Proteolysis of HCF-1 by Ser/Thr glycosylation-incompetent O-GlcNAc transferase:UDP-GlcNAc complexes

In complex with the cosubstrate UDP-N-acetylglucosamine (UDP-GlcNAc),O-linked-GlcNAc transferase (OGT) catalyzes Ser/ThrO-GlcNAcylation of many cellular proteins and proteolysis of the transcriptional coregulator HCF-1. Such a dual glycosyltransferase-protease activity, which occurs in the same active site, is unprecedented and integrates both reversible and irreversible forms of protein post-translational modification within one enzyme. Although occurring within the same active site, we show here that glycosylation and proteolysis occur through separable mechanisms. OGT consists of tetratricopeptide repeat (TPR) and catalytic domains, which, together with UDP-GlcNAc, are required for both glycosylation and proteolysis. Nevertheless, a specific TPR domain contact with the HCF-1 substrate is critical for proteolysis but not Ser/Thr glycosylation. In contrast, key catalytic domain residues and even a UDP-GlcNAc oxygen important for Ser/Thr glycosylation are irrelevant for proteolysis. Thus, from a dual glycosyltransferase-protease, essentially single-activity enzymes can be engineered both in vitro and in vivo. Curiously, whereas OGT-mediated HCF-1 proteolysis is limited to vertebrate species, invertebrate OGTs can cleave human HCF-1. We present a model for the evolution of HCF-1 proteolysis by OGT

The conserved threonine-rich region of the HCF-1PRO repeat activates promiscuous OGT:UDP-GlcNAc glycosylation and proteolysis activities

O-Linked GlcNAc transferase (OGT) possesses dual glycosyltransferase-protease activities. OGT thereby stably glycosylates serines and threonines of numerous proteins and, via a transient glutamate glycosylation, cleaves a single known substrate-the so-called HCF-1 <sub>PRO</sub> repeat of the transcriptional co-regulator host-cell factor 1 (HCF-1). Here, we probed the relationship between these distinct glycosylation and proteolytic activities. For proteolysis, the HCF-1 <sub>PRO</sub> repeat possesses an important extended threonine-rich region that is tightly bound by the OGT tetratricopeptide-repeat (TPR) region. We report that linkage of this HCF-1 <sub>PRO</sub> -repeat, threonine-rich region to heterologous substrate sequences also potentiates robust serine glycosylation with the otherwise poor R <sub>p</sub> -αS-UDP-GlcNAc diastereomer phosphorothioate and UDP-5S-GlcNAc OGT co-substrates. Furthermore, it potentiated proteolysis of a non-HCF-1 <sub>PRO</sub> -repeat cleavage sequence, provided it contained an appropriately positioned glutamate residue. Using serine- or glutamate-containing HCF-1 <sub>PRO</sub> -repeat sequences, we show that proposed OGT-based or UDP-GlcNAc-based serine-acceptor residue activation mechanisms can be circumvented independently, but not when disrupted together. In contrast, disruption of both proposed activation mechanisms even in combination did not inhibit OGT-mediated proteolysis. These results reveal a multiplicity of OGT glycosylation strategies, some leading to proteolysis, which could be targets of alternative molecular regulatory strategies

Analyzing Somatic DNA Repair in Arabidopsis Meiotic Mutants

Meiotic and somatic recombination share a common set of factors. Thus, the analysis of somatic DNA repair in meiotic mutant lines should be of special interest. Growth defects of mutant plants induced by specific genotoxins can thereby hint to DNA repair functions of the affected proteins. Here, we describe two kinds of approaches to characterize deficiencies in DNA repair in mutant lines of Arabidopsis thaliana, after genotoxin treatment

MiR-205-driven downregulation of cholesterol biosynthesis through SQLE-inhibition identifies therapeutic vulnerability in aggressive prostate cancer

Prostate cancer (PCa) shows strong dependence on the androgen receptor (AR) pathway. Here, we show that squalene epoxidase (SQLE), an enzyme of the cholesterol biosynthesis pathway, is overexpressed in advanced PCa and its expression correlates with poor survival. SQLE expression is controlled by micro-RNA 205 (miR-205), which is significantly downregulated in advanced PCa. Restoration of miR-205 expression or competitive inhibition of SQLE led to inhibition of de novo cholesterol biosynthesis. Furthermore, SQLE was essential for proliferation of AR-positive PCa cell lines, including abiraterone or enzalutamide resistant derivatives, and blocked transactivation of the AR pathway. Inhibition of SQLE with the FDA approved antifungal drug terbinafine also efficiently blocked orthotopic tumour growth in mice. Finally, terbinafine reduced levels of prostate specific antigen (PSA) in three out of four late-stage PCa patients. These results highlight SQLE as a therapeutic target for the treatment of advanced PCa

Measurement of the cross-section and charge asymmetry of WW bosons produced in proton-proton collisions at s=8\sqrt{s}=8 TeV with the ATLAS detector

This paper presents measurements of the $W^+ \rightarrow \mu^+\nu$ and $W^- \rightarrow \mu^-\nu$ cross-sections and the associated charge asymmetry as a function of the absolute pseudorapidity of the decay muon. The data were collected in proton--proton collisions at a centre-of-mass energy of 8 TeV with the ATLAS experiment at the LHC and correspond to a total integrated luminosity of 20.2~\mbox{fb^{-1}}. The precision of the cross-section measurements varies between 0.8% to 1.5% as a function of the pseudorapidity, excluding the 1.9% uncertainty on the integrated luminosity. The charge asymmetry is measured with an uncertainty between 0.002 and 0.003. The results are compared with predictions based on next-to-next-to-leading-order calculations with various parton distribution functions and have the sensitivity to discriminate between them.Comment: 38 pages in total, author list starting page 22, 5 figures, 4 tables, submitted to EPJC. All figures including auxiliary figures are available at https://atlas.web.cern.ch/Atlas/GROUPS/PHYSICS/PAPERS/STDM-2017-13

Quality of human-computer interaction - results of a national usability survey of hospital-IT in Germany

<p>Abstract</p> <p>Background</p> <p>Due to the increasing functionality of medical information systems, it is hard to imagine day to day work in hospitals without IT support. Therefore, the design of dialogues between humans and information systems is one of the most important issues to be addressed in health care. This survey presents an analysis of the current quality level of human-computer interaction of healthcare-IT in German hospitals, focused on the users' point of view.</p> <p>Methods</p> <p>To evaluate the usability of clinical-IT according to the design principles of EN ISO 9241-10 the IsoMetrics Inventory, an assessment tool, was used. The focus of this paper has been put on suitability for task, training effort and conformity with user expectations, differentiated by information systems. Effectiveness has been evaluated with the focus on interoperability and functionality of different IT systems.</p> <p>Results</p> <p>4521 persons from 371 hospitals visited the start page of the study, while 1003 persons from 158 hospitals completed the questionnaire. The results show relevant variations between different information systems.</p> <p>Conclusions</p> <p>Specialised information systems with defined functionality received better assessments than clinical information systems in general. This could be attributed to the improved customisation of these specialised systems for specific working environments. The results can be used as reference data for evaluation and benchmarking of human computer engineering in clinical health IT context for future studies.</p

Engolo and Capoeira. From Ethnic to Diasporic Combat Games in the Southern Atlantic

This article provides a re-examination of the main Afrocentric narrative of capoeira origins, the engolo or ‘Zebra Dance’, in light of historical primary sources and new ethnographic evidence gathered during fieldwork in south-west Angola. By examining engolo’s bodily techniques, its socio-historical context and cultural meanings, the piece emphasises its insertion into a pastoral lifestyle and highlights the relatively narrow ethnic character of the practice in Angola. This analysis and the comparison with capoeira helps us to develop certain hypotheses about the formation, migration, and re-invention of diasporic combat games between southern Angola and coastal Brazil, and more broadly, to increase our understanding of how African cultures spread across the southern Atlantic

Molecular Mechanism of a Green-Shifted, pH-Dependent Red Fluorescent Protein mKate Variant

Fluorescent proteins that can switch between distinct colors have contributed significantly to modern biomedical imaging technologies and molecular cell biology. Here we report the identification and biochemical analysis of a green-shifted red fluorescent protein variant GmKate, produced by the introduction of two mutations into mKate. Although the mutations decrease the overall brightness of the protein, GmKate is subject to pH-dependent, reversible green-to-red color conversion. At physiological pH, GmKate absorbs blue light (445 nm) and emits green fluorescence (525 nm). At pH above 9.0, GmKate absorbs 598 nm light and emits 646 nm, far-red fluorescence, similar to its sequence homolog mNeptune. Based on optical spectra and crystal structures of GmKate in its green and red states, the reversible color transition is attributed to the different protonation states of the cis-chromophore, an interpretation that was confirmed by quantum chemical calculations. Crystal structures reveal potential hydrogen bond networks around the chromophore that may facilitate the protonation switch, and indicate a molecular basis for the unusual bathochromic shift observed at high pH. This study provides mechanistic insights into the color tuning of mKate variants, which may aid the development of green-to-red color-convertible fluorescent sensors, and suggests GmKate as a prototype of genetically encoded pH sensors for biological studies