MuD: an interactive web server for the prediction of non-neutral substitutions using protein structural data

The discrimination between functionally neutral amino acid substitutions and non-neutral mutations, affecting protein function, is very important for our understanding of diseases. The rapidly growing amounts of experimental data enable the development of computational tools to facilitate the annotation of these substitutions. Here, we describe a Random Forests-based classifier, named Mutation Detector (MuD) that utilizes structural and sequence-derived features to assess the impact of a given substitution on the protein function. In its automatic mode, MuD is comparable to alternative tools in performance. However, the uniqueness of MuD is that user-reported protein-specific structural and functional information can be added at run-time, thereby enhancing the prediction accuracy further. The MuD server, available at http://mud.tau.ac.il, assigns a reliability score to every prediction, thus offering a useful tool for the prioritization of substitutions in proteins with an available 3D structure

A Myo6 Mutation Destroys Coordination between the Myosin Heads, Revealing New Functions of Myosin VI in the Stereocilia of Mammalian Inner Ear Hair Cells

Myosin VI, found in organisms from Caenorhabditis elegans to humans, is essential for auditory and vestibular function in mammals, since genetic mutations lead to hearing impairment and vestibular dysfunction in both humans and mice. Here, we show that a missense mutation in this molecular motor in an ENU-generated mouse model, Tailchaser, disrupts myosin VI function. Structural changes in the Tailchaser hair bundles include mislocalization of the kinocilia and branching of stereocilia. Transfection of GFP-labeled myosin VI into epithelial cells and delivery of endocytic vesicles to the early endosome revealed that the mutant phenotype displays disrupted motor function. The actin-activated ATPase rates measured for the D179Y mutation are decreased, and indicate loss of coordination of the myosin VI heads or ‘gating’ in the dimer form. Proper coordination is required for walking processively along, or anchoring to, actin filaments, and is apparently destroyed by the proximity of the mutation to the nucleotide-binding pocket. This loss of myosin VI function may not allow myosin VI to transport its cargoes appropriately at the base and within the stereocilia, or to anchor the membrane of stereocilia to actin filaments via its cargos, both of which lead to structural changes in the stereocilia of myosin VI–impaired hair cells, and ultimately leading to deafness

The Transcription and Translation Landscapes during Human Cytomegalovirus Infection Reveal Novel Host-Pathogen Interactions.

Viruses are by definition fully dependent on the cellular translation machinery, and develop diverse mechanisms to co-opt this machinery for their own benefit. Unlike many viruses, human cytomegalovirus (HCMV) does suppress the host translation machinery, and the extent to which translation machinery contributes to the overall pattern of viral replication and pathogenesis remains elusive. Here, we combine RNA sequencing and ribosomal profiling analyses to systematically address this question. By simultaneously examining the changes in transcription and translation along HCMV infection, we uncover extensive transcriptional control that dominates the response to infection, but also diverse and dynamic translational regulation for subsets of host genes. We were also able to show that, at late time points in infection, translation of viral mRNAs is higher than that of cellular mRNAs. Lastly, integration of our translation measurements with recent measurements of protein abundance enabled comprehensive identification of dozens of host proteins that are targeted for degradation during HCMV infection. Since targeted degradation indicates a strong biological importance, this approach should be applicable for discovering central host functions during viral infection. Our work provides a framework for studying the contribution of transcription, translation and degradation during infection with any virus

A systematic view on influenza induced host shutoff

DNA nanotechnology is an emerging field which utilizes the unique structural properties of nucleic acids in order to build nanoscale devices, such as logic gates, motors, walkers, and algorithmic structures. Predicting the structure and interactions of a DNA device requires effective modeling of both the thermodynamics and the kinetics of the DNA strands within the system. The kinetics of a set of DNA strands can be modeled as a continuous time Markov process through the state space of all secondary structures. The primary means of exploring the kinetics of a DNA system is by simulating trajectories through the state space and aggregating data over many such trajectories. We expand on previous work by extending the thermodynamics and kinetics models to handle multiple strands in a fixed volume, in a way that is consistent with previous models. We developed data structures and algorithms that allow us to take advantage of local properties of secondary structure, improving the efficiency of the simulator so that we can handle reasonably large systems. Finally, we illustrate the simulator’s analysis methods on a simple case study

The Transcription and Translation Landscapes during Human Cytomegalovirus Infection Reveal Novel Host-Pathogen Interactions

<div><p>Viruses are by definition fully dependent on the cellular translation machinery, and develop diverse mechanisms to co-opt this machinery for their own benefit. Unlike many viruses, human cytomegalovirus (HCMV) does suppress the host translation machinery, and the extent to which translation machinery contributes to the overall pattern of viral replication and pathogenesis remains elusive. Here, we combine RNA sequencing and ribosomal profiling analyses to systematically address this question. By simultaneously examining the changes in transcription and translation along HCMV infection, we uncover extensive transcriptional control that dominates the response to infection, but also diverse and dynamic translational regulation for subsets of host genes. We were also able to show that, at late time points in infection, translation of viral mRNAs is higher than that of cellular mRNAs. Lastly, integration of our translation measurements with recent measurements of protein abundance enabled comprehensive identification of dozens of host proteins that are targeted for degradation during HCMV infection. Since targeted degradation indicates a strong biological importance, this approach should be applicable for discovering central host functions during viral infection. Our work provides a framework for studying the contribution of transcription, translation and degradation during infection with any virus.</p></div

pUL135 contributes to ROCK1 degradation.

<p><b>A.</b> Fluorescent microscopy images of cells expressing UL135 (GFP positive) and stained with ROCK1 antibody (rhodamine red X) (left panel). ROCK1 levels in 20 cells were quantified using Imaris (*p-val < 0.05) (right panel). <b>B.</b> HFF cells transduced with lentiviral vector expressing pUL135 under doxycycline inducible promotor were analyzed for ROCK1 expression levels, with or without the addition of doxycycline, using western blot analysis (left panel). Differences in protein levels were quantified using the Licor program (right panel). Error bar indicate the standard deviation in the ratio of ROCK1 expression in cells with and without doxycycline addition between two independent experiments. <b>C.</b> Measurements of <i>rock1</i> mRNA levels in HFF transduced with pUL135 under doxycycline inducible promoter, 24 and 48 hours after addition of doxycycline. Each experiment was performed in triplicates and average results are presented. Error bars indicate standard deviation of the mean.</p

Integration between ribosome footprints and protein abundance allows detection of immune ligands that are degraded during infection.

<p><b>A.</b> Ribosome profiling measurements for HLA-A and the NK ligand PVRL2 compared with temporal protein expression measured [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005288#ppat.1005288.ref012" target="_blank">12</a>]. <b>B.</b> Expression of FAT1 and FAT4 measured by ribosome profiling, RNA-seq and real-time PCR analysis compared with protein abundance [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005288#ppat.1005288.ref012" target="_blank">12</a>]. <b>C and E.</b> Ribosome profiling measurements of BTN2A1 and IGSF8 compared with protein abundance [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005288#ppat.1005288.ref012" target="_blank">12</a>], respectively. <b>D and F.</b> Real-time PCR analysis of <i>btn2a1</i> (D) and <i>igsf8</i> (F) mRNA levels along HCMV infection (upper panels). HFF cells stably expressing BTN2A1-HA or IGSF8-HA were infected with HCMV. Protein levels were detected by western blot analysis using anti-HA antibody (lower panels). GFP levels (expressed from the same vector) were used as internal control. Real-time PCR data was normalized by the amount of <i>mfge8</i> mRNA. Each experiment was performed in triplicates.</p