Molecular quantum spin network controlled by a single qubit

Scalable quantum technologies will require an unprecedented combination of precision and complexity for designing stable structures of well-controllable quantum systems. It is a challenging task to find a suitable elementary building block, of which a quantum network can be comprised in a scalable way. Here we present the working principle of such a basic unit, engineered using molecular chemistry, whose control and readout are executed using a nitrogen vacancy (NV) center in diamond. The basic unit we investigate is a synthetic polyproline with electron spins localized on attached molecular sidegroups separated by a few nanometers. We demonstrate the readout and coherent manipulation of very few ($\leq 6$) of these $S=1/2$ electronic spin systems and access their direct dipolar coupling tensor. Our results show, that it is feasible to use spin-labeled peptides as a resource for a molecular-qubit based network, while at the same time providing simple optical readout of single quantum states through NV-magnetometry. This work lays the foundation for building arbitrary quantum networks using well-established chemistry methods, which has many applications ranging from mapping distances in single molecules to quantum information processing.Comment: Author name typ

Alternative splicing of MALT1 controls signalling and activation of CD4+ T cells

MALT1 channels proximal T-cell receptor (TCR) signalling to downstream signalling pathways. With MALT1A and MALT1B two conserved splice variants exist and we demonstrate here that MALT1 alternative splicing supports optimal T-cell activation. Inclusion of exon7 in MALT1A facilitates the recruitment of TRAF6, which augments MALT1 scaffolding function, but not protease activity. Naive CD4+ T cells express almost exclusively MALT1B and MALT1A expression is induced by TCR stimulation. We identify hnRNP U as a suppressor of exon7 inclusion. Whereas selective depletion of MALT1A impairs T-cell signalling and activation, downregulation of hnRNP U enhances MALT1A expression and T-cell activation. Thus, TCR-induced alternative splicing augments MALT1 scaffolding to enhance downstream signalling and to promote optimal T-cell activation

IκBβ acts to inhibit and activate gene expression during the inflammatory response

The activation of pro-inflammatory gene programs by nuclear factor-κB (NF-κB) is primarily regulated through cytoplasmic sequestration of NF-κB by the inhibitor of κB (IκB) family of proteins1. IκBβ, a major isoform of IκB, can sequester NF-κB in the cytoplasm2, although its biological role remains unclear. Although cells lacking IκBβ have been reported3, 4, in vivo studies have been limited and suggested redundancy between IκBα and IκBβ5. Like IκBα, IκBβ is also inducibly degraded; however, upon stimulation by lipopolysaccharide (LPS), it is degraded slowly and re-synthesized as a hypophosphorylated form that can be detected in the nucleus6, 7, 8, 9, 10, 11. The crystal structure of IκBβ bound to p65 suggested this complex might bind DNA12. In vitro, hypophosphorylated IκBβ can bind DNA with p65 and c-Rel, and the DNA-bound NF-κB:IκBβ complexes are resistant to IκBα, suggesting hypophosphorylated, nuclear IκBβ may prolong the expression of certain genes9, 10, 11. Here we report that in vivo IκBβ serves both to inhibit and facilitate the inflammatory response. IκBβ degradation releases NF-κB dimers which upregulate pro-inflammatory target genes such as tumour necrosis factor-α (TNF-α). Surprisingly, absence of IκBβ results in a dramatic reduction of TNF-α in response to LPS even though activation of NF-κB is normal. The inhibition of TNF-α messenger RNA (mRNA) expression correlates with the absence of nuclear, hypophosphorylated-IκBβ bound to p65:c-Rel heterodimers at a specific κB site on the TNF-α promoter. Therefore IκBβ acts through p65:c-Rel dimers to maintain prolonged expression of TNF-α. As a result, IκBβ^(−/−) mice are resistant to LPS-induced septic shock and collagen-induced arthritis. Blocking IκBβ might be a promising new strategy for selectively inhibiting the chronic phase of TNF-α production during the inflammatory response

Regulation von Bcl10 und Malt1 durch Ubiquitinierung und SUMOylierung und deren Bedeutung für die NF-kappaB Aktivierung in Lymphozyten

Title page and table of contents 1\. Introduction 1 2\. Results 31 3\. Discussion 65 4\. Summary 87 5\. Materials 91 6\. Methods 102 7\. Abbreviations 119 8\. References 122 9\. Appendix 134Since T cells play a crucial role in orchestrating the immune response, a detailed knowledge of TCR signaling is pivotal for understanding aberrant T or B cell responses in autoimmunity or cancer. The transcription factor NF-kappaB triggers the expression of many target genes involved in antigen-specific proliferation, differentiation and survival of T cells, thus representing a central mediator of T cell activation. At a molecular level, the IkB kinase (IKK) complex represents the gatekeeper of NF-kappaB activation in many pathways. In T cells, formation of a Carma1/Bcl10/Malt1 (CBM) complex was shown to be essential for IKK activation. However, the molecular mechanisms, which connect CBM complex formation to IKK activation, had remained elusive. In the presented work Malt1 was identified as a novel target for regulatory ubiquitination in the course of TCR signaling. The ubiquitin ligase TRAF6 (TNF receptor associated factor 6) was shown to associate with Malt1 and to mediate Malt1 ubiquitination in vitro and in vivo by attachment of lysine 63-linked ubiquitin chains to multiple lysine residues in the Malt1 C-terminus. Through RNAi evidence and the finding that the TRAF6 binding sites in Malt1 are required for NF-kappaB and T cell activation, the presented work strongly supports a critical role for TRAF6 in TCR signaling to NF-kappaB. By reconstitution experiments using Malt1 deficient primary T cells, a crucial function for C-terminal Malt1 ubiquitination in NF-kappaB and T cell activation could be demonstrated. Finally, Malt1-attached ubiquitin chains were shown to provide the interaction surface for recruitment of the regulatory IKK complex component IKKgamma through its ubiquitin-binding domain. Furthermore, binding of ubiquitin chains by IKKgamma was demonstrated to be essential for TCR triggered NF-kB activation. Based on the presented findings, a new model for CBM dependent IKK activation can be suggested: Upon CBM complex formation, TRAF6 associates with the C-terminus of Malt1 and mediates ubiquitination of multiple lysines in the vicinity. IKKgamma is then recruited to ubiquitin-chains attached to Malt1, what could either induce IKK complex activation by induced proximity or by recruitment of an IKK activating kinase. Both mechanisms are not mutually exclusive. Thus, Malt1 ubiquitination represents the missing link between IKK and CBM complexes, thereby directing TCR proximal signals to IKK activation. In addition, a second, ubiquitin-like protein modification was investigated. It was demonstrated that Bcl10 can be a target of sumoylation and that attachment of SUMO occurs specifically at lysine 110. The presented data suggest an interplay between Bcl10 sumoylation and subcellular localization. Sumoylation is enhanced upon nuclear localization of Bcl10 and SUMO fusion to Bcl10 leads to its nuclear import and retention. Furthermore, Bcl10-SUMO fusion results in an enhanced NF-kappaB activation potential compared to wildtype Bcl10. Aberrant nuclear localization of Bcl10 is observed in many MALT lymphomas, which critically depend on constitutive NF-kappaB activation, and correlates with advanced stages. The molecular mechanisms underlying this phenomenon are poorly understood. Thus, sumoylation could represent a novel mechanism to mediate nuclear localization of Bcl10. Additionally, the data suggest that sumoylation of nuclear Bcl10 could influence NF-kappaB activity by a yet uncharacterized mechanism.Stimulation des T-Zell-Rezeptors (TZR) durch Pathogene führt zur Einleitung einer spezifischen Immunantwort, in deren Koordination T-Zellen eine zentrale Rolle einnehmen. Der Transkriptionsfaktor NF-kappaB reguliert eine Vielzahl von Zielgenen, die für Vermehrung, Differenzierung und Überleben von T-Zellen entscheidend sind, und stellt daher einen wichtigen Faktor für die T-Zell- Aktivierung dar. Auf molekularer Ebene repräsentiert der IkB Kinase (IKK) Komplex die zentrale Schaltstelle der NF-kappaB Signalkaskade. Für T-Zellen wurde gezeigt, dass die Formierung eines Carma1/Bcl10/Malt1 (CBM) Komplexes entscheidend für die Aktivierung des IKK Komplexes ist. Die molekularen Mechanismen, die eine Weiterleitung des Signals vom CBM zum IKK Komplex steuern, sind allerdings bisher weitgehend unverstanden. Im Zuge dieser Doktorarbeit wurde Malt1 als neues Substrat für regulative Ubiquitinierung in der TZR Signalkaskade identifiziert. Es konnte gezeigt werden, dass die Ubiquitin-Ligase TRAF6 (TNF receptor associated factor 6) mit Malt1 assoziiert und sowohl in vitro als auch in vivo die Ubiquitinierung von Malt1 vermitteln kann. Dabei werden mehrere Lysine im C-terminalen Teil von Malt1 mit über Lysin-63 vernetzten Ubiquitin-Ketten modifiziert. Der Befund, dass die TRAF6 Bindungsstellen in Malt1 für NF-kappaB und T-Zell-Aktivierung benötigt werden, weisen auf eine entscheidende Rolle von TRAF6 in der T-Zell-Rezeptor Signalkaskade hin. Rekonstitutionsexperimente mit primären T-Zellen konnten eine essentielle Funktion der C-terminalen Malt1 Ubiquitinierung für NF-kappaB und T-Zell Aktivierung nachweisen. Weiterhin wurde gezeigt, dass die an Malt1 gebundenen Ubiquitin-Ketten eine molekulare Interaktionsoberfläche für die Rekrutierung der regulatorischen IKK Komplex Untereinheit IKKgamma darstellen. Durch Mutation des Ubiquitin-Bindungsmotivs konnte demonstriert werden, dass diese Proteindomäne in IKKgamma für die NF-kappaB Aktivierung in T-Zellen essentiell ist. Basierend auf diesen Ergebnissen kann ein neues Modell für die CBM induzierte IKK Aktivierung vorgeschlagen werden: Nach Bildung des CBM Komplexes assoziiert TRAF6 mit dem C-Terminus von Malt1 und bewirkt die Ubiquitinierung von Lysinen in der Nähe der Bindungsstelle. Die gebildeten Ubiquitin-Ketten führen dann zu einer über das Ubiquitin-Bindungsmotiv in IKKgamma vermittelten Rekrutierung des IKK Komplexes. Dies könnte durch induzierte Nähe die Autoaktivierung des IKK Komplexes oder die Rekrutierung einer IKK aktivierenden Kinase verursachen. Die Ubiquitinierung von Malt1 stellt folglich die bisher unbekannte Verbindung zwischen CBM und IKK Komplexen dar und leitet so vom TZR initiierte Signale an den IKK Komplex weiter. Zusätzlich wurde gezeigt, dass Bcl10 durch Sumoylierung an Lysin 110 modifiziert werden kann. Die präsentierten Daten belegen ein Zusammenspiel von Sumoylierung und intrazellulärer Lokalisation von Bcl10, wobei eine nukleäre Bcl10 Lokalisation die Sumoylierung fördert. Eine Fusion von SUMO und Bcl10 wiederum resultiert in einer Akkumulation des Proteins im Zellkern und zu einer im Vergleich zu Bcl10 verstärkten NF-kappaB Aktivierung. Eine abnormale Lokalisation von Bcl10 im Zellkern wurde in vielen Malt Lymphomen beobachtet, für die konstitutive NF-kappaB Aktivität ist charakteristisch ist. Sumoylierung von Bcl10 könnte daher einen bisher unerkannten Mechanismus repräsentieren, der diese vermittelt, und die NF-kappaB Aktivität über einen noch unbekannten Mechanismus beeinflussen

Control of Expression of Key Cell Cycle Enzymes Drives Cell Line-Specific Functions of CDK7 in Human PDAC Cells

Inhibition of the dual function cell cycle and transcription kinase CDK7 is known to affect the viability of cancer cells, but the mechanisms underlying cell line-specific growth control remain poorly understood. Here, we employed a previously developed, highly specific small molecule inhibitor that non-covalently blocks ATP binding to CDK7 (LDC4297) to study the mechanisms underlying cell line-specific growth using a panel of genetically heterogeneous human pancreatic tumor lines as model system. Although LDC4297 diminished both transcription rates and CDK T-loop phosphorylation in a comparable manner, some PDAC lines displayed significantly higher sensitivity than others. We focused our analyses on two well-responsive lines (Mia-Paca2 and Panc89) that, however, showed significant differences in their viability upon extended exposure to limiting LDC4297 concentrations. Biochemical and RNAseq analysis revealed striking differences in gene expression and cell cycle control. Especially the downregulation of a group of cell cycle control genes, among them CDK1/2 and CDC25A/C, correlated well to the observed viability differences in Panc89 versus Mia-Paca2 cells. A parallel downregulation of regulatory pathways supported the hypothesis of a feedforward programmatic effect of CDK7 inhibitors, eventually causing hypersensitivity of PDAC lines

κB-Ras and Ral GTPases regulate acinar to ductal metaplasia during pancreatic adenocarcinoma development and pancreatitis

The molecular mechanisms of acinar-to-ductal metaplasia (ADM) in the course of pancreatitis and cancer development are unclear. Here, the authors show that loss of κB-Ras and consequent Ral activation promotes tumour initiation and progression through persistent ADM and enhanced cell proliferatio