RavN is a member of a previously unrecognized group of Legionella pneumophila E3 ubiquitin ligases

The eukaryotic ubiquitylation machinery catalyzes the covalent attachment of the small protein modifier ubiquitin to cellular target proteins in order to alter their fate. Microbial pathogens exploit this post-translational modification process by encoding molecular mimics of E3 ubiquitin ligases, eukaryotic enzymes that catalyze the final step in the ubiquitylation cascade. Here, we show that the Legionella pneumophila effector protein RavN belongs to a growing class of bacterial proteins that mimic host cell E3 ligases to exploit the ubiquitylation pathway. The E3 ligase activity of RavN was located within its N-terminal region and was dependent upon interaction with a defined subset of E2 ubiquitin-conjugating enzymes. The crystal structure of the N-terminal region of RavN revealed a U-box-like motif that was only remotely similar to other U-box domains, indicating that RavN is an E3 ligase relic that has undergone significant evolutionary alteration. Substitution of residues within the predicted E2 binding interface rendered RavN inactive, indicating that, despite significant structural changes, the mode of E2 recognition has remained conserved. Using hidden Markov model-based secondary structure analyses, we identified and experimentally validated four additional L. pneumophila effectors that were not previously recognized to possess E3 ligase activity, including Lpg2452/SdcB, a new paralog of SidC. Our study provides strong evidence that L. pneumophila is dedicating a considerable fraction of its effector arsenal to the manipulation of the host ubiquitylation pathway.Funding: This work was funded by the Intramural Research Program of the National Institutes of Health (to MPM)(Project Number: 1ZIAHD008893-07) and by the Spanish Ministry of Economy and Competitiveness Grant (to AH)(BFU2014-59759-R) and the Severo Ochoa Excellence Accreditation (to AH)(SEV-2016-0644). This study made use of the Diamond Light Source beamline I04 (Oxfordshire, UK) and ALBA synchrotron beamline BL13-XALOC, funded in part by the Horizon 2020 programme of the European Union, iNEXT (H2020 Grant # 653706). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript

Cohesin is needed for bipolar mitosis in human cells

11p.-6 fig.Multi-polar mitosis is strongly linked with aggressive cancers and it is a histological diagnostic of tumor-grade. However, factors that cause chromosomes to segregate to more than two spindle poles are not well understood. Here we show that cohesins Rad21, Smc1 and Smc3 are required for bipolar mitosis in human cells. After Rad21 depletion, chromosomes align at the metaphase plate and bipolar spindles assemble in most cases, but in anaphase the separated chromatids segregate to multiple poles. Time-lapse microscopy revealed that the spindle poles often become split in Rad21-depleted metaphase cells. Interestingly, exogenous expression of non-cleavable Rad21 results in multi-polar anaphase. Since cohesins are present at the spindle poles in mitosis, these data are consistent with a non-chromosomal function of cohesin.L.A.D.M. was partially funded by CONACyT, J.F.G.A. by BFU2008-03579/BMC and P.E. by BFU2008-02947-C02-02/BMC, and D.J.C. by a seed grant from the University of Minnesota Academic Health Center.Peer reviewe

Comparison of degree, clustering coefficient, and betweeness centrality for each condition.

<p>Conditions marked with different letters are significantly different. The bars show standard error.</p

Results from the 2-way ANOVA comparing Condition and Channels.

<p>Results from the 2-way ANOVA comparing Condition and Channels.</p

Δ Accuracy.

<p>Conditions marked with different letters are significantly different.</p

Regional Link Strengths.

<p>Each condition is normalized against None. The significances are shown for each link individually. Conditions marked with different letters are significantly different.</p

Theta band PLV connectivity network weight matrices.

<p>For each weight matrix, the diagonal is always equal to one.</p

Mean clustering coefficient of EEG nodes in cortical networks for all six acoustic stimulation conditions.

<p>Bars indicate standard error.</p

Delayed match-to-sample visuospatial working memory task.

<p>Delayed match-to-sample visuospatial working memory task.</p