terminal region including L0 fully restored MRP1 function. Unexpectedly, a truncated MRP1 mutant lacking the entire TMD0 region but still containing L0 behaved like wild-type MRP1 in vesicle uptake and nucleotide trapping experiments. We also expressed the MRP1 constructs in polarized canine kidney derived MDCKII cells. Like wild-type MRP1, the MRP1 protein without the TMD0 region was routed to the lateral plasma membrane and transported dinitrophenyl glutathione and daunorubicin. The TMD0L0 and the MRP1 minus TMD0L0 remained in an intracellular compartment. Taken together, these experiments strongly suggest that the TMD0 region is neither required for the transport function of MRP1 nor for its proper routing to the plasma membrane.

Principles governing amino acid composition of integral membrane proteins: application to topology prediction.

A new method is suggested here for topology prediction of helical transmembrane proteins. The method is based on the hypothesis that the localizations of the transmembrane segments and the topology are determined by the difference in the amino acid distributions in various structural parts of these proteins rather than by specific amino acid compositions of these parts. A hidden Markov model with special architecture was developed to search transmembrane topology corresponding to the maximum likelihood among all the possible topologies of a given protein. The prediction accuracy was tested on 158 proteins and was found to be higher than that found using prediction methods already available. The method successfully predicted all the transmembrane segments in 143 proteins out of the 158, and for 135 of these proteins both the membrane spanning regions and the topologies were predicted correctly. The observed level of accuracy is a strong argument in favor of our hypothesis.

Membrane topology distinguishes a subfamily of the ATP-binding cassette (ABC) transporters.

A group of ATP-binding cassette (ABC) transporters, including the yeast cadmium transporter (YCF1), the mammalian multidrug resistance-associated protein (MRP), the multispecific organic anion transporter and its congener (MOAT and EBCR), as well as the sulfonylurea receptor (SUR), group into a subfamily by sequence comparison. We suggest that these MRP-related proteins are also characterized by a special, common membrane topology pattern. The most studied ABC transporters, the cystic fibrosis transmembrane conductance regulator (CFTR) and the multidrug resistance (MDR) proteins, were shown to contain a tandem repeat of six transmembrane helices, each set followed by an ATP-binding domain. According to the present study, in contrast to various membrane topology predictions proposed for the different MRP-related proteins, they all seem to have a CFTR/MDR-like core structure, and an additional, large, N-terminal hydrophobic region. This latter domain is predicted to contain 4-6 (most probably 5) transmembrane helices, and is occasionally glycosylated on the cell surface. Since all the MRP-related transporters were shown to interact with anionic compounds, the N-terminal membrane-bound domain may have a key role in these interactions.

Membrane topology and glycosylation of the human multidrug resistance-associated protein.

The membrane topology of the human multidrug resistance-associated protein (MRP) was examined by flow cytometry phenotyping, immunoblotting, and limited proteolysis in drug-resistant human and baculovirus-infected insect cells, expressing either the glycosylated or the underglycosylated forms of this protein. Inhibition of N-linked glycosylation in human cells by tunicamycin did not inhibit the transport function or the antibody recognition of MRP, although its apparent molecular mass was reduced from 180 kDa to 150 kDa. Extracellular addition of trypsin or chymotrypsin had no effect either on the function or on the molecular mass of MRP, while in isolated membranes limited proteolysis produced three large membrane-bound fragments. These experiments and the alignment of the MRP sequence with the human cystic fibrosis transmembrane conductance regulator (CFTR) suggest that human MRP, similarly to CFTR, contains a tandem repeat of six transmembrane helices, each followed by a nucleotide binding domain, and that the C-terminal membrane-bound region is glycosylated. However, the N-terminal region of MRP contains an additional membrane-bound, glycosylated area with four or five transmembrane helices, which seems to be a characteristic feature of MRP-like ATP-binding cassette transporters.

Independence divergence-generated binary trees of amino acids.

The discovery of the relationship between amino acids is important in terms of the replacement ability, as used in protein engineering homology studies, and gaining a better understanding of the roles which various properties of the residues play in the creation of a unique, stable, 3-D protein structure. Amino acid sequences of proteins edited by evolution are anything but random. The measure of nonrandomness, i.e. the level of editing, can be characterized by an independence divergence value. This parameter is used to generate binary tree relationships between amino acids. The relationships of residues presented in this paper are based on protein building features and not on the physico-chemical characteristics of amino acids. This approach is not biased by the tautology present in all sequence similarity-based relationship studies. The roles which various physico-chemical characteristics play in the determination of the relationships between amino acids are also discussed.

Chaos game representation of protein structures.

Chaos game representation (CGR) was proposed recently to visualize nucleotide sequences as one of the first applications of this technique in the field of biochemistry. In this paper we would like to demonstrate that representations similar to CGR can be generalized and applied for visualizing and analyzing protein databases. Examples of applications will be presented for investigating regularities, and motifs in the primary structure of proteins, and for analyzing possible structural attachments on the super-secondary structure level of proteins. A further application will be presented for testing structure prediction methods using CGR.