Der Elongationsfaktor PAF1 aus S. cerevisiae: Aufbau, Funktion und Interaktionen

Modulation of chromatin structure and regulation of transcription are strongly dependent on posttranslational modifications of core histones. Especially, the importance of cotranscriptional histone methylation as a regulatory signal for progression through the transcription cycle has been realised over the past years. All three histone methyltransferases in yeast are functionally dependent on elongation factor PAF1. Additionally, the PAF1 complex has a role in coupling 3'-end processing of premature mRNA with transcription elongation and termination. This work investigates the composition of the elongation factor PAF1 in yeast and the protein-protein interactions between members within the complex. On the basis of these data a model for the assembly of PAF1 is suggested. In addition, the individual effects of its components on elongation and telomeric silencing are analysed genetically and physiologically. Furthermore, the interactions between PAF1 complex, elongation factor yFACT, and Casein Kinase II are analyzed and a physical interaction between PAF1 complex and Bromodomainfactor 1 is decribed. The data demonstrate that the elongation factor PAF1 is a homogeneously composed pentameric complex of the proteins Ctr9, Rtf1, Leo1, Paf1 and Cdc73. The association between Paf1 and Ctr9 is crucial for stability of the complex. Ctr9 acts as a binding partner for most of the other components due to its arrangement of at least 10 serial TPR motives. The location of binding domains for Paf1 and Cdc73 can be mapped to 2-3 consecutive TPR motives on the N or C-terminal end of Ctr9, respectively. The importance of Paf1 and Ctr9 for the stability of the PAF1 complex is reflected in their specific importance for transcriptional elongation and their influence on telomeric silencing. The study of individual yeast mutants suggested that both Rtf1 and Cdc73 negatively regulate the activity of Paf1. This could indicate that the function of elongation factor PAF1 is subject to negative autoregulation. The interaction between PAF1 complex, yFACT, and Casein Kinase II (CKII) are also determined by Paf1 and Ctr9. Ctr9, Rtf1, Leo1, and Paf1 were found to be phosphorylated by Casein Kinase II in vitro and the phosphorylation site(s) in Ctr9 could be mapped to its the C terminal end. Furthermore, this work demonstrates a physical interaction between PAF1 complex and Bromodomainfactor 1 (Bdf1) in yeast. On the basis of the observed interactions between the elongation factors PAF1 and yFACT as well as CKII a model for a combined participation of all four factors on the transcriptional capping checkpoint is suggested and discussed.An der Modulation der Chromatinstruktur und der Regulation der Transkription ist die reversible posttranslationale Modifikation der in den Nukleosomen enthaltenen Core-Histone maßgeblich beteiligt. Besonders die Funktion der co-transkriptionellen Histon-Methylierung als regulatorisches Signal für das Durchlaufen des Transkriptionszyklus konnte innerhalb der letzten Jahre immer genauer aufgeklärt werden. Der Elongationsfaktor PAF1 erweist sich durch seinen regulatorischen Einfluss auf alle drei bekannten Histon-Methyltransferasen der Hefe als entscheidender Faktor für die co-transkriptionelle Methylierung von Histon H3 und besitzt zudem eine Funktion bei der auf den Transkriptionsverlauf abgestimmten Prozessierung des 3' Endes der naszierenden mRNA. In der vorliegenden Arbeit wird die Zusammensetzung des Elongationsfaktors PAF1 sowie die Protein-Protein Interaktionen zwischen den Komponenten des Komplexes eingehend untersucht und anhand der Daten ein Modell für den Aufbau des PAF1-Komplexes vorgeschlagen. Außerdem wird der Einfluss der einzelnen Bestandteile des PAF1-Komplexes auf die Elongation und das telomere Silencing phänotypisch und funktionell charakterisiert. Zudem werden die Interaktionen des Komplexes mit dem Elongationsfaktor yFACT sowie der Casein-Kinase II untersucht und eine physikalische Wechselwirkung mit dem Bromodomainfactor 1 aufgeklärt. Der Elongationsfaktor PAF1 erweist sich dabei als pentamerer Proteinkomplex, der homogen aus den Proteinen Ctr9, Rtf1, Leo1, Paf1 und Cdc73 aufgebaut ist. Dabei zeigt sich, dass die direkte Assoziation von Paf1 und Ctr9 für die Stabilität des PAF1-Komplexes maßgeblich ist. Besonders Ctr9 wirkt aufgrund seiner seriellen Aneinanderreihung von insgesamt mindestens 10 TPR-Motiven als Bindungspartner für die übrigen Komponenten, wobei die Position der Bindungsdomänen für Paf1 und Cdc73 auf diskrete Anordnungen aus jeweils 2-3 N- bzw. C-terminal gelegenen TPR-Motiven lokalisiert werden kann. Die Voraussetzung der Paf1-Ctr9-Bindung für die Stabilität des Elongationsfaktors spiegelt sich unmittelbar in dem Befund wider, dass beide Proteine die Funktion des PAF1-Komplexes während der Elongation und auch den Einfluss auf das Telomersilencing entscheidend vermitteln. Besonders anhand der phänotypischen Untersuchung der Elongationsdefekte von Deletionsmutanten wird jedoch deutlich, dass Rtf1 und Cdc73 teilweise antagonistisch zu Paf1 wirken, was auf eine regulatorische Selbstinhibierung des Elongationsfaktors hinweisen könnte. Auch die Interaktion zwischen dem Elongationsfaktor PAF1 und yFACT sowie der Casein-Kinase II (CKII) wird durch Paf1 und Ctr9 bestimmt. Zudem konnte in vitro die CKII-abhängige Phosphorylierung von Ctr9, Rtf1, Leo1 und Paf1 gezeigt und die Lokalisation der Phosphorylierung von Ctr9 auf dessen C-Terminus begrenzt werden. Daneben konnte erstmalig die physikalische Interaktion zwischen dem Elongationsfaktor PAF1 und dem Bromo-domainfactor 1 demonstriert werden. Aufgrund der beobachteten Interaktion zwischen den Elongationsfaktoren PAF1 und yFACT sowie der Casein-Kinase II und des Bromodomainfactor 1 und anhand von Literaturdaten wird ein Modell für eine ggf. gemeinsame Beteiligung aller vier Faktoren am Capping-Kontrollpunkt vorgeschlagen und diskutiert

Efficacy of cupping therapy in patients with the fibromyalgia syndrome-a randomised placebo controlled trial

© 2016 The Author(s). This study aimed to test the efficacy of cupping therapy to improve symptoms and quality of life in patients diagnosed with the fibromyalgia syndrome. Participants were randomly assigned to cupping therapy, sham or usual care. Cupping was administered five times at twice weekly intervals on the upper and lower back. The primary outcome measure was pain intensity at day 18. Secondary outcomes included functional disability, quality of life, fatigue and sleep quality as well as pressure pain sensitivity, satisfaction and safety at day 18 and 6 months. Altogether 141 patients were included in this study (139 females, 55.8 ± 9.1 years). After 18 days patients reported significant less pain after cupping compared to usual care (difference-12.4; 95% CI:-18.9;-5.9, p < 0.001) but not compared to sham (difference-3.0; 95% CI:-9.9, 3.9, p = 0.396). Further effects were found for quality of life compared to usual care. Patients were mildly satisfied with cupping and sham cupping; and only minor side effects were observed. Despite cupping therapy being more effective than usual care to improve pain intensity and quality of life, effects of cupping therapy were small and comparable to those of a sham treatment, and as such cupping cannot be recommended for fibromyalgia at the current time

Diazoxide-responsive hyperinsulinemic hypoglycemia caused by HNF4A gene mutations

Objective: The phenotype associated with heterozygous HNF4A gene mutations has recently been extended to include diazoxide responsive neonatal hypoglycemia in addition to maturity-onset diabetes of the young (MODY). To date, mutation screening has been limited to patients with a family history consistent with MODY. In this study, we investigated the prevalence of HNF4A mutations in a large cohort of patients with diazoxide responsive hyperinsulinemic hypoglycemia (HH). Subjects and methods: We sequenced the ABCC8, KCNJ11, GCK, GLUD1, and/or HNF4A genes in 220 patients with HH responsive to diazoxide. The order of genetic testing was dependent upon the clinical phenotype. Results: A genetic diagnosis was possible for 59/220 (27%) patients. KATP channel mutations were most common (15%) followed by GLUD1 mutations causing hyperinsulinism with hyperammonemia (5.9%), and HNF4A mutations (5%). Seven of the 11 probands with a heterozygous HNF4A mutation did not have a parent affected with diabetes, and four de novo mutations were confirmed. These patients were diagnosed with HI within the first week of life (median age 1 day), and they had increased birth weight (median +2.4 SDS). The duration of diazoxide treatment ranged from 3 months to ongoing at 8 years. Conclusions: In this large series, HNF4A mutations are the third most common cause of diazoxide responsive HH. We recommend that HNF4A sequencing is considered in all patients with diazoxide responsive HH diagnosed in the first week of life irrespective of a family history of diabetes, once KATP channel mutations have been excluded

Cystathionine beta synthase deficiency and brain edema associated with methionine excess under betaine supplementation: Four new cases and a review of the evidence

CBS deficient individuals undergoing betaine supplementation without sufficient dietary methionine restriction can develop severe hypermethioninemia and brain edema. Brain edema has also been observed in individuals with severe hypermethioninemia without concomitant betaine supplementation. We systematically evaluated reports from 11 published and 4 unpublished patients with CBS deficiency and from additional four cases of encephalopathy in association with elevated methionine. We conclude that, while betaine supplementation does greatly exacerbate methionine accumulation, the primary agent causing brain edema is methionine rather than betain

Monte Carlo investigations of phase transitions: status and perspectives

Using the concept of finite-size scaling, Monte Carlo calculations of various models have become a very useful tool for the study of critical phenomena, with the system linear dimension as a variable. As an example, several recent studies of Ising models are discussed, as well as the extension to models of polymer mixtures and solutions. It is shown that using appropriate cluster algorithms, even the scaling functions describing the crossover from the Ising universality class to the mean-field behavior with increasing interaction range can be described. Additionally, the issue of finite-size scaling in Ising models above the marginal dimension (d*=4) is discussed.Comment: 23 pages, including 14 PostScript figures. Presented at StatPhys-Taiwan, August 9-16, 1999. Also available as PDF file at http://www.cond-mat.physik.uni-mainz.de/~luijten/erikpubs.htm

Improving polymeric microemulsions with block copolymer polydispersity

Recent experiments have demonstrated that block copolymers are capable of stabilizing immiscible homopolymer blends producing bicontinuous microemulsion. The stability of these polymeric alloys requires the copolymer to form flexible, nonattractive monolayers along the homopolymer interfaces. We predict that copolymer polydispersity can substantially and simultaneously improve the monolayers in both of these respects. Furthermore, polydispersity should provide similar improvements in systems, such as colloidal suspensions and polymer/clay composites, that utilize polymer brushes to suppress attractive interactions

Bulk and Boundary Critical Behavior at Lifshitz Points

Lifshitz points are multicritical points at which a disordered phase, a homogeneous ordered phase, and a modulated ordered phase meet. Their bulk universality classes are described by natural generalizations of the standard $\phi^4$ model. Analyzing these models systematically via modern field-theoretic renormalization group methods has been a long-standing challenge ever since their introduction in the middle of the 1970s. We survey the recent progress made in this direction, discussing results obtained via dimensionality expansions, how they compare with Monte Carlo results, and open problems. These advances opened the way towards systematic studies of boundary critical behavior at $m$-axial Lifshitz points. The possible boundary critical behavior depends on whether the surface plane is perpendicular to one of the $m$ modulation axes or parallel to all of them. We show that the semi-infinite field theories representing the corresponding surface universality classes in these two cases of perpendicular and parallel surface orientation differ crucially in their Hamiltonian's boundary terms and the implied boundary conditions, and explain recent results along with our current understanding of this matter.Comment: Invited contribution to STATPHYS 22, to be published in the Proceedings of the 22nd International Conference on Statistical Physics (STATPHYS 22) of the International Union of Pure and Applied Physics (IUPAP), 4--9 July 2004, Bangalore, Indi

ETISEQ – an algorithm for automated elution time ion sequencing of concurrently fragmented peptides for mass spectrometry-based proteomics

<p>Abstract</p> <p>Background</p> <p>Concurrent peptide fragmentation (i.e. shotgun CID, parallel CID or MS^E) has emerged as an alternative to data-dependent acquisition in generating peptide fragmentation data in LC-MS/MS proteomics experiments. Concurrent peptide fragmentation data acquisition has been shown to be advantageous over data-dependent acquisition by providing greater detection dynamic range and providing more accurate quantitative information. Nevertheless, concurrent peptide fragmentation data acquisition remains to be widely adopted due to the lack of published algorithms designed specifically to process or interpret such data acquired on any mass spectrometer.</p> <p>Results</p> <p>An algorithm called Elution Time Ion Sequencing (ETISEQ), has been developed to enable automated conversion of concurrent peptide fragmentation data acquisition data to LC-MS/MS data. ETISEQ generates MS/MS-like spectra based on the correlation of precursor and product ion elution profiles. The performance of ETISEQ is demonstrated using concurrent peptide fragmentation data from tryptic digests of standard proteins and whole influenza virus. It is shown that the number of unique peptides identified from the digests is broadly comparable between ETISEQ processed concurrent peptide fragmentation data and the data-dependent acquired LC-MS/MS data.</p> <p>Conclusion</p> <p>The ETISEQ algorithm has been designed for easy integration with existing MS/MS analysis platforms. It is anticipated that it will popularize concurrent peptide fragmentation data acquisition in proteomics laboratories.</p