Abstract
Chemoproteomics is a key technology to characterize the mode of action of drugs, as it directly identifies the protein targets of bioactive compounds and aids in the development of optimized ...small-molecule compounds. Current approaches cannot identify the protein targets of a compound and also detect the interaction surfaces between ligands and protein targets without prior labeling or modification. To address this limitation, we here develop LiP-Quant, a drug target deconvolution pipeline based on limited proteolysis coupled with mass spectrometry that works across species, including in human cells. We use machine learning to discern features indicative of drug binding and integrate them into a single score to identify protein targets of small molecules and approximate their binding sites. We demonstrate drug target identification across compound classes, including drugs targeting kinases, phosphatases and membrane proteins. LiP-Quant estimates the half maximal effective concentration of compound binding sites in whole cell lysates, correctly discriminating drug binding to homologous proteins and identifying the so far unknown targets of a fungicide research compound.
Understanding how proteins and their complex interaction networks convert the genomic information into a dynamic living organism is a fundamental challenge in biological sciences. As an important ...step towards understanding the systems biology of a complex eukaryote, we cataloged 63% of the predicted Drosophila melanogaster proteome by detecting 9,124 proteins from 498,000 redundant and 72,281 distinct peptide identifications. This unprecedented high proteome coverage for a complex eukaryote was achieved by combining sample diversity, multidimensional biochemical fractionation and analysis-driven experimentation feedback loops, whereby data collection is guided by statistical analysis of prior data. We show that high-quality proteomics data provide crucial information to amend genome annotation and to confirm many predicted gene models. We also present experimentally identified proteotypic peptides matching approximately 50% of D. melanogaster gene models. This library of proteotypic peptides should enable fast, targeted and quantitative proteomic studies to elucidate the systems biology of this model organism.
Automatic gene prediction is one of the major challenges in computational sequence analysis. Traditional approaches to gene finding rely on statistical models derived from previously known genes. By ...contrast, a new class of comparative methods relies on comparing genomic sequences from evolutionary related organisms to each other. These methods are based on the concept of phylogenetic footprinting: they exploit the fact that functionally important regions in genomic sequences are usually more conserved than non-functional regions. We created a WWW-based software program for homology-based gene prediction at BiBiServ (Bielefeld Bioinformatics Server). Our tool takes pairs of evolutionary related genomic sequences as input data, e.g. from human and mouse. The server runs CHAOS and DIALIGN to create an alignment of the input sequences and subsequently searches for conserved splicing signals and start/stop codons near regions of local sequence conservation. Genes are predicted based on local homology information and splice signals. The server returns predicted genes together with a graphical representation of the underlying alignment. The program is available at http://bibiserv.TechFak.Uni-Bielefeld.DE/agenda/.
To characterize the quantitative properties of the optokinetic response (OKR) in zebrafish larvae as a tool to test visual performance in genetically modified larvae.
Horizontal OKR was triggered in ...5-day-old zebrafish larvae by stimulation with projected computer-generated gratings of varying contrast, angular velocity, temporal and spatial frequency, and brightness. Eye movements were analyzed by a custom-made eye tracker based on image analysis.
The gain of the OKR slow phase was dependent on angular velocity, spatial frequency, and contrast of a moving grating, but largely independent on brightness. Eye velocity was a logarithmically linear function of grating contrast with a slope of approximately 0.8 per log unit contrast.
The OKR of the larval zebrafish is not scaled for stimulus contrast and spatial frequency. These properties make the OKR a valuable tool to quantify behavioral visual performance such as visual acuity, contrast sensitivity, and light adaptation. This behavioral paradigm will be useful for analyzing visual performance in mutant and gene-knockdown larval zebrafish.
Phosphorylation of rhodopsin by rhodopsin kinase GRK1 is an important desensitization mechanism in scotopic vision. For cone vision GRK1 is not essential. However, cone opsin is phosphorylated ...following light stimulation. In cone-dominant animals as well as in humans, but not in rodents, GRK7, a cone-specific homolog of GRK1, has been identified in cone outer segments. To investigate the function of GRK7 in vivo, we cloned two orthologs of
grk7 in zebrafish and knocked down gene expression of
grk7a in zebrafish larvae by morpholino antisense nucleotides. Photoresponse recovery in Grk7a-deficient larvae was delayed in electroretinographic measurements, and temporal contrast sensitivity was reduced, particularly under bright-light conditions. These results show that function of a cone-specific kinase is essential for cone vision in the zebrafish retina and argue that pigment bleaching and spontaneous decay alone are not sufficient for light adaptation and rapid cone response inactivation.
Adaptation to a steady background has a profound effect on both color appearance and discrimination. We determined the temporal characteristics of chromatic adaptation for appearance and ...discrimination along different color directions. Subjects were adapted to a large uniform background made up of a CRT screen and a 45×64° wall, illuminated by computer controlled lamps. After an instant change in background color along a red–green or blue–yellow color axis, we measured thresholds for the detection of increments along the same axes at fixed times between 25 ms and 121 s. Analogously, color appearance was determined using achromatic matching. Three components of adaptation could be identified by their temporal characteristics. A slow exponential time course of adaptation with a half-life of about 20 s was common to appearance and discrimination. A faster component with a half-life of 40–70 ms — probably due to photoreceptor adaptation — was also common to both. Exclusive for color appearance, there was a third, extremely rapid mechanism with a half-life faster than 10 ms. This instantaneous process explained more than 50% of total adaptation for color appearance and could be shown to act in a multiplicative manner. We conclude that this instantaneous adaptation mechanism for color appearance is situated at a later processing stage, after mechanisms common to appearance and discrimination, and is based on multiplicative spatial interactions rather than on local, temporal adaptational processes. Color appearance, and thus color constancy, seems to be determined in large part by cortical computations.
Electroretinographic (ERG) method records a sum field potential of the retina in response to light. It mainly arises in the outer retina and is used as a non-invasive measure in both animal ...experiments and the clinic. Since it is a comprehensive method to assess outer retinal function, it is becoming increasingly useful in genetic studies of vision. Here we present a simple in-house built setup to measure ERGs of aquatic vertebrates. We have used this setup to efficiently and reliably measure intact larvae of zebrafish (
Danio rerio), Medaka fish (
Oryzias latipes), and
Xenopus laevis tadpoles. By slight modification of the setup, we were also able to measure adult zebrafish and Medaka, demonstrating the general versatility of the setup. We picked these organisms since they are increasingly used to study visual function with genetic means. This setup is easily built and will be particularly useful for laboratories setting up ERG measurements as a complement to their genetic studies.
We present a www server for homology-based gene prediction. The user enters a pair of evolutionary related genomic sequences, for example from human and mouse. Our software system uses CHAOS and ...DIALIGN to calculate an alignment of the input sequences and then searches for conserved splicing signals and start/stop codons around regions of local sequence similarity. This way, candidate exons are identified that are used, in turn, to calculate optimal gene models. The server returns the constructed gene model by email, together with a graphical representation of the underlying genomic alignment. Availability: http://bibiserv.TechFak.Uni-Bielefeld.DE/agenda/ Contact: ltaher@TechFak.Uni-Bielefeld.DE * To whom correspondence should be addressed.
Motivation: During evolution, functional regions in genomic sequences tend to be more highly conserved than randomly mutating ‘junk DNA’ so local sequence similarity often indicates biological ...functionality. This fact can be used to identify functional elements in large eukaryotic DNA sequences by cross-species sequence comparison. In recent years, several gene-prediction methods have been proposed that work by comparing anonymous genomic sequences, for example from human and mouse. The main advantage of these methods is that they are based on simple and generally applicable measures of (local) sequence similarity; unlike standard gene-finding approaches they do not depend on species-specific training data or on the presence of cognate genes in data bases. As all comparative sequence-analysis methods, the new comparative gene-finding approaches critically rely on the quality of the underlying sequence alignments. Results: Herein, we describe a new implementation of the sequence-alignment program DIALIGN that has been developed for alignment of large genomic sequences. We compare our method to the alignment programs PipMaker, WABA and BLAST and we show that local similarities identified by these programs are highly correlated to protein-coding regions. In our test runs, PipMaker was the most sensitive method while DIALIGN was most specific. Availability: The program is downloadable from the DIALIGN home page at http://bibiserv.techfak.uni-bielefeld.de/dialign/ Contact: burkhard@TechFak.Uni-Bielefeld.DE * To whom correspondence should be addressed at Universität Bielefeld, Technische Fakultät, Praktische Informatik, Postfach 100131, 33501 Bielefeld, Germany.
Colour constancy refers to the stable perception of object colour under changing illumination conditions. This problem has been reformulated as relational colour constancy, or the ability of the ...observer to discriminate between material changes and changes in illumination. It has been suggested that local cone excitation ratios play a prominent role in achieving such constancy. Here we show that perceptual colour constancy measured by achromatic adjustments is to a large part complete after 25 ms. This speaks against a prominent role for receptor adaptation, which takes significantly longer. We also found no difference in colour constancy between colour changes that were compatible with a change of illuminant, and between colour changes where local cone ratios were uncorrelated between the two illuminants. Our results show that constant cone ratios are not necessary for colour constancy.
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