Summary The mechanisms by which ubiquitin ligases are regulated remain poorly understood. Here we describe a series of molecular events that coordinately regulate CHIP, a neuroprotective E3 implicated in protein quality control. Through their opposing activities, the initiator E2, Ube2w, and the specialized deubiquitinating enzyme (DUB), ataxin-3, participate in initiating, regulating and terminating the CHIP ubiquitination cycle. Monoubiquitination of CHIP by Ube2w stabilizes the interaction between CHIP and ataxin-3, which through its DUB activity limits the length of chains attached to CHIP substrates. Upon completion of substrate ubiquitination ataxin-3 deubiquitinates CHIP, effectively terminating the reaction. Our results suggest that functional pairing of E3s with ataxin-3 or similar DUBs represents an important point of regulation in ubiquitin-dependent protein quality control. In addition, the results shed light on disease pathogenesis in SCA3, a neurodegenerative disorder caused by polyglutamine expansion in ataxin-3.

Evidence supports a role for epigenetic mechanisms in the pathogenesis of late-onset Alzheimer’s disease (LOAD), but little has been done on a genome-wide scale to identify potential sites involved in disease. This study investigates human postmortem frontal cortex genome-wide DNA methylation profiles between 12 LOAD and 12 cognitively normal age- and gender-matched subjects. Quantitative DNA methylation is determined at 27,578 CpG sites spanning 14,475 genes via the Illumina Infinium HumanMethylation27 BeadArray. Data are analyzed using parallel linear models adjusting for age and gender with empirical Bayes standard error methods. Gene-specific technical and functional validation is performed on an additional 13 matched pair samples, encompassing a wider age range. Analysis reveals 948 CpG sites representing 918 unique genes as potentially associated with LOAD disease status pending confirmation in additional study populations. Across these 948 sites the subtle mean methylation difference between cases and controls is 2.9%. The CpG site with a minimum false discovery rate located in the promoter of the gene Transmembrane Protein 59 (TMEM59) is 7.3% hypomethylated in cases. Methylation at this site is functionally associated with tissue RNA and protein levels of the TMEM59 gene product. The TMEM59 gene identified from our discovery approach was recently implicated in amyloid-β protein precursor post-translational processing, supporting a role for epigenetic change in LOAD pathology. This study demonstrates widespread, modest discordant DNA methylation in LOAD-diseased tissue independent from DNA methylation changes with age. Identification of epigenetic biomarkers of LOAD risk may allow for the development of novel diagnostic and therapeutic targets.

Background: Some proteins are ubiquitinated on their N terminus, yet the enzymes that facilitate N-terminal ubiquitination are unknown. Results: Ube2w has a novel active site and ubiquitinates the N terminus of substrates. Conclusion: Ube2w is an N terminus-ubiquitinating E2. Significance: Ube2w is the first identified E2 that ubiquitinates the N terminus of substrates.

Background and Purpose-Prehospital activation of in-hospital stroke response hastens treatment but depends on accurate emergency medical services (EMS) stroke recognition. We sought to measure EMS stroke recognition accuracy and identify clinical factors associated with correct stroke identification. Methods-Using EMS and hospital records, we assembled a cohort of EMS-transported suspect, confirmed, or missed ischemic stroke or transient ischemic attack cases. The sensitivity and positive predictive value (PPV) for EMS stroke recognition were calculated using the hospital discharge diagnosis as the gold standard. We used multivariable logistic regression analysis to determine the association between Cincinnati Prehospital Stroke Scale use and EMS stroke recognition. Results-During a 12-month period, 441 EMS-transported patients were enrolled; of which, 371 (84.1%) were EMSsuspected strokes and 70 (15.9%) were EMS-missed strokes. Overall, 264 cases (59.9%) were confirmed as either ischemic stroke (n=186) or transient ischemic attack (n=78). The sensitivity of EMS stroke recognition was 73.5% (95% confidence interval, 67.7-78.7), and PPV was 52.3% (95% confidence interval, 47.1-57.5). Sensitivity (84.7% versus 30.9%; P<0.0001) and PPV (56.2% versus 30.4%; P=0.0004) were higher among cases with Cincinnati Prehospital Stroke Scale documentation. In multivariate analysis, Cincinnati Prehospital Stroke Scale documentation was independently associated with EMS sensitivity (odds ratio, 12.0; 95% confidence interval, 5.7-25.5) and PPV (odds ratio, 2.5; 95% confidence interval, 1.3-4.7). Conclusions-EMS providers recognized 3 quarters of the patients with ischemic stroke and transient ischemic attack;however, half of EMS-suspected strokes were false positives. Documentation of a Cincinnati Prehospital Stroke Scale was associated with higher EMS stroke recognition sensitivity and PPV.