central carbohydrate metabolism in Sulfolobus species

Die Crenarchaea Sulfolobus solfataricus und S. acidocaldarius sind model Organismen der dritten Domäne des Lebens, der Archaea. Der Zentrale Kohlenhydrat Metabolismus (ZKM) von Sulfolobus spp. wurde in der Vergangenheit ausführlich untersucht mittels klassischer Biochemischer verfahren als auch durch moderner hoch Durchsatz Verfahren (Bsp. Proteomics, Genomics und Transcriptomics). Jedoch ist die Regulation des ZKMs in Sulfolobus spp. weitest gehend unbekannt. Wie in anderen Archaea sind in Sulfolobus die "klassischen" Kontrollpunkten, welche in Eukaryoten und Bakterien vorhanden sind, abwesend und der bisher einzige bekannte Kontrollpunkt ist die nicht phosphorylierende Glycerinaldehyd-3-phosphat Dehydrogenase (GAPN), welche durch Glukose 1-phosphat allosterisch reguliert wird. Im Rahmen dieser Thesis wurde untersucht welchen Einfluss Protein Phosphorylierung in Sulfolobus spp. hat. In S. solfataricus wird das Phosphoproteom untersucht, von Zellen die entweder auf D-Glukose oder Trypton gezogen wurden mittels der precursor aquisition independent from ion count (PAcIFIC) Technik. Die Phosphoproteom Analyse zeigte das in S. solfataricus eine sehr hohe Anzahl an Phosphoproteinen vorhanden ist mit einer ungewöhnlichen pSer/pThr/pTyr Verteilung. Desweiteren wurde gezeigt dass die Identifizierten Phosphoproteine in allen Zellulären Prozessen involviert sind. Besonders der ZKM scheint stark reguliert zu werden durch Proteinphosphorylierung und die verwendete Kohlenstoff Quelle hat einen starken Einfluss darauf. Ähnliche Experimente wurden auch mit S. acidocaldarius durchgeführt in Kombination mit Transcriptomics, Biochemie und Wachstums Studien um den Effekt der Protein Phosphatasen Deletion zu untersuchen. The entsprechenden Gene wurden kloniert und die rekombinanten Enzyme Biochemisch untersucht. Diese Untersuchungen zeigten das S. acidocaldarius eine Protein Phosphatase besitzt die spezifisch für pSer/Thr ist (PP2A) und eine Protein Phosphatase besitzt die eine Duale Substrat Spezifität aufweise, jedoch pTyr effizienter umsetzten kann (PTP). Die Untersuchung der Protein Phosphatase Deletionsstämme (Δsaci_ptp, Δsaci_pp2a) und des Eltern Stammes (MW001) zeigt das durch die Deletion die gesamt Anzahl an Phosphoprotein erhöht wird und auch die pSer/Thr/Tyr Verteilung beeinträchtigt wird. Ähnlich wie in S. solfataricus konnten Phosphoproteine aus allen Zellulären Prozessen identifiziert werden. Desweiteren zeigte die Phänotypische Charakterisierung der drei Stämme dass Zellen des Δsaci_pp2a Stammes eine andere Zellmorphologie besitzen und dass der Δsaci_pp2a Stamm sich wie ein Hyperbeweglicher Stamm verhält, verglichen mit MW001 und Δsaci_ptp. Die Untersuchung des Transkriptoms der drei Stämme durch RNA deep seq. zeigt das besonders Gene involviert in der Atmungskette und der Zell Bewegung reguliert sind. Desweiteren wurden Enzyme aus dem ZKM aus beiden Sulfolobus spp. Untersucht, wie die Glukose-1-Dehydrogenase (GDH), drei Aldehyd Dehydrogenasen (ALDHs) und die Triose-3-phosphat Isomerase (TPI). Die GDH von S. acidocaldarius weist ähnliche Biochemische Eigenschaften auf wie ihr Homolog aus S. solfataricus, sie besitzt eine breite Substrate Spezifität und kann sowohl NAD+ als auch NADP+ als Cosubstrate verwenden. Ihr physiologisches Cosubstrat ist NADP+ und das physiologische Substrat ist D-Xylose. Beide GDHs wurden als Phosphoproteine identifiziert mit einer konservierten Phosphorylierungsstelle, deren Einfluss bisher jedoch unbekannt ist. Die Untersuchung der ALDH Superfamilie aus S. solfataricus zeigte das SSO1218 eine Methylmalonat Semialdehyd Dehydrogenase ist, welche involviert ist im Abbau von Verzweigten Aminosäuren. Wohin gegen SSO1629 und SSO1842 Succinat Semialdehyd Dehydrogenase (SSADH) Isoenzyme sind. Beide haben sehr ähnliche Eigenschaften und sind für den γ-Aminobutyrat Metabolismus, Polyamin Metabolismus und den Stickstoff Metabolismus von Bedeutung. Die Biochemische Untersung der TPI von S. solfataricus zeigt das dieses Enzym sowohl in der Glykolyse als auch der Glukoneogenese aktiv ist, jedoch eine höhere Aktivität für die Glukoneogenetische Reaktion hat. Desweiteren ist diese TPI die erste Archaeale TPI die durch 3-Phosphoglycerat und Phosphoenolpyruvat Inhibiert wird. Manche der Untersuchten Enzyme (SSO1842, Saci_1079) wurden als Phosphoproteine Identifiziert und zukünftige Studien werden sich mit der Untersuchung der Phosphorylierung befassen um die verantwortlichen Signal Transduktions Kaskaden zu identifizieren.The Crenarchaea Sulfolobus solfataricus and S. acidocaldarius are among the best investigated Archaea and are well established as thermoacidophilic model organisms. The central carbohydrate metabolism (CCM) of Sulfolobus species has been well studied using classical biochemical and modern high throughput approaches such as genomics, proteomics and transcriptomics. However, the regulation of the CCM is still far from being understood. Like other archaea, Sulfolobus species lack the “classical” control points known from their eukaryotic or bacterial brethren and the only well established control point is the non-phosphorylating glyceraldehyde-3-phosphate dehydrogenase, which is activated at low levels of the metabolite glucose 1-phosphate. Furthermore, the complexity of the CCM in Sulfolobus species as well as other Archaea is still unsolved and there are often several paralogs in the archaeal genomes with unknown function. In the first part of this thesis the impact of regulation by post translational modifications via reversible protein phosphorylation was analyzed in S. solfataricus and S. acidocaldarius. Crenarchaea like Sulfolobus species lack bacterial type one/two-component systems (phosphorylation on His and Asp) but rely on eukaryal like protein phosphorylation on Ser, Thr and Tyr. In S. solfataricus we investigated how the offered carbon source glucose (glycolytic growth) or tryptone (gluconeogenic growth) alter the global phosphorylation pattern. Analysis by precursor aquisition independent from ion count (PAcIFIC) revealed a high number of p-proteins (540) with a so far unique enrichment for p-Tyr (>50%) compared to previous studies in all three domains of life (Table 1). Furthermore, p-proteins were detected in all functional categories indicating a major impact on all cellular processes. In response to the carbon source dramatic changes in the phosphorylation pattern were observed. In the CCM especially proteins at the beginning of various sugar degradation pathways and gluconeogenic EMP and TCA and at branching points to other metabolic routs were targeted by protein phosphorylation. In S. acidocaldarius we used a similar experimental approach which combined transcriptomics, biochemistry and growth studies to investigate the effect of deletion of the two protein phosphatases. The respective genes of the putative protein phosphatases were cloned and the recombinant enzymes in regard to their substrate specificity investigated. The catalytic subunit of the protein Ser/Thr phosphatase (PP2Ac) resembles their eukaryotic counterparts and was inhibited by okadaic acid and requires Mn2+-ions for catalytic activity and showed strict activity towards p-Ser/p-Thr peptides. Whereas the protein tyrosine phosphatase (PTP) revealed dual substrate specificity with p-Tyr and p-Thr peptides. The phenotypic characterization of the three strains (MW001, Δsaci_pp2a, Δsaci_ptp) revealed that the parant strain MW001 and Δsaci_ptp resembled each other, where as the Δsaci_pp2a strain showed a differences in growth and cell size variation. The number of p-proteins in the background strain MW001 was lower (69) compared to S. solfataricus, but similar to that reported for other prokaryotic and eukaryotic organisms (Table 1). According to the expectation the number of p-proteins was increased in the two phosphatase deletion strains (Δsaci_pp2a (143), Δsaci_ptp (407)) and comparable to the number determined in S. solfataricus. However, the deletion of the two protein phosphatases not only resulted in an increase of p-proteins, the whole phosphorylation pattern was significant changed. The combination of the three data sets revealed that similar to S. solfataricus all cellular processes are targeted by protein phosphorylation. Furthermore, the investigation of the transcriptome, via RNA deep sequencing analysis, revealed a major impact on the transcription of genes of the respiratory chain as well as genes involved in cell motility (e.g. archaellum). These results were further supported by the identification of respective p-proteins and by the finding that the Δsaci_pp2a strain showed a different motility behavior compared to the strains MW001 and Δsaci_ptp. The CCM showed also a complete different phosphorylation pattern in MW001 compared to the two deletion strains. Especially enzymes of the tricarboxylic acid cycle as well as enzymes located at branching points (e.g. amino acid metabolism) seem to be targeted by protein phosphorylation in the Δsaci_pp2a strain. The high amount of p-Tyr detected in both Sulfolobus species let suggest that phosphorylation on Tyr is a crenarchaeal feature. The high growth temperature seems not to be the main reason of the high p-Tyr amount since only a low amount of p-Tyr was identified in the thermophilic gram-negative bacterium Thermus thermophilus (grown at 70°C, 10% p-Tyr). Also in the Euryarchaeota Halobacterium salinarium only one phosphorylation site on Tyr was identified, which excluds that this is a typical archaeal feature. However, compared to Sulfolobus species, T. thermophilus and H. salinarium also possess His kinases, which might provide alternative possibilities to regulate cellular functions by reversible protein phosphorylation. Therefore, suggesting that the high amount of p-Tyr is a typical feature of the crenarchaeal Sulfolobus species. The second part of this thesis focused on unraveling the complexity and regulation of the CCM in Sulfolobus species. From S. solfataricus were the triose-3-phosphate isomerase (TPI) and members of the aldehyde dehydrogenase super family characterized and from S. acidocaldarius the putative glucose-1-dehydrogenase (GDH). To unravel the function of the S. solfataricus TPI in gluconeogenesis kinetic parameters for both triose-3-phosphates glyceraldehydes-3-phosphate and dihydroxyacetone-3-phosphate were determined. The determined catalytic efficiencies were rather small compared to previously characterized archaeal TPIs. The reason is still unclear and we expect further insight from the available crystal structure. The S. solfataricus TPI showed inhibition by phosphoenolpyruvate and 3-phosphoglycerate which was not reported for any archaeal TPI so far but is well established for eukaryotic TPIs. For the two so far uncharacterized members of the aldehyde dehydrogenase super family succinic semialdehyde dehydrogenase activity was demonstrated (SSO1629, SSO1842) and a major role in polyamine and γ-aminobutyric acid metabolism was proposed based on bioinformatic analysis. For SSO1218 methylmalonate semialdehyde dehydrogenase activity demonstrated and a role in the degradation of branched amino acids was proposed. The characterization of the GDH from S. acidocaldarius (Saci_1079), one of the 12 of the medium-chain alcohol/polyol dehydrogenase/reductase branch of the pyridine nucleotide-dependent alcohol/polyol/sugar dehydrogenase superfamily in S. acidocaldarius, revealed that this enzyme exhibits broad substrate specificity a broad range of different sugars and might play an important role not only in D-glucose, but also in D-xylose, D-galactose and D-fucose degradation. The enzyme shows highest similarity to GDH-1 (SSO3003) of S. solfataricus and resembles the enzyme in respect to its broad substrate specificity, kinetic parameters as well as structural features. Notably, some of the enzymes (e.g. SSO1842, Saci_1079) have been identified as p-proteins and future studies aim to unravel the impact of phosphorylation on enzyme function in order to resolve the respective signal transduction pathways

Protein phosphorylation and its role in archaeal signal transduction

Reversible protein phosphorylation is the main mechanism of signal transduction that enables cells to rapidly respond to environmental changes by controlling the functional properties of proteins in response to external stimuli. However, whereas signal transduction is well studied in Eukaryotes and Bacteria, the knowledge in Archaea is still rather scarce. Archaea are special with regard to protein phosphorylation, due to the fact that the two best studied phyla, the Euryarchaeota and Crenarchaeaota, seem to exhibit fundamental differences in regulatory systems. Euryarchaeota (e.g. halophiles, methanogens, thermophiles), like Bacteria and Eukaryotes, rely on bacterial-type two-component signal transduction systems (phosphorylation on His and Asp), as well as on the protein phosphorylation on Ser, Thr and Tyr by Hanks-type protein kinases. Instead, Crenarchaeota (e.g. acidophiles and (hyper)thermophiles) only depend on Hanks-type protein phosphorylation. In this review, the current knowledge of reversible protein phosphorylation in Archaea is presented. It combines results from identified phosphoproteins, biochemical characterization of protein kinases and protein phosphatases as well as target enzymes and first insights into archaeal signal transduction by biochemical, genetic and polyomic studie

Insights into the evolutionary conserved regulation of Rio ATPase activity

Department of Biochemistry III ‘House of the Ribosome’ and by the DFG Collaborative Research Center [SFB960-AP1] ‘Ribosome formation: principles of RNP biogenesis and control of their function’ (to S.F.-C.).; Work in the MacNeill laboratory was funded by Forskningsrådet for Natur og Univers (FNU) [sagsnr. 272-05-0446]; Scottish Universities Life Sciences Alliance (SULSA); Research in the Medenbach laboratory is supported by the Bavarian Research Network for Molecular Biosystems (BioSysNet); German Research Foundation (DFG) [ME4238/1-1]; DFG Collaborative Research Center [SFB960-B11] ‘Ribosome formation: principles of RNP biogenesis and control of their function’; German Federal Ministry of Education and Research (BMBF) within the framework of the e:Med research and funding concept [01ZX1401D]; Work in the Siebers laboratory was funded by a grant from the German Science Foundation (DFG) [SI642/10-1] from the Federal Ministry of Education and Research (BMBF) [0316188A]; Work in the LaRonde laboratory was funded by National Science Foundation [MCB0953493]; Publishing of this work was supported by the German Research Foundation (DFG) within the funding program Open Access Publishing. Funding for open access charge: DFG—Open Access program.Eukaryotic ribosome biogenesis is a complex dynamic process which requires the action of numerous ribosome assembly factors. Among them, the eukaryotic Rio protein family members (Rio1, Rio2 and Rio3) belong to an ancient conserved atypical protein kinase/ ATPase family required for the maturation of the small ribosomal subunit (SSU). Recent structure-function analyses suggested an ATPase-dependent role of the Rio proteins to regulate their dynamic association with the nascent pre-SSU. However, the evolutionary origin of this feature and the detailed molecular mechanism that allows controlled activation of the catalytic activity remained to be determined. In this work we provide functional evidence showing a conserved role of the archaeal Rio proteins for the synthesis of the SSU in archaea. Moreover, we unravel a conserved RNA-dependent regulation of the Rio ATPases, which in the case of Rio2 involves, at least, helix 30 of the SSU rRNA and the P-loop lysine within the shared RIO domain. Together, our study suggests a ribosomal RNA-mediated regulatory mechanism enabling the appropriate stimulation of Rio2 catalytic activity and subsequent release of Rio2 from the nascent pre- 40S particle. Based on our findings we propose a unified release mechanism for the Rio proteins.Publisher PDFPeer reviewe

Performance of the CMS Cathode Strip Chambers with Cosmic Rays

The Cathode Strip Chambers (CSCs) constitute the primary muon tracking device in the CMS endcaps. Their performance has been evaluated using data taken during a cosmic ray run in fall 2008. Measured noise levels are low, with the number of noisy channels well below 1%. Coordinate resolution was measured for all types of chambers, and fall in the range 47 microns to 243 microns. The efficiencies for local charged track triggers, for hit and for segments reconstruction were measured, and are above 99%. The timing resolution per layer is approximately 5 ns

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The operation and general performance of the CMS electromagnetic calorimeter using cosmic-ray muons are described. These muons were recorded after the closure of the CMS detector in late 2008. The calorimeter is made of lead tungstate crystals and the overall status of the 75848 channels corresponding to the barrel and endcap detectors is reported. The stability of crucial operational parameters, such as high voltage, temperature and electronic noise, is summarised and the performance of the light monitoring system is presented

“Hot standards” for the thermoacidophilic archaeon Sulfolobus solfataricus

Within the archaea, the thermoacidophilic crenarchaeote Sulfolobus solfataricus has become an important model organism for physiology and biochemistry, comparative and functional genomics, as well as, more recently also for systems biology approaches. Within the Sulfolobus Systems Biology (“SulfoSYS”)-project the effect of changing growth temperatures on a metabolic network is investigated at the systems level by integrating genomic, transcriptomic, proteomic, metabolomic and enzymatic information for production of a silicon cell-model. The network under investigation is the central carbohydrate metabolism. The generation of high-quality quantitative data, which is critical for the investigation of biological systems and the successful integration of the different datasets, derived for example from high-throughput approaches (e.g., transcriptome or proteome analyses), requires the application and compliance of uniform standard protocols, e.g., for growth and handling of the organism as well as the “–omics” approaches. Here, we report on the establishment and implementation of standard operating procedures for the different wet-lab and in silico techniques that are applied within the SulfoSYS-project and that we believe can be useful for future projects on Sulfolobus or (hyper)thermophiles in general. Beside established techniques, it includes new methodologies like strain surveillance, the improved identification of membrane proteins and the application of crenarchaeal metabolomics

IL-17 and neutrophils: unexpected players in the type 2 immune response

The study of immunity to helminth infection has been central to understanding the function of type 2 cytokines and their targets. Although type 2 cytokines are considered anti-inflammatory and promote tissue repair, they also contribute to allergy and fibrosis. Here, we utilise data from helminth infection models, to illustrate that IL-17 and neutrophils, typically associated with pro-inflammatory responses, are intimately linked with type 2 immunity. Neutrophils work with IL-4Rα-activated macrophages to control incoming larvae but this comes at a cost of enhanced tissue damage. Chitinase like proteins (CLPs) bridge these diverse outcomes, inducing both protective IL-17 and reparative Th2 responses. Dysregulation of CLPs, IL-17 and neutrophils likely contribute to disease severity and pathology associated with type 2 immunity

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CMS Data Processing Workflows during an Extended Cosmic Ray Run

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