65 research outputs found
Molecular motions at the 5 stem-loop of U4 snRNA: Implications for U4/U6 snRNP assembly
Das humane 15.5K Protein bindet an die 5' Schleife
der U4 snRNA (KtU4). Es begĂźnstigt den Aufbau des
Spliceosoms U4/U6 snRNP und ist notwendig fĂźr die
ZusammenfĂźhrung des 61K Proteins und des 20/60/90K
Proteinkomplexes mit der U4-snRNA. In der
kristallografischen Struktur des 15.5K-U4 snRNA
Komplexes gehĂśrt die RNA Faltung zu der Familie der so
genannten kink-turn (K-turn) Motive. Dieses Motiv ist
durch einen scharfen Knick im PhosphodiesterrĂźckgrat
gekennzeichnet. Zwei Helixstämme sind durch eine
purinreiche, asymmetrische, innere Schleife verbunden.
Diese enthält eine herausgeklappte Uridinbase und zwei
aufeinander folgende verschobene G-A Basenpaare. Der
kĂźrzere Helixstamm ist mit einer externen fĂźnffachen
Schleife verbunden. Mit Hilfe von
Molekulardynamiksimulationen konnte ich zeigen, dass
die Faltung von KtU4 durch die Proteinbindung
unterstßtzt wird. Konformationsänderungen wie die
Umwandlung zwischen alternativen Purin-Stapelschemata,
der Verlust von G-A Basenpaaren und die Ăffnung des
K-turns (k-e Bewegung) trat nur in den Simulationen
der ungebundenen RNA auf. Diese Simulationen zeigen zum
ersten Mal die Dynamik des K-turn Motivs mit atomarer
AuflĂśsung und finden sich in hervorragender
Ăbereinstimmung mit experimentellen Resultaten, die
durch chemische Erprobung und FRET-Studien an
EinzelmolekĂźlen der RNA gesammelt wurden. In der
ungebundenen RNA wurde die k-e Bewegung gleichermaĂen
durch den Verlust der G-A Basenpaare in der inneren
Schleife und durch Flexibilität im Phosphatrßckgrat der
beiden Helixstämme hervorgerufen. Jedoch war der
Verlust der G-A Basenpaare allein nicht ausreichend, um
eine groĂe Entfaltung der ungebundenen RNA zu
erreichen. Eine Untersuchung der Eigenmodi der RNA
Bewegung konnte zeigen, dass der Verlust der G-A
Basenpaare nur mit dem ersten Eigenmodus korreliert ist
aber nicht mit dem dritten Eigenmodus, der die k-e
Bewegung beschreibt. Aufgrund dieser Ergebnisse komme
ich zu dem Schluss, dass sich die G-A Basenpaare erst
bei der Bindung an das 15.5K Protein bilden und d
adurch selektiv die Ausrichtung der Helixstämme
stabilisieren. Die externe Schleife konnte in der
Kristallstruktur des 15.5K-KtU4 Komplexes nicht
aufgelĂśst werden. In den Simulationen des Komplexes
nahm sie eine bestimmte Orientierung ein, die nicht in
der ungebundenen RNA beobachtet wurde. Sie trat auch
dann nicht auf, wenn die natĂźrliche Sequenz durch
andere externe Schleifen ersetzt oder die Helixstämme
verlängert wurden. Auf Grund der Simulationsergebnisse
schlage ich vor, dass die nicht vorhandene Stapelung
zwischen dem letzten Basenpaar des Helixstamms und dem
benachbarten Nukleotid in der externen Schleife wichtig
ist fĂźr die korrekte Faltung der RNA und eine wichtige
Rolle in der nachfolgenden Bindung des 61K Proteins an
die U4 snRNA spielt
Loss of GâA base pairs is insufficient for achieving a large opening of U4 snRNA K-turn motif
Upon binding to the 15.5K protein, two tandem-sheared GâA base pairs are formed in the internal loop of the kink-turn motif of U4 snRNA (Kt-U4). We have reported that the folding of Kt-U4 is assisted by protein binding. Unstable interactions that contribute to a large opening of the free RNA (âkâe motionâ) were identified using locally enhanced sampling molecular dynamics simulations, results that agree with experiments. A detailed analysis of the simulations reveals that the kâe motion in Kt-U4 is triggered both by loss of GâA base pairs in the internal loop and backbone flexibility in the stems. Essential dynamics show that the loss of GâA base pairs is correlated along the first mode but anti-correlated along the third mode with the kâe motion. Moreover, when enhanced sampling was confined to the internal loop, the RNA adopted an alternative conformation characterized by a sharper kink, opening of GâA base pairs and modified stacking interactions. Thus, loss of GâA base pairs is insufficient for achieving a large opening of the free RNA. These findings, supported by previously published RNA structure probing experiments, suggest that GâA base pair formation occurs upon protein binding, thereby stabilizing a selective orientation of the stems
Variability of Biological Parameters in Blood Samples Between Two Consecutive Schedules of Hemodialysis
DNA-mediated cooperativity facilitates the co-selection of cryptic enhancer sequences by SOX2 and PAX6 transcription factors
Š The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research. Sox2 and Pax6 are transcription factors that direct cell fate decision during neurogenesis, yet the mechanism behind how they cooperate on enhancer DNA elements and regulate gene expression is unclear. By systematically interrogating Sox2 and Pax6 interaction on minimal enhancer elements, we found that cooperative DNA recognition relies on combinatorial nucleotide switches and precisely spaced, but cryptic composite DNA motifs. Surprisingly, all tested Sox and Pax paralogs have the capacity to cooperate on such enhancer elements. NMR and molecular modeling reveal very few direct protein-protein interactions between Sox2 and Pax6, suggesting that cooperative binding is mediated by allosteric interactions propagating through DNA structure. Furthermore, we detected and validated several novel sites in the human genome targeted cooperatively by Sox2 and Pax6. Collectively, we demonstrate that Sox- Pax partnerships have the potential to substantially alter DNA target specificities and likely enable the pleiotropic and context-specific action of these cell-lineage specifiers.Link_to_subscribed_fulltex
Dissecting the role of distinct OCT4-SOX2 heterodimer configurations in pluripotency
The transcription factors OCT4 and SOX2 are required for generating induced pluripotent stem cells (iPSCs) and for maintaining embryonic stem cells (ESCs). OCT4 and SOX2 associate and bind to DNA in different configurations depending on the arrangement of their individual DNA binding elements. Here we have investigated the role of the different OCT4-SOX2-DNA assemblies in regulating and inducing pluripotency. To this end, we have generated SOX2 mutants that interfere with specific OCT4-SOX2 heterodimer configurations and assessed their ability to generate iPSCs and to rescue ESC self-renewal. Our results demonstrate that the OCT4-SOX2 configuration that dimerizes on a Hoxb1-like composite, a canonical element with juxtaposed individual binding sites, plays a more critical role in the induction and maintenance of pluripotency than any other OCT4-SOX2 configuration. Overall, the results of this study provide new insight into the protein interactions required to establish a de novo pluripotent network and to maintain a true pluripotent cell fate.Link_to_subscribed_fulltex
The ubiquitination landscape of the influenza A virus polymerase.
During influenza A virus (IAV) infections, viral proteins are targeted by cellular E3 ligases for modification with ubiquitin. Here, we decipher and functionally explore the ubiquitination landscape of the IAV polymerase proteins during infection of human alveolar epithelial cells by applying mass spectrometry analysis of immuno-purified K-Îľ-GG (di-glycyl)-remnant-bearing peptides. We have identified 59 modified lysines across the three subunits, PB2, PB1Â and PA of the viral polymerase of which 17 distinctively affect mRNA transcription, vRNA replication and the generation of recombinant viruses via non-proteolytic mechanisms. Moreover, further functional and in silico analysis indicate that ubiquitination at K578 in the PB1 thumb domain is mechanistically linked to dynamic structural transitions of the viral polymerase that are required for vRNA replication. Mutations K578A and K578R differentially affect the generation of recombinant viruses by impeding cRNA and vRNA synthesis, NP binding as well as polymerase dimerization. Collectively, our results demonstrate that the ubiquitin-mediated charge neutralization at PB1-K578 disrupts the interaction to an unstructured loop in the PB2 N-terminus that is required to coordinate polymerase dimerization and facilitate vRNA replication. This provides evidence that IAV exploits the cellular ubiquitin system to modulate the activity of the viral polymerase for viral replication
Changing POU dimerization preferences converts Oct6 into a pluripotency inducer
� 2016 The Authors. Published under the terms of the CC BY 4.0 license The transcription factor Oct4 is a core component of molecular cocktails inducing pluripotent stem cells (iPSCs), while other members of the POU family cannot replace Oct4 with comparable efficiency. Rather, group III POU factors such as Oct6 induce neural lineages. Here, we sought to identify molecular features determining the differential DNA-binding and reprogramming activity of Oct4 and Oct6. In enhancers of pluripotency genes, Oct4 cooperates with Sox2 on heterodimeric SoxOct elements. By re-analyzing ChIP-Seq data and performing dimerization assays, we found that Oct6 homodimerizes on palindromic OctOct more cooperatively and more stably than Oct4. Using structural and biochemical analyses, we identified a single amino acid directing binding to the respective DNA elements. A change in this amino acid decreases the ability of Oct4 to generate iPSCs, while the reverse mutation in Oct6 does not augment its reprogramming activity. Yet, with two additional amino acid exchanges, Oct6 acquires the ability to generate iPSCs and maintain pluripotency. Together, we demonstrate that cell type-specific POU factor function is determined by select residues that affect DNA-dependent dimerization.Link_to_subscribed_fulltex
The Relationship of Cytokines IL-13 and IL-17 with Autoantibodies Profile in Early Rheumatoid Arthritis
Aims. In the present study, we aimed to assess the concentrations of IL-13 and IL-17 in serum of patients with early rheumatoid arthritis (eRA), the investigation of correlation between the concentrations of these cytokines and disease activity score, and the concentration of some autoantibodies and the evaluation of the utility of IL-13 and -17 concentration measurements as markers of disease activity. Materials and Methods. Serum samples were collected from 30 patients and from 28 controls and analysed parameters. Results. The serum concentrations of IL-13, IL-17, anti-CCP, and IgM-RF were statistically significantly higher in patients with eRA, compared to the controls. IL-13 concentrations in the severe and moderate groups with eRA were statistically higher than in the mild and control groups. Also, in the case of IL-17, serum concentrations increased proportionally with the disease activity of eRA. We observe that concentrations of IL-13 and -17 did not correlate with autoantibodies. IL-17 concentration significantly positively correlated with CRP, while IL-13 concentration significantly negatively correlated with CRP. Disease activity score, DAS28, was strongly positively correlated with levels of ESR and weakly positively correlated with concentrations of anti-RA33 autoantibodies. IL-13 has a higher diagnostic utility than IL-17, CRP, ESR, IgM-RF, and anti-CCP as markers of disease activity. Conclusions. The presence of higher IL-13 and IL-17 serum levels in patients, compared with those of controls, confirms that these markers, found with high specificity, might be involved in the pathogenesis of eRA. IL-13 and IL-17 might be of better usefulness in the prediction of eRA activity status than IgM-RF and anti-CCP
Nerve compression due to benign tumors or ganglion cysts in the upper limb â case series
Tumor nerve compressions in the upper limb are relatively rare, usually involving ganglion cysts and benign tumors. We present a case series of five patients with peripheral nerve compression in the upper limb due to tumor or cystic masses- ulnar nerve compression in the Guyonâs tunnel due to a ganglion cyst, large median nerve schwannoma compressing anterior interosseous nerve and median nerve, voluminous lipoma compressing median nerve in the proximal forearm, superficial branch of radial nerve compression by a synovial cyst and elbow region lipoma compressing radial nerve. In the beginning, those benign lesions are asymptomatic but, as they continue to grow adjacent to a peripheral nerve clinical manifestations appear progressively as compressive neuropathies. After a preoperative imagistic analysis, tumor resection with careful dissection, in order to preserve the neurovascular structures, is the elective surgical procedure in order to obtain an optimal functional recovery
Toponymes et appellatifs gĂŠographiques
Cojocaru Vlad. Toponymes et appellatifs gÊographiques. In: Nouvelle revue d'onomastique, n°47-48, 2007. pp. 185-197
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