On the TRAIL to Overcome BRAF-Inhibitor Resistance

BRAF inhibition has been an instant, although short-lasting, success in BRAF-mutated melanoma treatment. Novel data by Berger et al. now suggest that BRAF-inhibitor-mediated “priming to death” facilitates tumor necrosis factor–related apoptosis-inducing ligand–mediated apoptosis. We give an overview about the importance of the crosstalk of extrinsic and mitochondrial apoptotic signaling and propose other combination therapies that may prevent or overcome secondary resistance in melanoma

Cellular IAPs inhibit a cryptic CD95-induced cell death by limiting RIP1 kinase recruitment

cIAPs keep RIP1 from getting to the DISC complex and complex II; when cIAPs are repressed, signaling is modulated by the cFLIP isoform

A functional analysis of the pyrimidine catabolic pathway in Arabidopsis

Reductive catabolism of pyrimidine nucleotides occurs via a three-step pathway in which uracil is degraded to β-alanine, CO2 and NH3 through sequential activities of dihydropyrimidine dehydrogenase (EC 1.3.1.2, PYD1), dihydropyrimidinase (EC 3.5.2.2, PYD2) and β-ureidopropionase (EC 3.5.1.6, PYD3).A proposed function of this pathway, in addition to the maintenance of pyrimidine homeostasis, is the recycling of pyrimidine nitrogen to general nitrogen metabolism. PYD expression and catabolism of [2-14C]-uracil are markedly elevated in response to nitrogen limitation in plants, which can utilize uracil as a nitrogen source.PYD1, PYD2 and PYD3 knockout mutants were used for functional analysis of this pathway in Arabidopsis. pyd mutants exhibited no obvious phenotype under optimal growing conditions. pyd2 and pyd3 mutants were unable to catabolize [2-14C]-uracil or to grow on uracil as the sole nitrogen source. By contrast, catabolism of uracil was reduced by only 40% in pyd1 mutants, and pyd1 seedlings grew nearly as well as wild-type seedlings with a uracil nitrogen source. These results confirm PYD1 function and suggest the possible existence of another, as yet unknown, activity for uracil degradation to dihydrouracil in this plant.The localization of PYD-green fluorescent protein fusions in the plastid (PYD1), secretory system (PYD2) and cytosol (PYD3) suggests potentially complex metabolic regulation

cIAP1/2 Are Direct E3 Ligases Conjugating Diverse Types of Ubiquitin Chains to Receptor Interacting Proteins Kinases 1 to 4 (RIP1–4)

The RIP kinases have emerged as essential mediators of cellular stress that integrate both extracellular stimuli emanating from various cell-surface receptors and signals coming from intracellular pattern recognition receptors. The molecular mechanisms regulating the ability of the RIP proteins to transduce the stress signals remain poorly understood, but seem to rely only partially on their kinase activities. Recent studies on RIP1 and RIP2 have highlighted the importance of ubiquitination as a key process regulating their capacity to activate downstream signaling pathways. In this study, we found that XIAP, cIAP1 and cIAP2 not only directly bind to RIP1 and RIP2 but also to RIP3 and RIP4. We show that cIAP1 and cIAP2 are direct E3 ubiquitin ligases for all four RIP proteins and that cIAP1 is capable of conjugating the RIPs with diverse types of ubiquitin chains, including linear chains. Consistently, we show that repressing cIAP1/2 levels affects the activation of NF-κB that is dependent on RIP1, -2, -3 and -4. Finally, we identified Lys51 and Lys145 of RIP4 as two critical residues for cIAP1-mediated ubiquitination and NF-κB activation

A Non-Canonical Function of Zebrafish Telomerase Reverse Transcriptase Is Required for Developmental Hematopoiesis

Although it is clear that telomerase expression is crucial for the maintenance of telomere homeostasis, there is increasing evidence that the TERT protein can have physiological roles that are independent of this central function. To further examine the role of telomerase during vertebrate development, the zebrafish telomerase reverse transcriptase (zTERT) was functionally characterized. Upon zTERT knockdown, zebrafish embryos show reduced telomerase activity and are viable, but develop pancytopenia resulting from aberrant hematopoiesis. The blood cell counts in TERT-depleted zebrafish embryos are markedly decreased and hematopoietic cell differentiation is impaired, whereas other somatic lineages remain morphologically unaffected. Although both primitive and definitive hematopoiesis is disrupted by zTERT knockdown, the telomere lengths are not significantly altered throughout early development. Induced p53 deficiency, as well as overexpression of the anti-apoptotic proteins Bcl-2 and E1B-19K, significantly relieves the decreased blood cells numbers caused by zTERT knockdown, but not the impaired blood cell differentiation. Surprisingly, only the reverse transcriptase motifs of zTERT are crucial, but the telomerase RNA-binding domain of zTERT is not required, for rescuing complete hematopoiesis. This is therefore the first demonstration of a non-canonical catalytic activity of TERT, which is different from “authentic” telomerase activity, is required for during vertebrate hematopoiesis. On the other hand, zTERT deficiency induced a defect in hematopoiesis through a potent and specific effect on the gene expression of key regulators in the absence of telomere dysfunction. These results suggest that TERT non-canonically functions in hematopoietic cell differentiation and survival in vertebrates, independently of its role in telomere homeostasis. The data also provide insights into a non-canonical pathway by which TERT functions to modulate specification of hematopoietic stem/progenitor cells during vertebrate development. (276 words

Identification of Slfn-(Schlafen)-protein function and relevance in the cell cycle control and T cell ontogeny

Zusammenfassung 1\. Inhaltsverzeichniss I 2\. Einleitung 1 3\. Material 23 4\. Methoden 38 5\. Ergebnisse Teil 1 64 6\. Ergebnisse Teil 2 97 7\. Diskussion 136 8\. Literaturverzeichniss 170 9\. Anhang 182 10\. Danksagung 184 10\. ErklärungDie 10 Mitglieder der Slfn-Gen-Familie repräsentieren eine neue Gruppe von Proteinen mit bisher unbekannter Funktion. Die im Rahmen dieser Arbeit durchgeführten Analysen konnten einen neuen Einblick in die potentielle Bedeutung der individuellen Slfn-Proteine in der Zellzykluskontrolle und der T-Zellentwicklung geben. Die zu Beginn der Arbeit postulierte antiproliferative Aktivität der Slfn-Proteine ist keine generelle Aufgabe dieser Proteinfamilie. Die weitaus komplexere und Zelltyp-spezifische Funktionalität der verschiedenen Repräsentanten der Slfn-Protein-Familie ergibt sich: 1\. durch die unterschiedlichen Grössen der individuellen Slfn- Proteine und durch die Anzahl und Variationen der Proteindomänen am carboxy- terminalen Bereich, 2\. durch die differentielle subzelluläre Lokalisation in ektopisch Slfn-exprimierenden NIH3T3 Fibroblasten, 3\. durch die präferentielle Expression der slfn-Gene in hämatopoietischen Zelllinien, z. B. in T-Zellen, 4\. durch das differentielle Expressionsprofil der individuellen slfn-Gene in Thymozyten und T-Zellen während den verschiedenen Phasen der Entwicklung, Differenzierung und Aktivierung und 5\. durch die unterschiedlichen Auswirkungen einer ektopischen Slfn-Expression auf das Wachstum und dem Überleben von Fibroblasten, sowie auf die Entwicklung von hämatopoietischen Zelllinien, z. B. von T- und B-Zellen. Slfn1, das Gründungsmitglied der Slfn-Protein-Familie, dass zusammen mit dem Slfn2-Protein aus zwei Proteindomänen besteht und von dem präferentiell eine antiproliferative Aktivität beobachtet wurde, ist endogen in positiv selektierten CD4- und CD8-Thymozyten und in ruhenden peripheren CD4- und CD8-T-Zellen exprimiert. In zellulären Wachstumsphasen, z. B. in aktivierten T-Zellen, ist die Slfn1-Expression signifikant vermindert. Eine ektopische Expression des kompletten Slfn1-Proteins in proliferierenden Fibroblasten, die in ihrem Lebenszyklus kein endogenes Slfn1-Protein exprimieren, resultiert in einer ER-Stress-vermittelten Reduktion des zellulären Wachstums und in der Induktion des programmierten Zelltods. Der Grund für die Stressinduktion ist bislang nicht geklärt. Lediglich ein IRE1/Xbp-1-abhängiger Mechanismus wird eingeleitet, der für die transkriptionelle Erhöhung von BiP, ein potenter Gegenspieler der ER-Stressreaktion, und von CHOP, ein wichtiger Vermittler der Wachstumsinhibition und der Apoptoseinduktion, verantwortlich ist. Mechanistisch ist die ER-Stressreaktion durch regulative Elemente der Zellzykluskontrolle, z. B. mit p21CIP/WAF und mit p53, und folgend mit der Apoptose durch die proapoptotischen Proteine der Bcl-2-Familie, der Caspasen und der p38-Signalkaskade verbunden. Die transgene Slfn1-Expression im T- und B-Zellkompartiment beeinträchtigt deren Entwicklung nachhaltig, induziert Apoptose und vermindert die zelluläre Proliferation der DN-Thymozyten. Der Mechanismus der Slfn1-vermittelten Blockierung der T-Zellentwicklung im DN3-Thymozytenstadium ist unabhängig von den p53-regulierten Kontrollmechanismen und weiterhin ungeklärt. Die wenigen peripheren T-Zellen in der Slfn1-transgenen Maus expandieren wahrscheinlich durch homöostatische Proliferation, wodurch T-Zellen, präferentiell die CD8+-T-Zellen, mit einem Gedächtnis-ähnlichen Phänotyp entstehen. Ohne mitogene Stimulation sind diese T-Zellen ex vivo verstärkt apoptotisch. Die Funktionalität der peripheren T-Zellen wird durch das Slfn1-Transgen nicht eingeschränkt. Die TZR- vermittelte Aktivierung der peripheren Slfn1-transgenen T-Zellen führt ex vivo zur Hyperproliferation und zur verstärkten Produktion von Typ I-Zytokinen, z. B. von IL-2 und IFNg. Die T-Zell-vermittelte Immunantwort gegen Listerien wird durch die transgene Slfn1-Expression in den peripheren T-Zellen ebenfalls nicht beeinträchtigt. Die Funktion des Slfn1-Proteins und deren Bedeutung in der Zellzykluskontrolle und in der T-Zellontogenie sind zu diesem Zeitpunkt nicht eindeutig verstanden. Das aus drei Proteindomänen bestehende Slfn4-Protein ist endogen verstärkt in aktivierten T-Zellen exprimiert und lässt daher eine Wachstums-fördernde Funktion dieses Proteins vermuten. Das retroviral exprimierte Slfn4-Protein ist präferentiell im Zytoplasma lokalisiert und hat keine Auswirkungen auf das proliferative Verhalten von transduzierten Fibroblasten. Eine Beteiligung von Slfn4 an der Zellzykluskontrolle in diesen Fibroblasten ist demzufolge nicht offensichtlich. Die Entwicklung der intrathymischen T-Zellen und deren proliferative Expansion werden durch die kontinuierliche Slfn4-Expression in den transgenen Mäusen nicht beeinträchtigt. Eine mögliche Wachstums- inhibierende Funktion des Slfn4-Proteins ist durch die verminderte Proliferation in den peripheren T-Zellen der transgenen Tiere nicht auszuschliessen. Die Ursache für das verringerte T-Zellwachstum nach TZR- vermittelter Stimulation kann zu diesem Zeitpunkt nicht erklärt werden. Die Slfn-Proteine der dritten Untergruppe beinhalten als einzige Repräsentanten dieser Proteinfamilie funktionelle Motive, die möglicherweise auf eine enzymatische Aktivität als ATPase-abhängige RNA-Helikasen hinweisen. Als wahrscheinliche Modulatoren des RNA-Metabolismus besteht die Möglichkeit einer regulativen Beteiligung von Slfn5, Slfn8, Slfn9 und Slfn10 an der Zellzykluskontrolle. Die ektopische Expression der vier homologen Slfn- Proteine der dritten Untergruppe hat auf die retroviral transduzierten Fibroblasten keine Wachstums-modulierende Effekte. Die differentielle subzelluläre Lokalisation in transfizierten Fibroblastenzellen und das unterschiedliche Expressionsprofil in differenzierten und aktivierten T-Zellen lässt jedoch eine komplexe Zelltyp-spezifische Funktionalität dieser Protein in der Zellzykluskontrolle und der T-Zellontogenie vermuten. Eine fehlregulierte Slfn8-Expression im T-Zellkompartiment beeinträchtigt die intrathymischen Thymozytenentwicklung und vermindert die T-Zellproliferation, wodurch eine Beteiligung des Slfn8-Proteins an der Zellzykluskontrolle von T-Zellen sehr wahrscheinlich ist. Die Wirkungsmechanismen, die durch das transgene Slfn8-Protein beeinflusst werden und die molekularen Mechanismen, sowie die stimulatorischen Signale, die für die Induktion der endogenen Slfn- Expression benötigt werden, sind weitestgehend unverstanden. Die Identifizierung der funktionellen Bedeutung der Slfn-Proteine durch weiterführende Analysen könnte das molekulare Verständnis dieser Proteinfamilie für die hämatopoietische Zellentwicklung und �homöostase, sowie für die Zellzykluskontrolle, verbessern.The SLFN gene family consists of ten members, which represent a new group of proteins of so far unknown function. Analysis carried out during this thesis gave new insights into a possible function of individuals SLFN protein in cell cycle control and T-cell activation. The anti-proliferative activity of SLFN proteins, which was postulated for the founding member of this protein family, is not a general function of all cognates. The more complex and cell-specific function of the individual representatives of SLFN protein family is determined by: 1\. the different sizes of individual SLFN proteins and by the number and variety of protein domains in the carboxy-terminal region; 2\. differential subcellular localization in NIH3T3 fibroblasts ectopically expressing various SLFN proteins; 3\. the preferential expression of SLFN genes in haematopoietic cells, e. g. T-cells; 4\. the differential expression profile of individual SLFN genes in developing thymocytes and peripheral T-cells; 5\. differential effects of ectopic SLFN expression on growth and survival of fibroblasts and on the development of haematopoietic cells, e. g. T- and B-cells. Both SLFN1, the prototype of the SLFN protein family, and SLFN2, harbour two throughout the protein family conserved domains. SLFN1 has been shown to have an antiproliferative activity. It is strongly expressed in positively selected CD4 and CD8 thymocytes and in resting peripheral CD4 and CD8 T-cells. In activated T-cells, e. g. SLFN1 expression is significantly reduced. Ectopic expression of the full-length SLFN1 protein and proliferating fibroblasts, which so not express SLFN1 endogenously, results in ER-stress mediated reduction of cellular growth, and in induction of programmed cell death. The molecular events eliciting this stress response remains relusive. Consistant with the induction of the ER-stress related unfolded protein response, an IRE/Xbp-1 dependent mechanism is initiated. This leads to an increased expression of BiP, known as a possible antagonist of the ER-stress response, and CHOP, an important mediator of growth inhibition and induction of apoptosis. The ER-stress response is interconnected to regulatory elements of the cell cycle control (e. g. p21CIP/WAF and p53) and subsequentially to apoptotic mechanisms. The proapoptotic proteins of Bcl2 family, caspases and the p38 signal cascade are involved. Transgenic SLFN1 expression in the T- and B-cell compartments strongly compromises the development, induces apoptosis and probably impairs proliferation of immature DN thymocytes. The mechanism of the SLFN1 mediated block of the pre-T-cell development at the DN3 thymocyte stage is independent from p53 regulated control mechanisms. The few peripheral T-cell developing in SLFN1 transgenic mice seem to increase in numbers by homeostatic proliferation. Thus T-cells, preferentially CD8 T-cells, which memory like phenotype are generated. Without mitogenic stimulation these cells show a decreased survival potential. T-cell receptor stimulated SLFN1 transgenic T-cells are hyper-proliferative. Consistant is the increased production of type 1 cytokines, e. g. IL-2 and IFN-g, by these transgenic T-cells. The T-cell mediated immune response against the intracellular pathogen Listeria monocytogenes is likewise not affected by trangenic SLFN1 expression in peripheral T-cells. The function of the SLFN1 protein and its role in the cell cycle control and during T-cell ontogenesis are at present not clearly understood. Endogenous expression of the SLFN4 protein, which consists of 3 domains, is increased in activated T-cells. The retrovirally expressed SLFN4 protein is preferentially localized in the cytoplasm and has no effect on the proliferation of the fibroblasts. Therefore, an involvement of SLFN4 in cell cycle control in these cells is not apparent. The development of intrathymic T-cells and there proliferative expansion is not affected by expression of a SLFN4 transgene in mice. Based on the decreased proliferative potential of peripheric T-cells are potential growth regulatory influence of the SLFN4 protein cannot be ruled out. SLFN proteins belonging to the 3rd subgroup are the only respresentatives of the SLFN protein family that harbour known functional motifs, indicating that they act potentially as ATPase dependent RNA helicases. As probable modulators of RNA metabolism, SLFN5, SLFN8, SLFN9 and SLFN10 could be involved in regulatory aspects of cell cycle. Ectopic expression of these four homologous SLFN members has no growth modulating effects in retrovirally transduced fibroblasts. The differentially localization in transfected fibroblasts cells and differential expression profiles in resting and activated T-cells, respectively, point to a complex cell type specific function of these proteins in cell cycle control and T-cell ontogenesis. Transgenic T-cell specific expression of SLFN8 modulates intrathymic development of thymocytes and reduces the proliferative potential of mature T-cells. The mechanisms underlying the effects of the transgenic SLFN8 protein expression are unknown. Further analysis are necessary to unravel the molecular function of the individual SLFN proteins. This will improve the understanding of regulatory circuits influencing haematopoietic cell development and homeostasis

Functions of Chloroplastic Adenylate Kinases in Arabidopsis1[W][OA]

Adenosine monophosphate kinase (AMK; adenylate kinase) catalyses the reversible formation of ADP by the transfer of one phosphate group from ATP to AMP, thus equilibrating adenylates. The Arabidopsis (Arabidopsis thaliana) genome contains 10 genes with an adenylate/cytidylate kinase signature; seven of these are identified as putative adenylate kinases. Encoded proteins of at least two members of this Arabidopsis adenylate kinase gene family are targeted to plastids. However, when the individual genes are disrupted, the phenotypes of both mutants are strikingly different. Although absence of AMK2 causes only 30% reduction of total adenylate kinase activity in leaves, there is loss of chloroplast integrity leading to small, pale-looking plantlets from embryo to seedling development. In contrast, no phenotype for disruption of the second plastid adenylate kinase was found. From this analysis, we conclude that AMK2 is the major activity for equilibration of adenylates and de novo synthesis of ADP in the plastid stroma