Bioinformatic analyses of mammalian 5'-UTR sequence properties of mRNAs predicts alternative translation initiation sites-4

Eural Networks are a computational algorithm that uses layers of neurons with weighted edges connecting each layer to perform classification. To determine the specific ANN architecture, this study started with a static training set and modified the number of neurons in the hidden layer of the ANN as well as the activation function used for the neurons in each layer. The resulting ANN contained 10 neurons in the input layer, 20 neurons in the hidden layer and a single output neuron. Inputs to the ANN are normalized in order to negate the effect of measurements in different ranges. The output neuron provides values in the range [0, 1].<p><b>Copyright information:</b></p><p>Taken from "Bioinformatic analyses of mammalian 5'-UTR sequence properties of mRNAs predicts alternative translation initiation sites"</p><p>http://www.biomedcentral.com/1471-2105/9/232</p><p>BMC Bioinformatics 2008;9():232-232.</p><p>Published online 8 May 2008</p><p>PMCID:PMC2396638.</p><p></p

Bioinformatic analyses of mammalian 5'-UTR sequence properties of mRNAs predicts alternative translation initiation sites-5

Categories. These annotations were compiled via BLAST searches and subsequent Gene Ontology (GO) and protein family analysis. The chart depicts the protein functions represented by the identified aTIS sequences. The functions of these proteins are significant for biological regulation.<p><b>Copyright information:</b></p><p>Taken from "Bioinformatic analyses of mammalian 5'-UTR sequence properties of mRNAs predicts alternative translation initiation sites"</p><p>http://www.biomedcentral.com/1471-2105/9/232</p><p>BMC Bioinformatics 2008;9():232-232.</p><p>Published online 8 May 2008</p><p>PMCID:PMC2396638.</p><p></p

Bioinformatic analyses of mammalian 5'-UTR sequence properties of mRNAs predicts alternative translation initiation sites-0

Categories. These annotations were compiled via BLAST searches and subsequent Gene Ontology (GO) and protein family analysis. The chart depicts the protein functions represented by the identified aTIS sequences. The functions of these proteins are significant for biological regulation.<p><b>Copyright information:</b></p><p>Taken from "Bioinformatic analyses of mammalian 5'-UTR sequence properties of mRNAs predicts alternative translation initiation sites"</p><p>http://www.biomedcentral.com/1471-2105/9/232</p><p>BMC Bioinformatics 2008;9():232-232.</p><p>Published online 8 May 2008</p><p>PMCID:PMC2396638.</p><p></p

Bioinformatic analyses of mammalian 5'-UTR sequence properties of mRNAs predicts alternative translation initiation sites-3

Rated in this figure. The alternative start site, CUG, is circled in red. Features of this secondary structure served as inputs to the ANN. For each 50 base pair window surrounding the putative alternative initiation site (as shown in this figure), the local stability of the start codon itself and the free energy of the structure were recorded. The window size (50 bp) was experimentally determined as the minimum window size which produced consistent foldings through shifts in the folding window. Based on the scale of 0 to 3 scale, the stability would be measured as a 3 since the codon (all three bases) are present entirely in the loop structure.<p><b>Copyright information:</b></p><p>Taken from "Bioinformatic analyses of mammalian 5'-UTR sequence properties of mRNAs predicts alternative translation initiation sites"</p><p>http://www.biomedcentral.com/1471-2105/9/232</p><p>BMC Bioinformatics 2008;9():232-232.</p><p>Published online 8 May 2008</p><p>PMCID:PMC2396638.</p><p></p

Bioinformatic analyses of mammalian 5'-UTR sequence properties of mRNAs predicts alternative translation initiation sites-2

D a set of 'if-then' rules that allowed the sequences containing the aTISs to be classified independently from sequences that do not possess aTISs. The critical parameters for the classification tree consisted of number of upstream AUGs, 5'-UTR length, consensus sequences (at positions-6/-7), the presence or absence of the Internal Ribosome Entry Site (IRES) structure, as well as G/C ratio.<p><b>Copyright information:</b></p><p>Taken from "Bioinformatic analyses of mammalian 5'-UTR sequence properties of mRNAs predicts alternative translation initiation sites"</p><p>http://www.biomedcentral.com/1471-2105/9/232</p><p>BMC Bioinformatics 2008;9():232-232.</p><p>Published online 8 May 2008</p><p>PMCID:PMC2396638.</p><p></p

Bioinformatic analyses of mammalian 5'-UTR sequence properties of mRNAs predicts alternative translation initiation sites-1

stack indicates the sequence conservation at that position, while the height of nucleotide symbols within the stack indicates the relative frequency of each base at that position. The start site is indicated at positions 1, 2, and 3. . The relative abundance of nucleotides (A, T, C, G) at aTISs is shown for a window of -10 to +10 bases at the initiation codon, with the aTIS start codon in positions 1, 2, and 3. Conservation around all of the alternative start sites aTISs is illustrated. Note the strong conservation of (G/C) at the -6 position and C at the -7 position. . Graphical representation of relative nucleotide abundances at AUG sites is shown for bases in the region of -10 to +10 bases relative to the initiation codon, with the AUG codon in positions 1, 2, and 3. Conservation at the -3 and +4 locations are consistent with traditional Kozak consensus sequence. These features are distinguished from that of thte aTIS sequences which show conservation at positions -6 and -7 (Figure 2A).<p><b>Copyright information:</b></p><p>Taken from "Bioinformatic analyses of mammalian 5'-UTR sequence properties of mRNAs predicts alternative translation initiation sites"</p><p>http://www.biomedcentral.com/1471-2105/9/232</p><p>BMC Bioinformatics 2008;9():232-232.</p><p>Published online 8 May 2008</p><p>PMCID:PMC2396638.</p><p></p

Distinct Cleavage Properties of Cathepsin B Compared to Cysteine Cathepsins Enable the Design and Validation of a Specific Substrate for Cathepsin B over a Broad pH Range

The biological and pathological functions of cathepsin B occur in acidic lysosomes and at the neutral pH of cytosol, nuclei, and extracellular locations. Importantly, cathepsin B displays different substrate cleavage properties at acidic pH compared to neutral pH conditions. It is, therefore, desirable to develop specific substrates for cathepsin B that measure its activity over broad pH ranges. Current substrates used to monitor cathepsin B activity consist of Z-Phe-Arg-AMC and Z-Arg-Arg-AMC, but they lack specificity since they are cleaved by other cysteine cathepsins. Furthermore, Z-Arg-Arg-AMC monitors cathepsin B activity at neutral pH and displays minimal activity at acidic pH. Therefore, the purpose of this study was to design and validate specific fluorogenic peptide substrates that can monitor cathepsin B activity over a broad pH range from acidic to neutral pH conditions. In-depth cleavage properties of cathepsin B were compared to those of the cysteine cathepsins K, L, S, V, and X via multiplex substrate profiling by mass spectrometry at pH 4.6 and pH 7.2. Analysis of the cleavage preferences predicted the tripeptide Z-Nle-Lys-Arg-AMC as a preferred substrate for cathepsin B. Significantly, Z-Nle-Lys-Arg-AMC displayed the advantageous properties of measuring high cathepsin B specific activity over acidic to neutral pHs and was specifically cleaved by cathepsin B over the other cysteine cathepsins. Z-Nle-Lys-Arg-AMC specifically monitored cathepsin B activity in neuronal and glial cells which were consistent with relative abundances of cathepsin B protein. These findings validate Z-Nle-Lys-Arg-AMC as a novel substrate that specifically monitors cathepsin B activity over a broad pH range

The Marine Cyanobacterial Metabolite Gallinamide A Is a Potent and Selective Inhibitor of Human Cathepsin L

A number of marine natural products are potent inhibitors of proteases, an important drug target class in human diseases. Hence, marine cyanobacterial extracts were assessed for inhibitory activity to human cathepsin L. Herein, we have shown that gallinamide A potently and selectively inhibits the human cysteine protease cathepsin L. With 30 min of preincubation, gallinamide A displayed an IC₅₀ of 5.0 nM, and kinetic analysis demonstrated an inhibition constant of <i>k</i>_i = 9000 ± 260 M^–1 s^–1. Preincubation–dilution and activity-probe experiments revealed an irreversible mode of inhibition, and comparative IC₅₀ values display a 28- to 320-fold greater selectivity toward cathepsin L than closely related human cysteine cathepsin V or B. Molecular docking and molecular dynamics simulations were used to determine the pose of gallinamide in the active site of cathepsin L. These data resulted in the identification of a pose characterized by high stability, a consistent hydrogen bond network, and the reactive Michael acceptor enamide of gallinamide A positioned near the active site cysteine of the protease, leading to a proposed mechanism of covalent inhibition. These data reveal and characterize the novel activity of gallinamide A as a potent inhibitor of human cathepsin L

Functional categories of human DCSV soluble and membrane proteins.

<p>Pie charts illustrate the relative portion of proteins in each functional category for the soluble (panel A) and membrane (panel B) fractions of human DCSV. Each functional category, with name and percent of the total number of DCSV proteins, of the pie chart is shown as a distinct color. The proteins comprising each functional category are listed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0041134#pone.0041134.s006" target="_blank">Table S3</a>, and proteomics identification of soluble and membrane proteins of human DCSV are listed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0041134#pone.0041134.s004" target="_blank">Tables S1</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0041134#pone.0041134.s005" target="_blank">S2</a>.</p

Cytoscape systems biology analyses of the human DCSV proteome.

<p>Components of the DCSV proteomics data were analyzed by the Cytoscape systems biology program for predicting protein interaction networks. The functional protein categories are illustrated on the right hand side. Based on quantitative NASF data of the proteins, individual proteins are indicated as predominantly soluble (green circles), predominantly membrane (red circles), or present in both soluble and membrane at similar levels (yellow) (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0041134#pone-0041134-g003" target="_blank">Figure 3</a>). These color-coded protein symbols are those which were quantitated by NSAF. Proteins illustrated by grey circles are those which were identified, but not quantitated since they did not meet the criteria for quantitation in at least 3 out of 4 nano-LC-MS/MS runs.</p