RanBP9 controls the oligomeric state of CTLH complex assemblies.

The CTLH (C-terminal to lissencephaly-1 homology motif) complex is a multisubunit RING E3 ligase with poorly defined substrate specificity and flexible subunit composition. Two key subunits, muskelin and Wdr26, specify two alternative CTLH complexes that differ in quaternary structure, thereby allowing the E3 ligase to presumably target different substrates. With the aid of different biophysical and biochemical techniques, we characterized CTLH complex assembly pathways, focusing not only on Wdr26 and muskelin but also on RanBP9, Twa1, and Armc8ß subunits, which are critical to establish the scaffold of this E3 ligase. We demonstrate that the ability of muskelin to tetramerize and the assembly of Wdr26 into dimers define mutually exclusive oligomerization modules that compete with nanomolar affinity for RanBP9 binding. The remaining scaffolding subunits, Armc8ß and Twa1, strongly interact with each other and with RanBP9, again with nanomolar affinity. Our data demonstrate that RanBP9 organizes subunit assembly and prevents higher order oligomerization of dimeric Wdr26 and the Armc8ß-Twa1 heterodimer through its tight binding. Combined, our studies define alternative assembly pathways of the CTLH complex and elucidate the role of RanBP9 in governing differential oligomeric assemblies, thereby advancing our mechanistic understanding of CTLH complex architectures.

Structure of the ubiquitin-activating enzyme loaded with two ubiquitin molecules.

The activation of ubiquitin by the ubiquitin-activating enzyme Uba1 (E1) constitutes the first step in the covalent modification of target proteins with ubiquitin. This activation is a three-step process in which ubiquitin is adenylated at its C-terminal glycine, followed by the covalent attachment of ubiquitin to a catalytic cysteine residue of Uba1 and the subsequent adenylation of a second ubiquitin. Here, a ubiquitin E1 structure loaded with two ubiquitin molecules is presented for the first time. While one ubiquitin is bound in its adenylated form to the active adenylation domain of E1, the second ubiquitin represents the status after transfer and is covalently linked to the active-site cysteine. The covalently linked ubiquitin enables binding of the E2 enzyme without further modification of the ternary Uba1-ubiquitin2 arrangement. This doubly loaded E1 structure constitutes a missing link in the structural analysis of the ubiquitin-transfer cascade.

Structures of UBA6 explain its dual specificity for ubiquitin and FAT10.

The covalent modification of target proteins with ubiquitin or ubiquitin-like modifiers is initiated by E1 activating enzymes, which typically transfer a single modifier onto cognate conjugating enzymes. UBA6 is an unusual E1 since it activates two highly distinct modifiers, ubiquitin and FAT10. Here, we report crystal structures of UBA6 in complex with either ATP or FAT10. In the UBA6-FAT10 complex, the C-terminal domain of FAT10 binds to where ubiquitin resides in the UBA1-ubiquitin complex, however, a switch element ensures the alternate recruitment of either modifier. Simultaneously, the N-terminal domain of FAT10 interacts with the 3-helix bundle of UBA6. Site-directed mutagenesis identifies residues permitting the selective activation of either ubiquitin or FAT10. These results pave the way for studies investigating the activation of either modifier by UBA6 in physiological and pathophysiological settings.

Risk factors for bruxism

The aim of the present study was to summarize the risk factors for bruxism that were identified by a systematic search of the literature published between 2007 and 2016. Depending on the size of the odds ratios (ORs) and the lower limit of the 95% confidence intervals indicated by the reports, four risk categories were differentiated. Among others, emotional stress, consumption of tobacco, alcohol, or coffee, sleep apnea syndrome, and anxiety disorders were recognized as important factors among adults. In children and adolescents, apart from distress, behavioral abnormalities and sleep disturbances predominated. Knowledge of the identified risk factors may be useful when taking the medical history of bruxing patients. Although many of the described variables cannot be influenced by prophylactic or therapeutic means, we recommend the following patient-centered approach ("SMS therapy"): self-observation, muscle relaxation, stabilization (Michigan) splint.

VASP-dependent regulation of actin cytoskeleton rigidity, cell adhesion, and detachment.

Enabled/vasodilator-stimulated phosphoprotein (Ena/VASP) proteins are established regulators of actin-based motility, platelet aggregation, and growth cone guidance. However, the molecular mechanisms involved essentially remain elusive. Here we report on a novel mechanism of VASP action, namely the regulation of tensile strength, contractility, and rigidity of the actin cytoskeleton. Compared to wild-type cells fibroblasts derived from VASP-deficient mice have thicker and more stable actin stress fibres. Furthermore focal adhesions are enlarged, myosin light chain phosphorylation is increased, and the rigidity of the filament-supported plasma membrane is elevated about three- to fourfold, as is evident from atomic force microscopy. Moreover, fibronectin-coated beads adhere stronger to the surface of VASP-deficient cells. The resistance of these beads to mechanical displacement by laser tweezers is dramatically increased in an F-actin-dependent mode. Cytoskeletal stabilization coincides with slower cell adhesion and detachment, while overall adhesion is increased. Interestingly, many of these effects observed in VASP (-/-) cells are recapitulated in VASP-overexpressing cells, hinting towards a balanced stoichiometry necessary for appropriate VASP function. Taken together, our results suggest that VASP regulates surface protrusion formation and cell adhesion through modulation of the mechanical properties of the actin cytoskeleton.