Stress-induced O-GlcNAcylation: an adaptive process of injured cells.

In the 30 years, since the discovery of nucleocytoplasmic glycosylation, O-GlcNAc has been implicated in regulating cellular processes as diverse as protein folding, localization, degradation, activity, post-translational modifications, and interactions. The cell co-ordinates these molecular events, on thousands of cellular proteins, in concert with environmental and physiological cues to fine-tune epigenetics, transcription, translation, signal transduction, cell cycle, and metabolism. The cellular stress response is no exception: diverse forms of injury result in dynamic changes to the O-GlcNAc subproteome that promote survival. In this review, we discuss the biosynthesis of O-GlcNAc, the mechanisms by which O-GlcNAc promotes cytoprotection, and the clinical significance of these data.

Enhanced lysis and accelerated establishment of viscoelastic properties of fibrin clots are associated with pulmonary embolism.

The factors that contribute to pulmonary embolism (PE), a potentially fatal complication of deep vein thrombosis (DVT), remain poorly understood. Whereas fibrin clot structure and functional properties have been implicated in the pathology of venous thromboembolism and the risk for cardiovascular complications, their significance in PE remains uncertain. Therefore, we systematically compared and quantified clot formation and lysis time, plasminogen levels, viscoelastic properties, activated factor XIII cross-linking, and fibrin clot structure in isolated DVT and PE subjects. Clots made from plasma of PE subjects showed faster clot lysis times with no differences in lag time, rate of clot formation, or maximum absorbance of turbidity compared with DVT. Differences in lysis times were not due to alterations in plasminogen levels. Compared with DVT, clots derived from PE subjects showed accelerated establishment of viscoelastic properties, documented by a decrease in lag time and an increase in the rate of viscoelastic property formation. The rate and extent of fibrin cross-linking by activated factor XIII were similar between clots from DVT and PE subjects. Electron microscopy revealed that plasma fibrin clots from PE subjects exhibited lower fiber density compared with those from DVT subjects. These data suggest that clot structure and functional properties differ between DVT and PE subjects and provide insights into mechanisms that may regulate embolization.

Mass Spectrometry-Based Protein Footprinting Defines the Binding Pocket of Crotonylated H3K14 in the PHD1 Domain of BAF45D within the BAF Chromatin Remodeling Complex.

The BRG-/BRM-associated factor (BAF) chromatin remodeling complex is a central actor in transcription. One mechanism by which BAF affects gene expression is via its various histone mark readers, including double plant homeodomains (DPF), located in the BAF45D subunit. DPF domains recognize lysine acetyl and acylations, including crotonylation, localized at promoters and enhancers. Despite a significant degree of conservation between DPF domains, attempts to crystallize BAF45D with a crotonylated histone 3 peptide (H3K14Cr) were unsuccessful. In addition, recent cryoEM and modeled structures failed to define the Req domain of BAF45D, which is responsible for reading lysine modifications. Thus, the precise mechanism of crotonyl group recognition and binding by BAF45D within the BAF complex remains unclear. We turned to protein footprinting mass spectrometry to map the binding interface between H3K14Cr and BAF45D. This technique is able to demarcate protein-binding interfaces by modifying surface-accessible residues and is not limited by protein size or composition. Experiments performed in the isolated DPF domain of BAF45D (BAF45DDPF)-delineated H3K14Cr peptide binding across the PHD1 and PHD2 pockets. We observed markedly similar effects on the BAF45D subunit when assessing H3K14Cr binding in the purified full BAF complex. The ATPase motor, BRM, also displayed H3K14Cr-protected peptides in two separate domains that were subsequently evaluated in direct binding assays. These data confirm the BAF45D-crotonylamide interaction within its obligate complex and are the first to demonstrate H3K14Cr direct binding to BRM.