Axotrophin/MARCH7 acts as an E3 ubiquitin ligase and ubiquitinates tau protein in vitro impairing microtubule binding

AbstractTau is the major microtubule-associated protein in neurons involved in microtubule stabilization in the axonal compartment. Changes in tau gene expression, alternative splicing and posttranslational modification regulate tau function and in tauopathies can result in tau mislocalization and dysfunction, causing tau aggregation and cell death. To uncover proteins involved in the development of tauopathies, a yeast two-hybrid system was used to screen for tau-interacting proteins. We show that axotrophin/MARCH7, a RING-variant domain containing protein with similarity to E3 ubiquitin ligases interacts with tau. We defined the tau binding domain to amino acids 552–682 of axotrophin comprising the RING-variant domain. Co-immunoprecipitation and co-localization confirmed the specificity of the interaction. Intracellular localization of axotrophin is determined by an N-terminal nuclear targeting signal and a C-terminal nuclear export signal. In AD brain nuclear localization is lost and axotrophin is rather associated with neurofibrillary tangles. We find here that tau becomes mono-ubiquitinated by recombinant tau-interacting RING-variant domain, which diminishes its microtubule-binding. In vitro ubiquitination of four-repeat tau results in incorporation of up to four ubiquitin molecules compared to two molecules in three-repeat tau. In summary, we present a novel tau modification occurring preferentially on 4-repeat tau protein which modifies microtubule-binding and may impact on the pathogenesis of tauopathies

Characterizing Protein Interactions Employing a Genome-Wide siRNA Cellular Phenotyping Screen

<div><p>Characterizing the activating and inhibiting effect of protein-protein interactions (PPI) is fundamental to gain insight into the complex signaling system of a human cell. A plethora of methods has been suggested to infer PPI from data on a large scale, but none of them is able to characterize the effect of this interaction. Here, we present a novel computational development that employs mitotic phenotypes of a genome-wide RNAi knockdown screen and enables identifying the activating and inhibiting effects of PPIs. Exemplarily, we applied our technique to a knockdown screen of HeLa cells cultivated at standard conditions. Using a machine learning approach, we obtained high accuracy (82% AUC of the receiver operating characteristics) by cross-validation using 6,870 known activating and inhibiting PPIs as gold standard. We predicted <i>de novo</i> unknown activating and inhibiting effects for 1,954 PPIs in HeLa cells covering the ten major signaling pathways of the Kyoto Encyclopedia of Genes and Genomes, and made these predictions publicly available in a database. We finally demonstrate that the predicted effects can be used to cluster knockdown genes of similar biological processes in coherent subgroups. The characterization of the activating or inhibiting effect of individual PPIs opens up new perspectives for the interpretation of large datasets of PPIs and thus considerably increases the value of PPIs as an integrated resource for studying the detailed function of signaling pathways of the cellular system of interest.</p></div

Clustering of the chemokine receptors and their interactors.

<p>Clustering of the chemokine receptors and their interactors.</p

Workflow.

<p>Images of a genome-wide cellular RNAi knockdown screen (the screening data was derived from the Mitocheck project, <a href="http://www.mitocheck.org" target="_blank">www.mitocheck.org</a>) were segmented and their features extracted to compile pairwise phenotype descriptors for a large set of gene pairs. These descriptors were used to train a machine learning system to discriminate activating and inhibiting PPIs taken from a reference. The performance was evaluated using cross-validation. The trained SVM models were used to predict the effects of uncharacterized PPIs. In addition, the SVM models were used to estimate similarity of the effects of proteins for all combinations of protein pairs in the network. Subsequently, this Effect Similarity Rate (ESR) was exemplarily used for clustering of functionally related protein sub-networks.</p

a) Receiver Operating Characteristics curves for the predictions of activation.

<p>Cross-validation results for all pathways combined (AUC  = 0.75, dashed line) and when training and validation was done for each set of pathways separately (AUC  = 0.82, solid line). <b>b</b>) Histogram of the votes for activating PPIs (green) and inhibiting PPIs (blue) when training and validation was done for each set of major signaling pathways separately. The thresholds for 80% confidence were set at 920 and 88 votes for activation and inhibition, respectively (dashed lines).</p

Characterization of phenotypic similarity by linear discrimination.

<p>(<b>a-c</b>) Images of cells in which <i>sfrp1, dvl2</i> or <i>fzd7</i> were knocked down, respectively. (<b>d</b>) First two principal components (PC 1 and PC 2) of the features for cells with knockdown of <i>sfrp1</i> and <i>dvl2</i>. (<b>e</b>) First two principal components of the features for cells with knockdown of <i>dvl2</i> and <i>fzd7</i>. Dotted lines sketch a linear separation.</p

Pairs of Pfam domain sets showing significant^* enrichment of predicted interactions.

<p>^*p≤0.1 only; t≥1 for both pos/neg - no zeroes.</p><p>Pairs of Pfam domain sets showing significant^{<a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1003814#nt101" target="_blank">*</a>} enrichment of predicted interactions.</p

Incidence of severe critical events in paediatric anaesthesia (APRICOT): a prospective multicentre observational study in 261 hospitals in Europe

Background Little is known about the incidence of severe critical events in children undergoing general anaesthesia in Europe. We aimed to identify the incidence, nature, and outcome of severe critical events in children undergoing anaesthesia, and the associated potential risk factors. Methods The APRICOT study was a prospective observational multicentre cohort study of children from birth to 15 years of age undergoing elective or urgent anaesthesia for diagnostic or surgical procedures. Children were eligible for inclusion during a 2-week period determined prospectively by each centre. There were 261 participating centres across 33 European countries. The primary endpoint was the occurence of perioperative severe critical events requiring immediate intervention. A severe critical event was defined as the occurrence of respiratory, cardiac, allergic, or neurological complications requiring immediate intervention and that led (or could have led) to major disability or death. This study is registered with ClinicalTrials.gov, number NCT01878760. Findings Between April 1, 2014, and Jan 31, 2015, 31 127 anaesthetic procedures in 30 874 children with a mean age of 6.35 years (SD 4.50) were included. The incidence of perioperative severe critical events was 5.2% (95% CI 5.0-5.5) with an incidence of respiratory critical events of 3.1% (2.9-3.3). Cardiovascular instability occurred in 1.9% (1.7-2.1), with an immediate poor outcome in 5.4% (3.7-7.5) of these cases. The all-cause 30-day in-hospital mortality rate was 10 in 10 000. This was independent of type of anaesthesia. Age (relative risk 0.88, 95% CI 0.86-0.90; p<0.0001), medical history, and physical condition (1.60, 1.40-1.82; p<0.0001) were the major risk factors for a serious critical event. Multivariate analysis revealed evidence for the beneficial effect of years of experience of the most senior anaesthesia team member (0.99, 0.981-0.997; p<0.0048 for respiratory critical events, and 0.98, 0.97-0.99; p=0.0039 for cardiovascular critical events), rather than the type of health institution or providers. Interpretation This study highlights a relatively high rate of severe critical events during the anaesthesia management of children for surgical or diagnostic procedures in Europe, and a large variability in the practice of paediatric anaesthesia. These findings are substantial enough to warrant attention from national, regional, and specialist societies to target education of anaesthesiologists and their teams and implement strategies for quality improvement in paediatric anaesthesia