Graded inhibition of oncogenic Ras-signaling by multivalent Ras-binding domains

BACKGROUND: Ras is a membrane-associated small G-protein that funnels growth and differentiation signals into downstream signal transduction pathways by cycling between an inactive, GDP-bound and an active, GTP-bound state. Aberrant Ras activity as a result of oncogenic mutations causes de novo cell transformation and promotes tumor growth and progression. RESULTS: Here, we describe a novel strategy to block deregulated Ras activity by means of oligomerized cognate protein modules derived from the Ras-binding domain of c-Raf (RBD), which we named MSOR for multivalent scavengers of oncogenic Ras. The introduction of well-characterized mutations into RBD was used to adjust the affinity and hence the blocking potency of MSOR towards activated Ras. MSOR inhibited several oncogenic Ras-stimulated processes including downstream activation of Erk1/2, induction of matrix-degrading enzymes, cell motility and invasiveness in a graded fashion depending on the oligomerization grade and the nature of the individual RBD-modules. The amenability to accurate experimental regulation was further improved by engineering an inducible MSOR-expression system to render the reversal of oncogenic Ras effects controllable. CONCLUSION: MSOR represent a new tool for the experimental and possibly therapeutic selective blockade of oncogenic Ras signals

Entwicklung von multivalenten Sonden zur gezielten Analyse und Beeinflussung Ras-abhängiger Signalwege

Die kleine GTPase Ras ist ein zentrales Element bei der Regulation vieler zellulärer Prozesse. Ras ist über Lipidanker in Membranen eingebettet und wird intrazellulär durch das Zusammenspiel von GEF und GAP aktiviert bzw. deaktiviert. Aktiviertes, d.h. GTP-beladenes Ras rekrutiert diverse Effektorproteine über deren Ras-bindende Domäne (RBD) an die Membran, wo diese Proteine aktiviert werden und dann die biologischen Aktivitäten von Ras vermitteln. Aufgrund seiner initialen Rolle bei der zellulären Transformation und Tumorigenese wurden verschiedene Ansätze entwickelt, um die Funktionalität von Ras zu unterdrücken, und um insbesondere der transformierenden Wirkung infolge einer deregulierten Ras-Aktivität entgegenzuwirken. Dabei zielen bisherige Ansätze vorwiegend auf die Suppression der Expression oder der Prozessierung des Proteins ab. In dieser Arbeit wird die Etablierung eines neuartigen Ansatzes beschrieben, der es erlaubt die Aktivität des vollständig prozessierten, reifen Ras-Proteins zu modulieren. Hierfür wurden RBD des Ras-Effektors Raf-1 zu Oligomeren aus zwei bzw. drei Domänen verknüpft. Um die Variabilität dieser Konstrukte zu erhöhen, wurden Oligomere bestehend aus der wildtypischen oder mutierten RBD, die eine abgeschwächte Affinität gegenüber Ras-GTP aufweisen, entwickelt. Diese Konstrukte werden zusammenfassend als multivalent scavengers of oncogenic Ras(MSOR) bezeichnet. Durch in vitroVersuche mit rekombinant hergestellten MSOR konnte gezeigt werden, dass die mutierten Varianten eine geringere Affinität für aktives Ras besitzen als die wildtypischen. Diese Versuche demonstrieren aber vor allem, dass die Stärke der Bindung an Ras-GTP mit der Zahl der jeweils miteinander verknüpften Bindedomänen zunimmt, wodurch die Interaktion stabilisiert wird. Die Wechselwirkung von MSOR und Ras ließ sich auch in vivo durch Expression fluoreszenzmarkierter Varianten (eGFP-MSOR) demonstrieren. Durch ektopische Expression verschiedener Ras-Varianten in Cos-7 Zellen konnte die GTP-spezifische Interaktion der eGFP-MSOR mit verschiedenen Mitgliedern der Ras-Subfamilie einschließlich der Ras-Isoformen nachgewiesen werden. Die Modulation der Ras-Aktivität durch MSOR wurde nach Koexpression von MSOR und Ras in verschiedenen Zelllinien durch die Analyse ausgewählter Ras-vermittelter zellulärer Prozesse studiert. Es wurde gezeigt, dass das wildtypische trimere MSOR-Konstrukt (eGFP-WT-3) die Ras(G12V)-vermittelte ERK2-Phosphorylierung und MMP1-Genexpression deutlich supprimieren kann. In Übereinstimmung mit der blockierenden Wirkung der MSOR konnte auch die Invasivität sowie das substratunabhängige Wachstum im Soft-Agar mit eGFP-WT-3 unterdrückt werden. Die Befunde korrelieren mit der Fähigkeit dieses MSOR, einerseits die Ras(G12V)-vermittelte Genexpression matrix-degradierender Enzyme wie Cathepsin L oder uPA aufzuheben und andererseits die Expression pro-apoptotischer Faktoren zu fördern. In diesem Zusammenhang wurde zudem beobachtet, dass die Expression oligomerer wildtypischer MSOR in Cos-7 Zellen zur Apoptose dieser Zellen führt. Die Wirksamkeit der MSOR in den Transformationsassays verdeutlicht deren inhibitorisches Potential gegenüber Ras-vermittelten Effekten. Viele der publizierten Untersuchungen zur Visualisierung der Ras-Aktivität erfolgten ausschließlich unter Bedingungen der Ras-Überexpression. Dies führt jedoch zu unphysiologischen Effekten, weshalb die Inzidenz einiger Befunde für die in vivoSituation fraglich ist. Ein besonders wichtiges Ergebnis dieser Arbeit ist der Nachweis, dass MSOR aufgrund ihrer effektiven Bindung an Ras-GTP auch als Reporter endogener Ras-Aktivität eingesetzt werden können. Nach Expression von eGFP-R59A,N64D-3 konnte die EGF- und TPA-induzierte Aktivierung des endogenen Ras anhand der Translokation des Reporters mittels konfokaler Mikroskopie in Echtzeit verfolgt werden. Das endogene Ras wird nach Stimulation nahezu ausschließlich an der Plasmamembran aktiviert. Die in der Literatur postulierte stimulus-abhängige Aktivierung von heterolog exprimiertem Ras im perinuklären Bereich, respektive Golgi-Apparat oder in Endosomen konnte hingegen nicht beobachtet werden. Da eGFP-R59A,N64D-3 ein sensitiver Detektor für endogenes Ras ist, belegt dieses Ergebnis die unterschiedliche räumliche Aktivierung von endogenem gegenüber überexprimiertem Ras. Die entwickelten MSOR-Konstrukte bieten ein Spektrum neuartiger Moleküle, mit denen sich zum einen die Ras-Aktivierung in vivo verfolgen lässt, die anderseits aber auch wirksam mit Ras-spezifischen Effekten interferieren können. Durch den modularen Aufbau der Konstrukte aus einzelnen Domänen, deren Affinität für Ras-GTP variiert werden kann, wird eine zielorientierte Anwendung der MSOR möglich

Measurement of the photon+b+b-jet production differential cross section in ppˉp\bar{p} collisions at \sqrt{s}=1.96~\TeV

We present measurements of the differential cross section dsigma/dpT_gamma for the inclusive production of a photon in association with a b-quark jet for photons with rapidities |y_gamma|< 1.0 and 30<pT_gamma <300 GeV, as well as for photons with 1.5<|y_gamma|< 2.5 and 30< pT_gamma <200 GeV, where pT_gamma is the photon transverse momentum. The b-quark jets are required to have pT>15 GeV and rapidity |y_jet| < 1.5. The results are based on data corresponding to an integrated luminosity of 8.7 fb^-1, recorded with the D0 detector at the Fermilab Tevatron $p\bar{p}$ Collider at sqrt(s)=1.96 TeV. The measured cross sections are compared with next-to-leading order perturbative QCD calculations using different sets of parton distribution functions as well as to predictions based on the kT-factorization QCD approach, and those from the Sherpa and Pythia Monte Carlo event generators.Comment: 10 pages, 9 figures, submitted to Phys. Lett.

Search for a Narrow ttbar Resonance in ppbar Collisions at sqrt{s}=1.96 TeV

We report a search for a narrow ttbar resonance that decays into a lepton+jets final state based on an integrated luminosity of 5.3/fb of proton-antiproton collisions at sqrt{s}=1.96 TeV collected by the D0 Collaboration at the Fermilab Tevatron Collider. We set upper limits on the production cross section of such a resonance multiplied by its branching fraction to ttbar which we compare to predictions for a leptophobic topcolor Z' boson. We exclude such a resonance at the 95% confidence level for masses below 835 GeV.Comment: 7 pages, 3 figures, submitted to Physical Review Letter

Measurement of the inclusive and dijet cross-sections of b-jets in pp collisions at sqrt(s) = 7 TeV with the ATLAS detector

The inclusive and dijet production cross-sections have been measured for jets containing b-hadrons (b-jets) in proton-proton collisions at a centre-of-mass energy of sqrt(s) = 7 TeV, using the ATLAS detector at the LHC. The measurements use data corresponding to an integrated luminosity of 34 pb^-1. The b-jets are identified using either a lifetime-based method, where secondary decay vertices of b-hadrons in jets are reconstructed using information from the tracking detectors, or a muon-based method where the presence of a muon is used to identify semileptonic decays of b-hadrons inside jets. The inclusive b-jet cross-section is measured as a function of transverse momentum in the range 20 < pT < 400 GeV and rapidity in the range |y| < 2.1. The bbbar-dijet cross-section is measured as a function of the dijet invariant mass in the range 110 < m_jj < 760 GeV, the azimuthal angle difference between the two jets and the angular variable chi in two dijet mass regions. The results are compared with next-to-leading-order QCD predictions. Good agreement is observed between the measured cross-sections and the predictions obtained using POWHEG + Pythia. MC@NLO + Herwig shows good agreement with the measured bbbar-dijet cross-section. However, it does not reproduce the measured inclusive cross-section well, particularly for central b-jets with large transverse momenta.Comment: 10 pages plus author list (21 pages total), 8 figures, 1 table, final version published in European Physical Journal

Search for squarks and gluinos with the ATLAS detector in final states with jets and missing transverse momentum using √s=8 TeV proton-proton collision data

A search for squarks and gluinos in final states containing high-p T jets, missing transverse momentum and no electrons or muons is presented. The data were recorded in 2012 by the ATLAS experiment in s√=8 TeV proton-proton collisions at the Large Hadron Collider, with a total integrated luminosity of 20.3 fb−1. Results are interpreted in a variety of simplified and specific supersymmetry-breaking models assuming that R-parity is conserved and that the lightest neutralino is the lightest supersymmetric particle. An exclusion limit at the 95% confidence level on the mass of the gluino is set at 1330 GeV for a simplified model incorporating only a gluino and the lightest neutralino. For a simplified model involving the strong production of first- and second-generation squarks, squark masses below 850 GeV (440 GeV) are excluded for a massless lightest neutralino, assuming mass degenerate (single light-flavour) squarks. In mSUGRA/CMSSM models with tan β = 30, A 0 = −2m 0 and μ > 0, squarks and gluinos of equal mass are excluded for masses below 1700 GeV. Additional limits are set for non-universal Higgs mass models with gaugino mediation and for simplified models involving the pair production of gluinos, each decaying to a top squark and a top quark, with the top squark decaying to a charm quark and a neutralino. These limits extend the region of supersymmetric parameter space excluded by previous searches with the ATLAS detector

Search for direct production of charginos and neutralinos in events with three leptons and missing transverse momentum in √s = 7 TeV pp collisions with the ATLAS detector

A search for the direct production of charginos and neutralinos in final states with three electrons or muons and missing transverse momentum is presented. The analysis is based on 4.7 fb−1 of proton–proton collision data delivered by the Large Hadron Collider and recorded with the ATLAS detector. Observations are consistent with Standard Model expectations in three signal regions that are either depleted or enriched in Z-boson decays. Upper limits at 95% confidence level are set in R-parity conserving phenomenological minimal supersymmetric models and in simplified models, significantly extending previous results

Measurement of the cross-section and charge asymmetry of WW bosons produced in proton-proton collisions at s=8\sqrt{s}=8 TeV with the ATLAS detector

This paper presents measurements of the $W^+ \rightarrow \mu^+\nu$ and $W^- \rightarrow \mu^-\nu$ cross-sections and the associated charge asymmetry as a function of the absolute pseudorapidity of the decay muon. The data were collected in proton--proton collisions at a centre-of-mass energy of 8 TeV with the ATLAS experiment at the LHC and correspond to a total integrated luminosity of 20.2~\mbox{fb^{-1}}. The precision of the cross-section measurements varies between 0.8% to 1.5% as a function of the pseudorapidity, excluding the 1.9% uncertainty on the integrated luminosity. The charge asymmetry is measured with an uncertainty between 0.002 and 0.003. The results are compared with predictions based on next-to-next-to-leading-order calculations with various parton distribution functions and have the sensitivity to discriminate between them.Comment: 38 pages in total, author list starting page 22, 5 figures, 4 tables, submitted to EPJC. All figures including auxiliary figures are available at https://atlas.web.cern.ch/Atlas/GROUPS/PHYSICS/PAPERS/STDM-2017-13

Measurement of the production of a W boson in association with a charm quark in pp collisions at √s = 7 TeV with the ATLAS detector

The production of a W boson in association with a single charm quark is studied using 4.6 fb−1 of pp collision data at s√ = 7 TeV collected with the ATLAS detector at the Large Hadron Collider. In events in which a W boson decays to an electron or muon, the charm quark is tagged either by its semileptonic decay to a muon or by the presence of a charmed meson. The integrated and differential cross sections as a function of the pseudorapidity of the lepton from the W-boson decay are measured. Results are compared to the predictions of next-to-leading-order QCD calculations obtained from various parton distribution function parameterisations. The ratio of the strange-to-down sea-quark distributions is determined to be 0.96+0.26−0.30 at Q 2 = 1.9 GeV2, which supports the hypothesis of an SU(3)-symmetric composition of the light-quark sea. Additionally, the cross-section ratio σ(W + +c¯¯)/σ(W − + c) is compared to the predictions obtained using parton distribution function parameterisations with different assumptions about the s−s¯¯¯ quark asymmetry

Inclusive search for same-sign dilepton signatures in pp collisions at root s=7 TeV with the ATLAS detector

An inclusive search is presented for new physics in events with two isolated leptons (e or mu) having the same electric charge. The data are selected from events collected from p p collisions at root s = 7 TeV by the ATLAS detector and correspond to an integrated luminosity of 34 pb(-1). The spectra in dilepton invariant mass, missing transverse momentum and jet multiplicity are presented and compared to Standard Model predictions. In this event sample, no evidence is found for contributions beyond those of the Standard Model. Limits are set on the cross-section in a fiducial region for new sources of same-sign high-mass dilepton events in the ee, e mu and mu mu channels. Four models predicting same-sign dilepton signals are constrained: two descriptions of Majorana neutrinos, a cascade topology similar to supersymmetry or universal extra dimensions, and fourth generation d-type quarks. Assuming a new physics scale of 1 TeV, Majorana neutrinos produced by an effective operator V with masses below 460 GeV are excluded at 95% confidence level. A lower limit of 290 GeV is set at 95% confidence level on the mass of fourth generation d-type quarks