Synthese und Evaluierung stabilisierter Bindungsepitope zur Adressierung kleiner GTPasen

Kleine GTPasen dienen als molekulare Schalter bei der Kontrolle zahlreicher wichtiger zellulärer Prozesse. Die fehlerhafte Funktion kleiner GTPasen steht daher in Verbindung mit der Entstehung und Entwicklung diverser Erkrankungen. Allerdings konnte diese Proteinklasse mit konventionellen Methoden nur äußerst schwer direkt adressiert werden, da ihre Regulierung und biologische Signalwirkung über ausgedehnte und flache Protein‒Protein-Interaktionen (PPIs) vermittelt werden. Um Verbindungen zu generieren, die an kleine GTPasen binden und potentiell ihre Signalwirkung modulieren können, wurden deshalb im Rahmen dieser Arbeit Bindungsepitope natürlicher Interaktionspartner konformativ stabilisiert. Auf Basis struktureller Daten von GTPase‒Protein-Komplexen wurden dazu geeignete Peptidsequenzen identifiziert und chemisch modifiziert. Im ersten Teil der Arbeit wurden Ras-Proteine, die prominenteste Unterfamilie kleiner GTPasen, adressiert. Basierend auf einem α-helikalen Bindungsmotiv des Nukleotidaustauschfaktors SOS wurden Peptide mit α-methylierter Kohlenwasserstoffverbrückung (hydrocarbon stapled peptides) entwickelt, während eine vom Effektor c-Raf abgeleitete β-Haarnadel mittels unterschiedlicher Verbrückungsstrategien stabilisiert wurde. Aufgrund der sehr geringen Affinität der Ausgangssequenzen wurden jedoch keine Derivate erhalten, die für eine Anwendung als PPI-Inhibitoren geeignet wären. Im zweiten Teil der Arbeit wurde in einer breit angelegten Machbarkeitsstudie eine analoge Strategie für die größte Unterfamilie kleiner GTPasen, sog. Rab-Proteine, verfolgt. Ausgehend von diversen α-helikalen Bindungsmotiven wurde eine Bibliothek von stapled peptides generiert, die sich im Vergleich zu den Ausgangssequenzen durch deutlich gesteigerte Affinitäten auszeichneten. Obgleich die Peptide keinen Einfluss auf die GEF-vermittelte Nukleotidaustauschrate von Rab-GTPasen zeigten, konnte ein Peptid identifiziert werden, das selektiv an die aktivierte Form von Rab8a bindet und in der Lage ist, eine Rab‒Effektor-Wechselwirkung in vitro zu inhibieren. Abschließend konnte durch Variation der Verbrückungsarchitektur die Affinität dieses Peptids weiter gesteigert werden.Small GTPases work as molecular switches inside cells and play a key role in the control of numerous crucial cellular processes. The aberrant function of small GTPases has therefore been associated with a variety of different diseases. However, the direct targeting of small GTPases by means of conventional small molecular approaches has turned out to be extremely challenging as GTPase regulation and signaling is mediated by shallow and extended protein‒protein-interaction surfaces. In this thesis stabilized peptide binding epitopes derived from protein interaction sites of natural interaction partners were explored as modulators of GTPase signaling. Based on structural data of GTPase‒protein complexes suitable peptide sequences were selected and chemically modified. The first part of this thesis explored this strategy for Ras proteins, the most prominent subfamily of small GTPases. Based on an α-helix of the nucleotide exchange factor SOS a small library of hydrocarbon stapled peptides was generated, while a c-Raf derived β-hairpin was stabilized using different types of cross-links. However, none of the peptide derivative showed sufficient target affinity, probably due to very poor affinities of the corresponding wild type sequences. In the second part of the thesis the same approach was applied to Rab proteins, the largest subfamily of small GTPases. A library of hydrocarbon stapled peptides was designed based on a variety α-helical binding epitopes from different Rab interaction partners. These peptides showed significantly increased affinities. Though no influence on the GEF-catalyzed nucleotide exchange was observed, one of the stapled peptides binds selectively to activated Rab8a and inhibits a Rab8a‒effector-interaction in vitro. Target affinity was further improved by variation of the stapling architecture

Chemical profiling of DNA G-quadruplex-interacting proteins in live cells.

DNA-protein interactions regulate critical biological processes. Identifying proteins that bind to specific, functional genomic loci is essential to understand the underlying regulatory mechanisms on a molecular level. Here we describe a co-binding-mediated protein profiling (CMPP) strategy to investigate the interactome of DNA G-quadruplexes (G4s) in native chromatin. CMPP involves cell-permeable, functionalized G4-ligand probes that bind endogenous G4s and subsequently crosslink to co-binding G4-interacting proteins in situ. We first showed the robustness of CMPP by proximity labelling of a G4 binding protein in vitro. Employing this approach in live cells, we then identified hundreds of putative G4-interacting proteins from various functional classes. Next, we confirmed a high G4-binding affinity and selectivity for several newly discovered G4 interactors in vitro, and we validated direct G4 interactions for a functionally important candidate in cellular chromatin using an independent approach. Our studies provide a chemical strategy to map protein interactions of specific nucleic acid features in living cells

DNA G-quadruplex structures mould the DNA methylome

Control of DNA methylation level is critical for gene regulation, and the factors that govern hypomethylation at CpG islands (CGIs) are still being uncovered. Here, we provide evidence that G-quadruplex (G4) DNA secondary structures are genomic features that influence methylation at CGIs. We show that the presence of G4 structure is tightly associated with CGI hypomethylation in the human genome. Surprisingly, we find that these G4 sites are enriched for DNA methyltransferase 1 (DNMT1) occupancy, which is consistent with our biophysical observations that DNMT1 exhibits higher binding affinity for G4s as compared to duplex, hemi-methylated or single-stranded DNA. The biochemical assays also show that the G4 structure itself, rather than sequence, inhibits DNMT1 enzymatic activity. Based on these data, we propose that G4 formation sequesters DNMT1 thereby protecting certain CGIs from methylation and inhibiting local methylation.This work is supported by a core CRUK award (C14303/A17197). S.B. is a Senior Investigator of the Wellcome Trust (grant no. 099232/z/12/z). JS is a Marie Curie Fellow of the European Union (747297-QAPs-H2020-MSCA-IF-2016)

G-quadruplexes are transcription factor binding hubs in human chromatin

Abstract: Background: The binding of transcription factors (TF) to genomic targets is critical in the regulation of gene expression. Short, double-stranded DNA sequence motifs are routinely implicated in TF recruitment, but many questions remain on how binding site specificity is governed. Results: Herein, we reveal a previously unappreciated role for DNA secondary structures as key features for TF recruitment. In a systematic, genome-wide study, we discover that endogenous G-quadruplex secondary structures (G4s) are prevalent TF binding sites in human chromatin. Certain TFs bind G4s with affinities comparable to double-stranded DNA targets. We demonstrate that, in a chromatin context, this binding interaction is competed out with a small molecule. Notably, endogenous G4s are prominent binding sites for a large number of TFs, particularly at promoters of highly expressed genes. Conclusions: Our results reveal a novel non-canonical mechanism for TF binding whereby G4s operate as common binding hubs for many different TFs to promote increased transcription

G-quadruplexes are transcription factor binding hubs in human chromatin

Abstract: Background: The binding of transcription factors (TF) to genomic targets is critical in the regulation of gene expression. Short, double-stranded DNA sequence motifs are routinely implicated in TF recruitment, but many questions remain on how binding site specificity is governed. Results: Herein, we reveal a previously unappreciated role for DNA secondary structures as key features for TF recruitment. In a systematic, genome-wide study, we discover that endogenous G-quadruplex secondary structures (G4s) are prevalent TF binding sites in human chromatin. Certain TFs bind G4s with affinities comparable to double-stranded DNA targets. We demonstrate that, in a chromatin context, this binding interaction is competed out with a small molecule. Notably, endogenous G4s are prominent binding sites for a large number of TFs, particularly at promoters of highly expressed genes. Conclusions: Our results reveal a novel non-canonical mechanism for TF binding whereby G4s operate as common binding hubs for many different TFs to promote increased transcription

Differential cross section measurements for the production of a W boson in association with jets in proton–proton collisions at √s = 7 TeV

Measurements are reported of differential cross sections for the production of a W boson, which decays into a muon and a neutrino, in association with jets, as a function of several variables, including the transverse momenta (pT) and pseudorapidities of the four leading jets, the scalar sum of jet transverse momenta (HT), and the difference in azimuthal angle between the directions of each jet and the muon. The data sample of pp collisions at a centre-of-mass energy of 7 TeV was collected with the CMS detector at the LHC and corresponds to an integrated luminosity of 5.0 fb[superscript −1]. The measured cross sections are compared to predictions from Monte Carlo generators, MadGraph + pythia and sherpa, and to next-to-leading-order calculations from BlackHat + sherpa. The differential cross sections are found to be in agreement with the predictions, apart from the pT distributions of the leading jets at high pT values, the distributions of the HT at high-HT and low jet multiplicity, and the distribution of the difference in azimuthal angle between the leading jet and the muon at low values.United States. Dept. of EnergyNational Science Foundation (U.S.)Alfred P. Sloan Foundatio

Optimasi Portofolio Resiko Menggunakan Model Markowitz MVO Dikaitkan dengan Keterbatasan Manusia dalam Memprediksi Masa Depan dalam Perspektif Al-Qur`an

Risk portfolio on modern finance has become increasingly technical, requiring the use of sophisticated mathematical tools in both research and practice. Since companies cannot insure themselves completely against risk, as human incompetence in predicting the future precisely that written in Al-Quran surah Luqman verse 34, they have to manage it to yield an optimal portfolio. The objective here is to minimize the variance among all portfolios, or alternatively, to maximize expected return among all portfolios that has at least a certain expected return. Furthermore, this study focuses on optimizing risk portfolio so called Markowitz MVO (Mean-Variance Optimization). Some theoretical frameworks for analysis are arithmetic mean, geometric mean, variance, covariance, linear programming, and quadratic programming. Moreover, finding a minimum variance portfolio produces a convex quadratic programming, that is minimizing the objective function ðð¥with constraintsð ð 𥠥 ðandð´ð¥ = ð. The outcome of this research is the solution of optimal risk portofolio in some investments that could be finished smoothly using MATLAB R2007b software together with its graphic analysis

Juxtaposing BTE and ATE – on the role of the European insurance industry in funding civil litigation

One of the ways in which legal services are financed, and indeed shaped, is through private insurance arrangement. Two contrasting types of legal expenses insurance contracts (LEI) seem to dominate in Europe: before the event (BTE) and after the event (ATE) legal expenses insurance. Notwithstanding institutional differences between different legal systems, BTE and ATE insurance arrangements may be instrumental if government policy is geared towards strengthening a market-oriented system of financing access to justice for individuals and business. At the same time, emphasizing the role of a private industry as a keeper of the gates to justice raises issues of accountability and transparency, not readily reconcilable with demands of competition. Moreover, multiple actors (clients, lawyers, courts, insurers) are involved, causing behavioural dynamics which are not easily predicted or influenced. Against this background, this paper looks into BTE and ATE arrangements by analysing the particularities of BTE and ATE arrangements currently available in some European jurisdictions and by painting a picture of their respective markets and legal contexts. This allows for some reflection on the performance of BTE and ATE providers as both financiers and keepers. Two issues emerge from the analysis that are worthy of some further reflection. Firstly, there is the problematic long-term sustainability of some ATE products. Secondly, the challenges faced by policymakers that would like to nudge consumers into voluntarily taking out BTE LEI