Mapping the O-Mannose Glycoproteome in Saccharomyces cerevisiae

O-Mannosylation is a vital protein modification conserved from fungi to humans. Yeast is a perfect model to study this post-translational modification, because in contrast to mammals O-mannosylation is the only type of O-glycosylation. In an essential step toward the full understanding of protein O-mannosylation we mapped the O-mannose glycoproteome in baker's yeast. Taking advantage of an O-glycan elongation deficient yeast strain to simplify sample complexity, we identified over 500 O-glycoproteins from all subcellular compartments for which over 2300 O-mannosylation sites were mapped by electron-transfer dissociation (ETD)-based MS/MS. In this study, we focus on the 293 O-glycoproteins (over 1900 glycosylation sites identified by ETD-MS/MS) that enter the secretory pathway and are targets of ER-localized protein O-mannosyltransferases. We find that O-mannosylation is not only a prominent modification of cell wall and plasma membrane proteins, but also of a large number of proteins from the secretory pathway with crucial functions in protein glycosylation, folding, quality control, and trafficking. The analysis of glycosylation sites revealed that O-mannosylation is favored in unstructured regions and β-strands. Furthermore, O-mannosylation is impeded in the proximity of N-glycosylation sites suggesting the interplay of these types of post-translational modifications. The detailed knowledge of the target proteins and their O-mannosylation sites opens for discovery of new roles of this essential modification in eukaryotes, and for a first glance on the evolution of different types of O-glycosylation from yeast to mammals

Targeted silencing of the oncogenic transcription factor SOX2 in breast cancer

The transcription factor (TF) SOX2 is essential for the maintenance of pluripotency and self-renewal in embryonic stem cells. In addition to its normal stem cell function, SOX2 over-expression is associated with cancer development. The ability to selectively target this and other oncogenic TFs in cells, however, remains a significant challenge due to the 'undruggable' characteristics of these molecules. Here, we employ a zinc finger (ZF)-based artificial TF (ATF) approach to selectively suppress SOX2 gene expression in cancer cells. We engineered four different proteins each composed of 6ZF arrays designed to bind 18 bp sites in the SOX2 promoter and enhancer region, which controls SOX2 methylation. The 6ZF domains were linked to the Kruppel Associated Box (SKD) repressor domain. Three engineered proteins were able to bind their endogenous target sites and effectively suppress SOX2 expression (up to 95% repression efficiencies) in breast cancer cells. Targeted down-regulation of SOX2 expression resulted in decreased tumor cell proliferation and colony formation in these cells. Furthermore, induced expression of an ATF in a mouse model inhibited breast cancer cell growth. Collectively, these findings demonstrate the effectiveness and therapeutic potential of engineered ATFs to mediate potent and long-lasting down-regulation of oncogenic TF expression in cancer cells

Fires at Neumark-Nord 2, Germany: An analysis of fire proxies from a Last Interglacial Middle Palaeolithic basin site

Few sites with evidence for fire use are known from the Last Interglacial in Europe. Hearth features are rarely preserved, probably as a result of post-depositional processes. The small postglacial basins (<300 m in diameter) that dominate the sedimentary context of the Eemian record in Europe are high-resolution environmental archives often containing charcoal particles. This case study presents the macroscopic charcoal record of the Neumark-Nord 2 basin, Germany, and the correlation of this record with the distinct find levels of the basin margin that also contain thermally altered archaeological material. Increased charcoal quantities are shown to correspond to phases of hominin presence-a pattern that fits best with recurrent anthropogenic fires within the watershed. This research shows the potential of small basin localities in the reconstruction of local fire histories, where clear archaeological features like hearths are missing

The genetic interaction network of CCW12, a Saccharomyces cerevisiae gene required for cell wall integrity during budding and formation of mating projections

<p>Abstract</p> <p>Background</p> <p>Mannoproteins construct the outer cover of the fungal cell wall. The covalently linked cell wall protein Ccw12p is an abundant mannoprotein. It is considered as crucial structural cell wall component since in baker's yeast the lack of <it>CCW12 </it>results in severe cell wall damage and reduced mating efficiency.</p> <p>Results</p> <p>In order to explore the function of <it>CCW12</it>, we performed a Synthetic Genetic Analysis (SGA) and identified genes that are essential in the absence of <it>CCW12</it>. The resulting interaction network identified 21 genes involved in cell wall integrity, chitin synthesis, cell polarity, vesicular transport and endocytosis. Among those are <it>PFD1</it>, <it>WHI3</it>, <it>SRN2</it>, <it>PAC10</it>, <it>FEN1 </it>and <it>YDR417C</it>, which have not been related to cell wall integrity before. We correlated our results with genetic interaction networks of genes involved in glucan and chitin synthesis. A core of genes essential to maintain cell integrity in response to cell wall stress was identified. In addition, we performed a large-scale transcriptional analysis and compared the transcriptional changes observed in mutant <it>ccw12</it>Δ with transcriptomes from studies investigating responses to constitutive or acute cell wall damage. We identified a set of genes that are highly induced in the majority of the mutants/conditions and are directly related to the cell wall integrity pathway and cell wall compensatory responses. Among those are <it>BCK1</it>, <it>CHS3</it>, <it>EDE1</it>, <it>PFD1</it>, <it>SLT2 </it>and <it>SLA1 </it>that were also identified in the SGA. In contrast, a specific feature of mutant <it>ccw12</it>Δ is the transcriptional repression of genes involved in mating. Physiological experiments substantiate this finding. Further, we demonstrate that Ccw12p is present at the cell periphery and highly concentrated at the presumptive budding site, around the bud, at the septum and at the tip of the mating projection.</p> <p>Conclusions</p> <p>The combination of high throughput screenings, phenotypic analyses and localization studies provides new insight into the function of Ccw12p. A compensatory response, culminating in cell wall remodelling and transport/recycling pathways is required to buffer the loss of <it>CCW12</it>. Moreover, the enrichment of Ccw12p in bud, septum and mating projection is consistent with a role of Ccw12p in preserving cell wall integrity at sites of active growth.</p> <p>The microarray data produced in this analysis have been submitted to NCBI GEO database and GSE22649 record was assigned.</p

Epigenetic Regulation of a Murine Retrotransposon by a Dual Histone Modification Mark

Large fractions of eukaryotic genomes contain repetitive sequences of which the vast majority is derived from transposable elements (TEs). In order to inactivate those potentially harmful elements, host organisms silence TEs via methylation of transposon DNA and packaging into chromatin associated with repressive histone marks. The contribution of individual histone modifications in this process is not completely resolved. Therefore, we aimed to define the role of reversible histone acetylation, a modification commonly associated with transcriptional activity, in transcriptional regulation of murine TEs. We surveyed histone acetylation patterns and expression levels of ten different murine TEs in mouse fibroblasts with altered histone acetylation levels, which was achieved via chemical HDAC inhibition with trichostatin A (TSA), or genetic inactivation of the major deacetylase HDAC1. We found that one LTR retrotransposon family encompassing virus-like 30S elements (VL30) showed significant histone H3 hyperacetylation and strong transcriptional activation in response to TSA treatment. Analysis of VL30 transcripts revealed that increased VL30 transcription is due to enhanced expression of a limited number of genomic elements, with one locus being particularly responsive to HDAC inhibition. Importantly, transcriptional induction of VL30 was entirely dependent on the activation of MAP kinase pathways, resulting in serine 10 phosphorylation at histone H3. Stimulation of MAP kinase cascades together with HDAC inhibition led to simultaneous phosphorylation and acetylation (phosphoacetylation) of histone H3 at the VL30 regulatory region. The presence of the phosphoacetylation mark at VL30 LTRs was linked with full transcriptional activation of the mobile element. Our data indicate that the activity of different TEs is controlled by distinct chromatin modifications. We show that activation of a specific mobile element is linked to a dual epigenetic mark and propose a model whereby phosphoacetylation of histone H3 is crucial for full transcriptional activation of VL30 elements

Aberrant Processing of the WSC Family and Mid2p Cell Surface Sensors Results in Cell Death of Saccharomyces cerevisiae O-Mannosylation Mutants

Protein O mannosylation is a crucial protein modification in uni- and multicellular eukaryotes. In humans, a lack of O-mannosyl glycans causes congenital muscular dystrophies that are associated with brain abnormalities. In yeast, protein O mannosylation is vital; however, it is not known why impaired O mannosylation results in cell death. To address this question, we analyzed the conditionally lethal Saccharomyces cerevisiae protein O-mannosyltransferase pmt2 pmt4Δ mutant. We found that pmt2 pmt4Δ cells lyse as small-budded cells in the absence of osmotic stabilization and that treatment with mating pheromone causes pheromone-induced cell death. These phenotypes are partially suppressed by overexpression of upstream elements of the protein kinase C (PKC1) cell integrity pathway, suggesting that the PKC1 pathway is defective in pmt2 pmt4Δ mutants. Congruently, induction of Mpk1p/Slt2p tyrosine phosphorylation does not occur in pmt2 pmt4Δ mutants during exposure to mating pheromone or elevated temperature. Detailed analyses of the plasma membrane sensors of the PKC1 pathway revealed that Wsc1p, Wsc2p, and Mid2p are aberrantly processed in pmt mutants. Our data suggest that in yeast, O mannosylation increases the activity of Wsc1p, Wsc2p, and Mid2p by enhancing their stability. Reduced O mannosylation leads to incorrect proteolytic processing of these proteins, which in turn results in impaired activation of the PKC1 pathway and finally causes cell death in the absence of osmotic stabilization

«… faire sentir par la lecture des Auteurs le génie & le caractère de la langue Françoise» − Französische Grammatiken und Lehrbücher des 17. und 18. Jahrhunderts aus den Beständen der Universitätsbibliothek Augsburg: Eine Ausstellung des Lehrstuhls für Romanische Sprachwissenschaft (Französisch/Italienisch) der Universität Augsburg in Zusammenarbeit mit der Universitätsbibliothek Augsburg ; Katalog zur Ausstellung

Für die französische Sprache in Frankreich gilt in ganz besonderer Weise, dass sie seit mehreren Jahrhunderten kulturelle Symbolfunktion besitzt, also zu einem zentralen Ort im kulturellen Gedächtnis der Franzosen geworden ist. Diese Symbolfunktion ist an eine Sprachnorm gebunden, die auf der Vorbildrolle klassischer Literaten wie Molière und Racine fußt. Im französischen Sprachbewusstsein ist die klassische französische Literatur bis in die Gegenwart repräsentativ für den guten Sprachgebrauch – den "bon usage". Begründet, definiert und beschrieben wird diese Sprachnorm oder Modellsprache im 17. und 18. Jahrhundert in zahlreichen Grammatiken, Wörterbüchern und Lehrwerken, die bei der Verbreitung der französischen Sprache in Frankreich und über die Landesgrenzen hinaus eine wichtige Rolle spielen. Der historische Bestand der Universitätsbibliothek Augsburg, insbesondere die Oettingen-Wallersteinsche Sammlung, beherbergt eine Reihe der wichtigsten Dokumente französischer Sprachnormierung dieser Zeit und damit einen wertvollen Fundus für die sprachhistorische Forschung. Im Wintersemester 2013-2014 haben sich Studierende der Universität Augsburg in einem forschungsbezogenen Projektseminar unter Leitung von Prof. Dr. Sabine Schwarze, Lehrstuhl für Romanische Sprachwissenschaft, eingehend mit diesen Texten und deren Rolle für die französische Sprach- und Kulturgeschichte beschäftigt. Die Ausstellung gibt einen Einblick in das Lehrmaterial, das für den Französischunterricht in der Zeit der Normierung des "bon usage", also des guten französischen Sprachgebrauchs, entwickelt wurde und vermittelt Erklärungen zur Rolle der einzelnen Autoren und Texte für die französische Normierungsgeschichte und das französische Sprachbewusstsein bis in die Gegenwart