655 research outputs found

    The Cytoplasmic Tail of the Notch Ligand Jagged-1: Intrinsic Disorder, Induced Order and Molecular Interactions

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    Notch signaling plays a key role in cell differentiation in all metazoans. As both receptors and ligands are cell-surface proteins, Notch signaling is restricted to nearby interacting cells. Notch ligands are membrane-spanning proteins made of a large extracellular region, a transmembrane segment, and a 100\u2013200 residue cytoplasmic tail. The sequence of the intracellular region of Jagged-1, one of the five ligands to Notch receptors in man, is very well conserved throughout evolution but does not encode any globular domain. The cytoplasmic tail of Jagged-1 mediates protein\u2013protein interactions through the C-terminal PDZ binding motif, is involved in ligand endocytosis triggered by mono-ubiquitination, and, as a consequence of regulated intramembrane proteolysis, can be released into the cytosol as a signaling fragment. The intracellular region of Jagged-1 may then exist in at least two forms: as a membrane-tethered protein located at the interface between the membrane and the cytoplasm, and as a soluble nucleocytoplasmic protein. To investigate its structural properties, a recombinant protein corresponding to the human Jagged-1 intracellular region (J1_tmic) was expressed, purified, and characterized in different environments using various biophysical methods such as circular dichroism, tryptophan fluorescence, size-exclusion chromatography, and NMR. In solution, J1_tmic behaves as an intrinsically disordered protein, but displays a significant helical propensity. In the presence of SDS micelles or negatively charged phospholipid micelles and vesicles, used to mimick the interface between the plasma membrane and the cytosol, J1_tmic gains partial helical structure. The partial folding and association of the intracellular region of Jagged-1 with the membrane is expected to reduce its \u201ccapture radius\u201d towards target proteins and to make selected residues unavailable for post-translational modifications or binding. Binding of Jagged-1 intracellular region to the PDZ domain of afadin, a protein located at cell-cell adherens junctions, couples Notch signaling with the adhesion system and the cytoskeleton. The interaction between the PDZ domain of afadin (AF6_PDZ) and a series of polypeptides comprising the Jagged-1 PDZ-binding motif (EYIV) was investigated using NMR chemical shift perturbation and surface plasmon resonance. It was shown that binding of Jagged-1 intracellular region to AF6_PDZ is strictly local, involving only the last six residues of the binding motif and the PDZ binding groove, and that it does not trigger global folding of J1_tmic. In the C-terminal region of Jagged-1 cytoplasmic tail, four potential phosphorylation sites can be identified, one of them (Y1216) located in the PDZbinding motif. It was found that, while phosphorylation at any of these sites disrupts binding of the C-terminal peptides to lipid micelles, phosphorylation at (Y1216) also affects the interaction with AF6_PDZ, with a reduction in the binding affinity. Phosphorylation thus provides a potential way to modulate the interaction of Jagged-1 C-terminal region not only with the membrane but also with the partner PDZ. It was also shown that the R1213Q mutation in the PDZ binding motif associated with a congenital obstruction of the bile ducts, increases the affinity for AF6_PDZ. In summary, this work presents the first biochemical and structural characterization of Jagged-1 cytoplasmic tail in solution and in environments that mimic the membrane/cytoplasm interface, and the first biophysical study on its interaction with the afadin PDZ domain

    Dimerization of the Notch Intracellular Domain Results in Distinct Signaling Activity

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    The Notch signaling pathway is a core component of multicellularity; enabling cells to directly communicate with both their neighbors and the surrounding microenvironment. These signals are translated directly through the Notch proteins, where a fragment of Notch transitions into the nucleus to act as a co-transcription factor, setting into motion a host of physiological responses. Commonly involved in pathways that define a cell’s identity and fate decisions, what appears to be a simplistic pathway instead exists in a state of high-tunability and strict control. Missteps in this pathway are generally embryonically lethal or lead to a suite of congenital disorders and cancers. Therefore, it’s pertinent to understand the mechanisms of Notch that provide its flexibility and pleiotropic outcomes. One such property is its ability to homodimerize on DNA while within its transcriptional activation complex, resulting in an enhanced transcriptional signal of a select pool of Notch target genes. This dissertation reviews the general mechanics behind Notch signaling, discusses how the field of Notch dimerization came to be and where it stands currently, and finally, details my contributions to the understanding of this regulatory mechanism. Despite Notch’s ubiquitous function in metazoan life, there are still many mysteries behind this signaling pathway. The work detailed here describes my time spent as a basic science researcher, where my findings contribute a couple of puzzle pieces to the expansive Notch signaling field

    Critical analysis and comparison of data protection techniques for genomics data sets

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    This work reviews the current literature on protecting genomic information. The goal is to provide insight on how to define a secure file format for such data. We compare the published ideas to the requirements defined by MPEG. We also propose new ideas to secure such data

    Toward a complexity theory for randomized search heuristics : black-box models

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    Randomized search heuristics are a broadly used class of general-purpose algorithms. Analyzing them via classical methods of theoretical computer science is a growing field. While several strong runtime bounds exist, a powerful complexity theory for such algorithms is yet to be developed. We contribute to this goal in several aspects. In a first step, we analyze existing black-box complexity models. Our results indicate that these models are not restrictive enough. This remains true if we restrict the memory of the algorithms under consideration. These results motivate us to enrich the existing notions of black-box complexity by the additional restriction that not actual objective values, but only the relative quality of the previously evaluated solutions may be taken into account by the algorithms. Many heuristics belong to this class of algorithms. We show that our ranking-based model gives more realistic complexity estimates for some problems, while for others the low complexities of the previous models still hold. Surprisingly, our results have an interesting game-theoretic aspect as well.We show that analyzing the black-box complexity of the OneMaxn function class—a class often regarded to analyze how heuristics progress in easy parts of the search space—is the same as analyzing optimal winning strategies for the generalized Mastermind game with 2 colors and length-n codewords. This connection was seemingly overlooked so far in the search heuristics community.Randomisierte Suchheuristiken sind vielseitig einsetzbare Algorithmen, die aufgrund ihrer hohen Flexibilität nicht nur im industriellen Kontext weit verbreitet sind. Trotz zahlreicher erfolgreicher Anwendungsbeispiele steckt die Laufzeitanalyse solcher Heuristiken noch in ihren Kinderschuhen. Insbesondere fehlt es uns an einem guten Verständnis, in welchen Situationen problemunabhängige Heuristiken in kurzer Laufzeit gute Lösungen liefern können. Eine Komplexitätstheorie ähnlich wie es sie in der klassischen Algorithmik gibt, wäre wünschenswert. Mit dieser Arbeit tragen wir zur Entwicklung einer solchen Komplexitätstheorie für Suchheuristiken bei. Wir zeigen anhand verschiedener Beispiele, dass existierende Modelle die Schwierigkeit eines Problems nicht immer zufriedenstellend erfassen. Wir schlagen daher ein weiteres Modell vor. In unserem Ranking-Based Black-Box Model lernen die Algorithmen keine exakten Funktionswerte, sondern bloß die Rangordnung der bislang angefragten Suchpunkte. Dieses Modell gibt für manche Probleme eine bessere Einschätzung der Schwierigkeit. Wir zeigen jedoch auch, dass auch im neuen Modell Probleme existieren, deren Komplexität als zu gering einzuschätzen ist. Unsere Ergebnisse haben auch einen spieltheoretischen Aspekt. Optimale Gewinnstrategien für den Rater im Mastermindspiel (auch SuperHirn) mit n Positionen entsprechen genau optimalen Algorithmen zur Maximierung von OneMaxn-Funktionen. Dieser Zusammenhang wurde scheinbar bislang übersehen. Diese Arbeit ist in englischer Sprache verfasst

    A novel genome-wide approach to identify in vivo targets of Notch signalling

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    The Notch signalling pathway regulates many developmental processes in metazoan embryos and adults such as cell proliferation, stem cell maintenance, cell fate specification and apoptosis. Despite the importance of this pathway, few targets have been identified, with the Hes (Hairy and Enhancer-of-split) protein family being the best-characterised group of downstream effectors. I have established transgenic mice carrying Biotin Acceptor Peptide (BAP)- tagged versions of Notch1. The tagged protein is fully functional and is biotinylated after crossing to mice expressing the biotinylase from E. coli. Biotinylation was confirmed in a range of different tissues. However, streptavidin chromatin pull-down (bioChIP) experiments from these tissues showed no significant enrichment of known Notch1 target sequences. A possible explanation could be the indirect and transient nature of the interaction between Notch, its DNA binding partner CSL and the promoter of the target gene. A transgenic mouse line expressing a BAP-tagged version of the transcription factor Hes7, a downstream effector of Notch signalling and key regulator of somitogenesis, was similarly generated. Although the tagged Hes7 protein is functional and gets biotinylated in cell culture assays, the transgenic mice exhibit a severe somite/skeletal phenotype indicating that the tagged allele is hypomorphic. A detailed analysis of the phenotype revealed differential axial requirements for Hes7

    DNAコンピューティングシステムの設計支援 : DNAツールボックスとその拡張

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    学位の種別:課程博士University of Tokyo(東京大学

    Unconstitutional Exploitation Of Delegated Authority: How To Deter Prosecutors From Using Substantial Assistance To Defeat The Intent Of Federal Sentencing Laws

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    This Comment addresses whether the intent of the federal sentencing system is defeated when prosecutors reward high-level drug offenders with lenient sentences in exchange for testimony against less culpable co-conspirators. This Comment argues that prosecutors violate separation-of-powers principles when they move for downward departures on behalf of kingpins who provide substantial assistance in a case against less culpable co-defendants because Congress did not authorize such an exercise of prosecutorial discretion. In such instances where the intent of Congress is defeated, the prosecutor is essentially making law and thereby encroaching upon the law-making function of Congress. To cure this constitutional abuse of prosecutorial discretion, the trial court should suppress the testimony of high-level conspirators pursuant to the exclusionary rule

    Unconstitutional Exploitation Of Delegated Authority: How To Deter Prosecutors From Using Substantial Assistance To Defeat The Intent Of Federal Sentencing Laws

    Get PDF
    This Comment addresses whether the intent of the federal sentencing system is defeated when prosecutors reward high-level drug offenders with lenient sentences in exchange for testimony against less culpable co-conspirators. This Comment argues that prosecutors violate separation-of-powers principles when they move for downward departures on behalf of kingpins who provide substantial assistance in a case against less culpable co-defendants because Congress did not authorize such an exercise of prosecutorial discretion. In such instances where the intent of Congress is defeated, the prosecutor is essentially making law and thereby encroaching upon the law-making function of Congress. To cure this constitutional abuse of prosecutorial discretion, the trial court should suppress the testimony of high-level conspirators pursuant to the exclusionary rule
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