Une distribution lipidique hétérogène est essentielle à l’identité et fonction des organelles, mais l’échange par trafic vésiculaire tend à annuler cette distribution. Il existe donc des mécanismes qui assurent l’homéostasie des lipides. Les protéines Osh (S. cerevisiae) et les OSBP-Related Proteins (ORP, H. sapiens), sont des transporteurs de lipides. Osh4 est capable d’échanger de l’ergostérol contre le phosphatidylinositol-4-phosphate (PI4P), présent sur l’appareil de Golgi. Utilisant des outils fluorescents mesurant avec une précision inégalée le transport de stérol et de PI4P, nous démontrons qu’Osh4 transporte du stérol contre son gradient de concentration en utilisant l’énergie d’un gradient de PI4P. Un couplage au métabolisme du PI4P permettrait à Osh4 d’alimenter le Golgi avec du stérol, ainsi créant le gradient de stérol entre ces organelles. La protéine OSBP participe, via sa capacité à connecter la membrane du RE à celle du trans-Golgi, à la création de jonctions entre ces organelles. Nous avons montré qu’OSBP, par échange stérol/PI4P, utilise le PI4P pour transférer du cholestérol au Golgi, mais également pour autoréguler sa capacité à former les jonctions. Osh6 lie la phosphatidylsérine, nous permettant d’étudier un nouveau mécanisme d’échange. Nous avons résolu la structure cristallographique d’un complexe Osh6/PI4P et avons pu observer l’échange de ces deux ligands par Osh6 entre deux membranes. Cette étude nous permet de suggérer que l’échange de PI4P avec divers lipides, via les protéines Osh/ORP, serait un mécanisme général permettant aux cellules de maintenir le gradient lipidique entre le RE et les membranes tardives de la voie sécrétoire. / An uneven lipid distribution is essential for the function of eukaryotic organelles. However, exchange of material by vesicular trafficking has a tendency to perturb this distribution; mechanisms must though exist to ensure lipid homeostasis. Osh proteins (S. cerevisiae) and OSBP-Related Proteins (ORPs, H. sapiens), are lipid transfer proteins (LTPs). Osh4 is capable of exchanging ergosterol for phosphatidylinositol 4-phosphate (PI4P), found on the Golgi. Using novel fluorescent tools to measure with unprecedented precision the transport of sterol and PI4P, we find that Osh4 can transport sterol against its concentration gradient using the energy of a PI4P gradient. Coupled to phosphoinositide metabolism, this allows Osh4 to transport sterol to the trans-Golgi and create the sterol gradients observed between these organelles. OSBP participates in the creation of membrane contact sites (MCSs) via its capacity to connect ER membranes to those of the trans-Golgi. We have shown that it uses PI4P for transporting cholesterol from the ER to the trans-Golgi by sterol/PI4P counterexchange, hence also autoregulating its tethering activity. Finally, the identification of phosphatidylserine as a ligand for Osh6 allowed us to analyze the possible extrapolation of the PI4P counterexchange mechanism. We have solved the crystal structure of Osh6 in complex with PI4P and have been able to follow counterexchange of PI(4)P and PS in vitro. Concluding, our studies allow us to suggest a general mechanism for ORP/Osh-mediated counterexchange of PI4P for other lipids to maintain lipid gradients between the ER and late membranes of the secretory pathway.

Les cellules eucaryotes possèdent un territoire membranaire dit « électrostatique » qui est définit par la présence de phospholipides négativement chargés sur la face cytosolique des membranes. Cette propriété permet le recrutement de protéine cytosolique contenant des motifs/domaines positivement chargés au niveau des membranes via des interactions électrostatiques. Nous nous sommes demandés si le territoire électrostatique est présent chez les cellules végétales et quel est son organisation ? Quels sont le(s) lipide(s) anionique(s) impliqués dans son maintien ? Et quel est son (ces) rôle(s) dans la signalisation et le développement des plantes ? Premièrement, nous avons mis en avant que la membrane plasmique est le compartiment intracellulaire le plus électronégativement chargé (Simon, Platre et al., 2016 Nature Plants). Ce champ électrostatique est gouverné par trois lipides anioniques différents, l’acide phosphatidique, la phosphatidylserine et le phosphatidylinositol-4-phosphate. Nous avons montré que cette propriété unique de la membrane plasmique permet de réguler des voies de signalisation hormonale, tel que celle de l’auxine et des brassinostéroïdes. Notamment, la phosphatidylserine régule la dynamique spatiotemporelle des petites GTPases de la famille Rho. En réponse à l’auxine, ce lipide permet de regrouper les protéines Rho dans des domaines membranaires. La formation de ces domaines est requise pour l’activité de ces protéines permettant de contrôler l’endocytose, la dynamique du cytosquelette mais également régule la morphogenèse cellulaire ainsi que la réponse gravitropique de la racine. / The « electrostatic territory» is part of the eukaryotic membrane organization and is defined by the enrichment of negatively charged phospholipids at the membrane cytosolic face. This feature is involved in the membrane recruitment of cytosolic proteins, which contain positively charged motifs and/or domains. In this work, we used Arabidopsis thaliana as a model and explored the existence of an electrostatic territory in plant cells. We found that the plasma membrane is the most anionic intracellular membrane (Simon, Platre et al., 2016 Nature Plants). This electrostatic field is maintained by lipid cooperation between, phosphatidic acid, phosphatidylserine and phosphatidylinositol-4-phosphate. The cell surface unique feature is involved in the regulation of hormonal signalling such as auxin and brassinosteroids pathways. We found that phosphatidylserine tunes the spatiotemporal dynamics of small GTPases from the Rho family. During auxin response, PS is required to cluster Rho into specialized membrane domains. We show that nanocluster formation is required for Rho-mediated auxin signaling including the regulation of endocytosis, cytoskeleton organization, morphogenesis and the root gravitropic response.

Le virus de l’hépatite C (VHC) est l’un des principaux facteurs étiologiques du carcinome hépatocellulaire. Le traitement des hépatites virales C a été récemment amélioré grâce à une trithérapie (interféron, ribavirine et anti-protéase virale). Néanmoins l’importance des effets secondaires et l’émergence de mutants résistants nécessitent de découvrir de nouveaux antiviraux. Dans ce contexte, notre équipe a récemment découvert que les phosphatidylinositol 4-kinases de type III (PI4KIIIα et PI4KIIIβ) étaient indispensables à la propagation du virus dans une lignée hépatique humaine, et ce, à 2 étapes de son cycle biologique : l’entrée et la réplication. L’objectif du présent travail était de poursuivre la validation de ces nouvelles cibles thérapeutiques potentielles, en approfondissant nos connaissances sur la dépendance du virus à l’égard de ces kinases au cours de son entrée. Pour cela, nous avons utilisé le modèle des hépatocytes humains primaires, système in vitro plus proche du contexte physiologique que les modèles utilisés jusqu’à présent et qui étaient basés sur l’exploitation de lignées. Deux axes ont été développés : Vérification de l’importance de l’activité kinase des PI4KIIIs au cours de l’entrée du VHC dans les hépatocytes humains primaires, à travers une approche chimique ; Validation de l’implication de ces kinases et de leur activité enzymatique au cours de l’entrée virale grâce à une approche génétique basée sur l’ARN interférence et la restauration de phénotype. En parallèle, nous avons étudié l’expression de PI4KIIIα au cours de pathologies hépatiques. Nos résultats suggèrent une implication de PI4KIIIα au cours de l’entrée du VHC dans les hépatocytes humains primaires, mais restent à confirmer quant à l’implication de PI4KIIIβ. Par ailleurs, l’analyse de l’expression de PI4KIIIα dans le carcinome hépatocellulaire (CHC) conduit à proposer cette kinase comme un nouveau marqueur moléculaire, qui pourrait améliorer les modèles de pronostic déjà établis et pourrait conduire au développement de nouvelles approches thérapeutiques pour les patients atteints d’un CHC, quelque soit l’étiologie. / Hepatitis C virus (HCV) is one of the leading causes of hepatocellular carcinoma. Therapeutic treatment against the virus has been recently improved by a tritherapy including pegylated interferon, ribavirin and antiviral protease. Nevertheless, the importance of side effects and the emergence of resistant mutants require the development of new antivirals. In this context, our team has recently discovered that Type III phosphatidylinositol 4-kinases (PI4KIIIα and PI4KIIIβ) are essential for the propagation of the virus in a human hepatic cell line at the entry and replication steps. To further characterize these potential therapeutic targets, we investigate the implication of these kinases during the HCV entry, using primary human hepatocytes, a model closer to the in vivo conditions. Two lines of research were developed: Verification of the importance of the kinase activity of PI4KIIIs during HCV entry in primary human hepatocytes, through a chemical approach; Validation of the involvement of these kinases and their enzymatic activity during viral entry through a genetic approach based on RNA interference and phenotype rescue. In parallel, we studied the expression of PI4KIIIα in liver diseases. Our results suggest the involvement of PI4KIIIα in HCV entry; the involvement of PI4KIIIIβ needs to be confirmed. The analysis of PI4KIIIα expression in hepatocellular carcinoma led us to propose this kinase as a new molecular marker, which could improve the already established prognosis models and could lead to the development of new therapeutic approaches.

Phosphatidyl D-myo-inositol (3,4,5)-trisphosphate [PtdIns(3,4,5)P3] contributes to the activation of protein kinase B (PKB) by interacting with the PKB PH domain. PKB is known to be up-regulated in several cancer cell types. Compounds that can display selective inhibition of this kinase have promising chemotherapeutic potential, and inhibition of the PH domain of PKB represents a realistic means by which to achieve this. Analysis of the X-ray crystal structures of apo PKBαPH and PKBαPH bound to D-myo-inositol 1,3,4,5-tetrakisphosphate [InsP4, the inositol head group of PtdIns(3,4,5)P3] led to the design of PtdIns(3,4,5)P3 and InsP4 analogues as potential PKB PH domain inhibitors. The synthesis of PtdIns(3,4,5)P3 analogues modified at the C-4 position was investigated, but it was discovered that such compounds were prone to migration of the 1-position phosphate. Subsequently, a range of racemic InsP4 analogues, modified at the C-1 or C-4 position, were successfully synthesised. Advanced progress has also been made towards the synthesis of enantiomerically pure analogues of InsP4.

Thesis advisor: Mary F. Roberts / Secreted phosphatidylinositol-specific phospholipase Cs (PI-PLCs) are often virulence factors in pathogenic bacteria. Understanding how these enzymes interact with target membranes may provide novel methods to control bacterial infections. In this work, two typical PI-PLC enzymes, from Bacillus thuringiensis (Bt) and Staphylococcus aureus (Sa), were studied and their membrane binding properties were examined and correlated with enzymatic activity. BtPI-PLC is kinetically activated by allosteric binding of a phosphatidylcholine (PC) molecule. MD simulations of the protein in solution suggested correlated loop and helix motions around the active site could regulate BtPI-PLC activity. Vesicle binding and enzymatic studies of variants of two proline residues, Pro245 and Pro254, that were associated with these motions showed that loss of the correlated motions between the two halves of PI-PLC were more critical for enzymatic activity than for vesicle binding. Furthermore, loss of enzyme activity could be rescued to a large extent with PC present in a vesicle. This suggests that binding to PC changes the enzyme conformation to keep the active site accessible. SaPI-PLC shows 41.3% sequence similarity with BtPI-PLC but has very different ways its activity is regulated. While it is kinetically activated by PC it does not in fact bind to that phospholipid. Enzymatic and membrane interaction assays showed that SaPI-PLC has evolved a complex, apparently unique way to control its access to PI or GPI-anchored substrate. (i) An intramolecular cation-pi latch facilitates soluble product release under acidic conditions without dissociation from the membrane. (ii) There is a cationic pocket on the surface of enzyme that likely modulates the location of the protein. (iii) Dimerization of protein is enhanced in membranes containing phosphatidylcholine (PC), which acts not by specifically binding to the protein, but by reducing anionic lipid interactions with the cationic pocket that stabilizes monomeric protein. SaPI-PLC activity is modulated by competition between binding of soluble anions or anionic lipids to the cationic sensor and transient dimerization on the membrane depleted in anionic phospholipids. This protein also served as a way to test the hypothesis that a cation-pi box provides for PC recognition site. This structural motif was engineered into SaPI-PLC by forming N254Y/H258Y. This variant selectively binds PC-enriched vesicles and the enzyme binding behavior mimics that of BtPI-PLC. Itaconic acid (ITA) is a metabolite synthesized in macrophages and related cell lines by a cis-aconitate decarboxylase (cADC). cADC activity is dramatically increased upon macrophage stimulation. In this work, the cell line RAW264.7 was used to show that cADC activity upon stimulation requires de novo protein synthesis. MS analyses of partially purified RAW264.7 protein extracts from stimulated cells show a large increase for immunoresponsive gene 1 protein (IRG1) and siRNA knockdown of the IRG1 reduces cADC activity upon stimulation. Suspected active site residues of IRG1 were identified by mutagenesis studies of the recombinant protein based on a homology structure model of fungal cADC. The cloning and overexpression of this enzyme should help clarify the cofactor-independent decarboxylation mechanism of this mammalian enzyme as well as open up future studies into the specific role of ITA in the mammalian immune system and cancers. / Thesis (PhD) — Boston College, 2013. / Submitted to: Boston College. Graduate School of Arts and Sciences. / Discipline: Chemistry.

Thesis advisor: Mary F. Roberts / Thesis advisor: Steven D. Bruner / The bacterial phosphatidylinositol-specific phospholipase C (PI-PLC) from <italic>Bacillus thuringiensis</italic> is specifically activated by low concentrations of a non-substrate lipid, phosphatidylcholine (PC), presented as an interface. However, if the PC concentration in the interface is too high relative to substrate, the enzyme exhibits surface dilution inhibition. Understanding this bacterial enzyme, which shares many kinetic features with the larger and more complex mammalian PI-PLC enzymes, requires elucidating the mechanism for PC activation and inhibition. Various techniques were applied to study the interaction of the protein with vesicles composed of both the activator lipid PC and the substrate lipid (or a nonhydrolyzable analogue). Fluorescence correlation spectroscopy (FCS), used to monitor bulk partitioning of the enzyme on vesicles, revealed that both the PC and the substrate analogue are required for the tightest binding of the PI-PLC to vesicles. Furthermore, the tightest binding occurred at low mole fractions of substrate-like phospholipids. Field cycling <super>31</super>P NMR (fc-P-NMR) spin-lattice relaxation studies provided information on how bound protein affects the lipid dynamics in mixed substrate analogue/PC vesicles. The combination of the two techniques could explain the enzyme kinetic profile for the PC activation and surface dilution inhibition: small amounts of PC in an interface enhanced PI-PLC binding to substrate-rich vesicles while high fractions of PC tended to sequester the enzyme from the bulk of its substrate leading to reduced specific activity. FCS binding profiles of mutant proteins were particularly useful in determining if a specific mutation affected a single or both phospholipid binding modes. In addition, an allosteric PC binding site was identified by fc-P-NMR and site directed spin labeling. A proposed model for PC activation suggested surface-induced dimerization of the protein. Experiments in support of the model used cysteine mutations to create covalent dimers of this PI-PLC. Two of these disulfide linked dimers, formed from W242C or S250C, exhibited higher specific activities and tighter binding to PC surfaces. In addition, single molecule total internal reflection fluorescence microscopy was used to monitor the off-rate of PI-PLC from surface tethered vesicles, providing us with a direct measure of off-rates of the protein from different composition vesicles. / Thesis (PhD) — Boston College, 2009. / Submitted to: Boston College. Graduate School of Arts and Sciences. / Discipline: Chemistry.

Mosaic, activating mutations in PIK3CA, the gene encoding the catalytic p110α subunit of class IA phosphatidylinositol 3-kinase (PI3K), are the cause of rare, developmental growth disorders collectively known as PIK3CA-Related Overgrowth Spectrum (PROS). Given the pressing need for targeted therapy and evidence for tissue- and cell lineage-specific distribution of PIK3CA mutations in PROS, developmental models of this disease will be a key asset for preclinical drug testing and for a better understanding of PIK3CA activation in development. This PhD project addressed the lack of human, developmental PROS models by establishing isogenic series of human induced pluripotent stem cells (iPSCs) with endogenously expressed, activating PIK3CA mutations. This involved the optimisation of a CRISPR/Cas9 protocol for efficient knockin of different PIK3CA variants into human iPSCs. An isogenic iPSC series was established with cells expressing either wild-type PIK3CA or PIK3CA-H1047R, knocked into either one or both endogenous alleles. In parallel, mosaic patient- derived fibroblast cultures were reprogrammed to obtain isogenic wild-type and heterozygous iPSCs expressing PIK3CA-E418K. The models were used in comprehensive signalling studies, providing new insights into PI3K signalling in human iPSCs and how it is perturbed by genetic p110α activation. PIK3CA-E418K, a rare variant in both PROS and cancer, caused minimal pathway activation, in contrast to the highly recurrent variant PIK3CA-H1047R which induced strong PI3K signalling in both heterozygous and homozygous iPSCs according to a graded pattern. Studies of clinically relevant PI3K pathway inhibitors provided proof-of-concept that stem cell-based PROS models can be used for preclinical drug testing, and demonstrated that p110α is likely to be the main catalytic isoform mediating canonical PI3K signalling in human iPSCs. Differentiation assays revealed allele dose-dependent effects of PIK3CA-H1047R on stemness, with homozygous iPSCs exhibiting widespread transcriptome remodelling affect- ing genes implicated in cancer and development. Accordingly, these cells showed increased expression of pluripotency genes such as NANOG and NODAL, resulting in self-sustained "stemness" in embryoid body and teratoma assays. In comparison, heterozygous mutants behaved similar to wild-type controls under all differentiation paradigms. Furthermore, evidence was obtained that strong activation of PI3K signalling is fully compatible with definitive endoderm formation, arguing against cell-autonomous differentiation defects as the cause of endoderm sparing in PROS. In summary, these studies demonstrate the utility of human stem cell-based models of PROS for preclinical drug testing and for improved understanding of class IA PI3K signalling in human development. They are also likely to be useful in efforts to obtain a better understanding of PIK3CA-H1047R in human cancer.

As doenças alérgicas alcançaram proporções mundialmente epidêmicas. A ativação de receptores para IgE, Fc RI, em mastócitos, é o mecanismo chave para a iniciação e propagação das respostas patofisiológicas dos processos alérgicos. Após a interação destas células com um alérgeno, há a ativação de uma cascata de eventos de sinalização, a qual resulta na secreção de mediadores alérgicos pré-formados, através de um processo regulado de exocitose, além da síntese e secreção de mediadores lipídicos e citocinas. Desta forma, a inibição da responsividade dos mastócitos, quando ativados por um alérgeno, representa uma via importante para o desenvolvimento de novos candidatos a fármacos com indicação antialérgica. Neste sentido, este trabalho buscou, num primeiro momento, contribuir com a compreensão dos papéis desempenhados por um glicerofosfolipídeo de membrana, fosfatidilinositol 4,5-bifosfato (PtdIns(4,5)P2), em eventos de sinalização em mastócitos, mediados por IgE. Este trabalho permitiu destacar a importância de PtdIns(4,5)P2 como um regulador chave das respostas de Ca2+ e das alterações de morfologia de mastócitos estimulados por um alérgeno. Foi observado ainda que níveis reduzidos de PtdIns(4,5)P2 determinaram a inibição do processo de endocitose de receptores Fc RI ativados, um evento crucial para a redução da transdução de sinais. Estes resultados não somente trazem um ganho de conhecimento acerca dos detalhes que orquestram os eventos de sinalização em mastócitos estimulados por um alérgeno, como também apontam que a regulação dos níveis de PtdIns(4,5)P2 pode certamente ser apontada como alvo para o desenvolvimento de novas moléculas inibidoras da ativação mastocitária. A segunda etapa deste trabalho teve como objetivo avaliar o potencial inibitório de alguns compostos de origem natural e sintética sobre a degranulação de mastócitos, evento em que mediadores alérgicos, como a histamina, são secretados, em resposta ao estímulo celular. Inicialmente, avaliou-se o efeito inibitório de um conjunto de arilcumarinas sintéticas, estruturalmente relacionadas, sobre a degranulação. Um número significativo de moléculas foram ativas e, dentre elas, algumas substituições junto à estrutura do anel 3-fenilcumarínico, como as hidroxilações das posições 6, 2\'e 5\', puderam ser identificadas como importantes para o potencial bioativo. Finalmente, o trabalho apresentou uma molécula de origem natural, como um potente inibidor da degranulação de mastócitos e da secreção de citocinas. Trata-se da piridovericina, um metabólito secundário, isolado do fungo entomopatogênico Beauveria bassiana. Dessa forma, tanto as cumarinas, como a piridovericina podem ser apontadas como compostos de partida de grande potencial para o desenvolvimento de novos fármacos anti-alérgicos. / Allergic diseases have approached epidemic proportions worldwide. The activation of IgE receptors, Fc RI, from mast cells, is the key event for the initiation and propagation of pathophysiological responses involved in the allergic processes. The interaction between mast cells and allergens triggers a signaling cascade, which results in secretion of pre-formed allergic mediators, through regulated exocytosis, in addition to the synthesis and secretion of lipid mediators and cytokines. In this way, the inhibition of mast cell responsiveness, upon allergen stimulation, represents an important pathway for the development of new antiallergic drug candidates. Thus, the present work tried, firstly, to gain better insight of the roles played by phosphatidylinositol 4,5-biphosphate (PtdIns(4,5)P2) during IgE-mediated mast cell signaling. This work highlighted the importance of PtdIns(4,5)P2 as a key regulator of Ca2+ responses and mast cell morphological alteration, when activated by an allergen. It was observed that reduced levels of PtdIns(4,5)P2 determined the inhibition of activated Fc RI endocytosis, a crucial event for signal transduction termination. Those results not only improve the actual knowledge in mast cell signaling but also point out the regulation of PtsIns(4,5)P2 levels as a target to be pursued during the development of new inhibitors of mast cell activation. The second part of this work aimed to evaluate the capacity of both synthetic and natural compounds to inhibit mast cell degranulation, characterized by the release of granule-contained allergic mediators, such as histamine, upon cell stimulation. Initially, the inhibitory effect of a set of structurally related synthetic arylcoumarins was evaluated. A significant number of molecules were active, and a few substitutions within such molecules could be pointed as important for the biological activity, such as the hydroxylation of carbons 6, 2\' e 5\' of the 3-phenylcoumarin ring. Lastly, this work presents a natural compound as a potent inhibitor of mast cell degranulation and cytokine secretion. The refered compound is pyridovericin, a secondary metabolite isolated from the entomophatogenic fungus Beauveria bassiana. Therefore, both the coumarin derivatives and pyridovericin can be regarded as lead compounds for the development of new anti-allergic drugs.

Die Interaktion des Masernvirus mit T-Zellen stört die Aktivierung der TCR-Signalkaskade durch die Hemmung der Phosphatidylinositol-3-Kinase (PI3K), die zur Einstellung der T-Zell-Funktionen führt und dadurch nachgeschaltete (downstream) Signalwege sowie den Eintritt in den Zellzyklus, aber auch die Genexpression reguliert. Infolgedessen können die Aktivität spleißregulatorischer Faktoren sowie die Spleißmuster von mRNAs verändert werden, wie zum Beispiel bei der alternativ gespleißten SHIP1-Isoform SIP110, die eine T-Zell-inhibitorische Aktivität zeigt. Um alternativ gespleißte (AS) und differentiell regulierte (RG) Transkripte in T-Zellen infolge von PI3K-Inhibition zu erfassen, wurde ein Human Exon 1.0 ST Array an RNA-Proben von humanen T-Zellen, 24 h stimuliert und stimuliert/ PI3K-inhibiert, durchgeführt. Durch die Anwendung geeigneter bioinformatischer Algorithmen konnten spezifisch in PI3K-inhibierten Zellen angereicherte Transkripte nachgewiesen und in die Kategorien AS (2192 Gene) und RG (619 Gene) eingeteilt werden. Ausgewählte Gene wurde mittels RT-PCR und qPCR validiert, gefolgt von der funktionellen Annotation beider Genlisten. AS Gene konnten verstärkt in ECM-Rezeptor Interaktionen, fokaler Adhäsion, Proliferation, Zytoskelettorganisation und Tumorsignalwegen gefunden werden, während RG Gene eher in der DNA-Replikation, DNA-Reparatur und Stressantwort vertreten waren. Gene beider Gruppen konnten auf Signalwege bezogen werden, die essentiell für den TCR-Signalweg, die Zytoskelettdynamik und den Zellzykluseintritt waren. Das stützt die Annahme, dass die Außerkraftsetzung der PI3K-Schüsselprozesse der T-Zell-Aktivierung sowohl auf der Ebene der RG als auch der AS Gene wirkt. Über die Ingenuity Pathway Analyse konnten wir unsere Genlisten mit Genen vergleichen, die bereits auf solche Schlüsselprozesse und die Viabilität bezogen werden können. In der Überschneidung wurden z.B. die AS GTP-Austauschfaktoren Vav1 und Vav3 gefunden, die für die Übersetzung extrazellulärer Signale in Zytoskelettdynamik, Proteinphosphatasen und Adapter von Bedeutung sind. Ausgewählte Gene (AS - FBXO6 und LAT2, RG - SLFN5) wurden aus PI3K-arretierten T-Zellen kloniert und deren Effekt auf grundlegende zelluläre Funktionen durch Überexpression in HEK293T-Zellen überprüft. Die Fusionsproteine veränderten weder die Zellviabilität noch die Proliferation dieser Zellen. Über einen auf siRNA basierenden Knockdown wurde überprüft, ob das RG Gene SLFN5 als Suppressor auf die T-Zell-Aktivierung agiert. Der Knockdown in primären T-Zellen zeigte keinen Einfluss auf die Zellviabilität, Proliferation und Polarisation. Jedoch konnte ein signifikanter Effekt auf die T-Zell-Adhärenz auf Fibronektin gezeigt werden, was darauf schließen lässt, dass SLFN5 die T-Zell-Adhärenz negativ reguliert. Des Weiteren wurde die MV-induzierte Regulation selektierter Gene betrachtet und Unterschiede in der Regulation im Vergleich zur direkten PI3K-Inhibition festgestellt. Ein Grund dafür könnte sein, dass das MV eine Inhibition auf vielen Ebenen induziert, anstelle der alleinigen PI3K-Inhibition. Abschließend wurde untersucht, ob ausgewählte Gene an der Regulation in verschiedenen T-Zelllinien beteiligt sind und als Tumorsuppressoren agieren könnten. FBXO6 als Regulator der CHK1-Stabilität wurde in den meisten Zelllinien nicht exprimiert. Die Annahme, dass eine Stress-induzierte defekte Ubiquitinierungsmaschinerie an der Resistenz von Tumorzellen auf Chemotherapeutika beteiligt ist, macht FBXO6 zu einem interessanten Kandidaten als Biomarker für Tumorsensitivität gegenüber Krebsmedikamenten. Diese Annahme bedarf jedoch weiterer Untersuchungen. / The interaction of measles virus (MV) with T cells interferes with the activation of the TCR-signaling by the inhibition of the phosphatidylinositol-3-kinase (PI3K), leading to the termination of T cell functions and consequently to the regulation of downstream signaling as well as cell cycle entry. PI3K-inhibition also affects the activity of splice regulatory elements and the splicing pattern of mRNAs, as for example the alternatively spliced SHIP1 isoform SIP110 that shows T cell inhibitory activity. To integrate early alternatively spliced (AS) and differentially regulated (RG) transcripts in response to PI3K interference in T cells at a general level, we performed a Human Exon 1.0 ST Array analysis on RNAs isolated from human T cells PI3K-inhibited or not prior to 24h stimulation. Applying suitable bioinformatic algorithms, transcripts detected specifically in PI3K-inhibited cells were assigned to categories defining RG (619 genes) and AS species (2192 genes). A selection of genes was validated by RT-PCR and qPCR followed by functional annotation of both gene lists. AS genes were found to be enriched in ECM-receptor interactions, focal adhesion, proliferation, cytoskeleton organization and tumor signaling, while RG genes were rather related to processes as DNA-replication, DNA-repair and stress response. Some genes that belonged to both groups target pathways essential for TCR-signaling, cytoskeletal dynamics and cell cycle entry, strongly support the notion that PI3K abrogation interferes with key T cell activation processes at the level of differential regulation as well as alternative splicing. Using Ingenuity Pathway Analysis we compared our gene lists to genes already known to specifically relate to key T cell functions and viability. In the overlap we found for example AS GTP-exchange factors Vav1 and Vav3 important for translating extracellular signals into cytoskeletal dynamics, protein phosphatases and adaptors. Selected candidate genes (AS - FBXO6 und LAT2, RG - SLFN5) were cloned from PI3K-arrested T cells into the pEGFP-vector and tested by overexpression in HEK293T for their effect on basic cell functions. These fusion proteins did not affect viability and proliferation of these cells. Using siRNA-based knockdown the potential of the RG gene SLFN5 to act as suppressors of key steps in T cell activation was tested. Its knockdown in primary T cells did not affect cell viability, proliferation and polarization. However, T cell adherence on fibronectin was significantly enhanced indicating that SLFN5 negatively regulates T cell adhesion to the ECM. Additionally we had a look at the MV induced regulation of selected genes and found a difference of the regulation in comparison to direct PI3K-inhibition. A reason for these unexpected results could be that MV induces a multi-level inhibition, rather than PI3K-inhibition only. Finally we wanted to find out, if selected genes were also implicated in the regulation of different T cell lines and therefore could act as tumor suppressors. FBXO6, a regulator of CHK1 stability, for example was not expressed in most investigated cell lines. Assuming that a stress-induced defective ubiquitination complex is involved in the resistance of tumor cells to chemotherapeutic agents FBXO6 might be an interesting candidate as biomarker for tumor sensitivity to cancer medication. If some of these differences in regulation are the result of immortalization, corresponding genes could consequently act as tumor suppressor. This issue has to be subject to further research.

Das Multiple Myelom (MM) ist eine unheilbare Erkrankung, die aus einer klonalen Proliferation maligner Plasmazellen im Knochenmark hervorgeht. Dabei liegt ein komplexes Signalnetzwerk vor, das zum Überleben und Wachstum der MM-Zellen führt. Das MM ist durch eine enorme genetische und phänotypische Heterogenität gekennzeichnet. Die konstitutive Aktivierung des PI3K/Akt-Signalwegs spielt bei ungefähr der Hälfte der Patienten mit MM eine wichtige Rolle für das Überleben der MM-Zellen und ist daher ein potentieller therapeutischer Ansatzpunkt. Isoform-spezifische Untersuchungen der katalytischen Untereinheiten der Klasse I-PI3K (p110α, p110β, p110γ, p110δ) sollten zur Erkenntnis führen, welche dieser Isoformen für das MM Zellüberleben wichtig sind, um spezifischere Behandlungen mit möglichst geringen Nebenwirkungen zu erlauben. Dafür wurden zunächst Isoform-spezifische Knockdown-Experimente mit MM Zelllinien durchgeführt und sowohl deren Überleben als auch die Aktivierung der nachgeschalteten Komponenten im PI3K Signalweg untersucht. Zur Verifizierung der Ergebnisse wurden sowohl MM Zelllinien als auch Primärzellen mit Isoform-spezifischen PI3K-Inhibitoren behandelt (BYL 719 für p110α, TGX 221 für p110β, CAY10505 für p110γ und CAL 101 für p110δ) und in gleicher Weise untersucht. In beiden Versuchsansätzen stellte sich die katalytische Untereinheit p110α als wichtigste Isoform für das Überleben von MM Zellen mit konstitutiv phosphoryliertem Akt Signal heraus. Weder der Knockdown noch die pharmakologische Inhibition der anderen drei Isoformen (p110β, p110γ, p110δ) führten in MM-Zelllinien zur Beeinträchtigung des Zellüberlebens. Auch reagierten die Primärzellen von MM Patienten größtenteils nicht mit Apoptose auf eine Behandlung mit TGX 221, CAY10505 oder CAL 101. Aufbauend auf der postulierten Bedeutung von p110α, wurde der dafür spezifische Inhibitor BYL 719 mit bereits klinisch etablierten Therapeutika in Kombination verwendet, woraus eine im Vergleich zur Einzelbehandlung verstärkte Apoptose resultierte. Insgesamt deuten diese Daten darauf hin, dass PI3K/p110α eine therapeutisch nutzbare Zielstruktur zur Behandlung des Multiplen Myeloms darstellt. Daher scheinen weitergehende prä-klinische Studien mit p110α Inhibitoren erfolgversprechend. / Multiple myeloma (MM) is an incurable disease, which results from clonal proliferation of malignant plasma cells in the bone marrow. Thereby, a complex signaling network regulates the survival and growth of MM cells. This malignant hematological disease is characterized by profound genetic and phenotypical heterogeneity. The PI3K/Akt signaling pathway is constitutively activated in about 50% of patients with MM and therefore plays an important role for the survival of MM cells. Accordingly, treatment of MM patients with the most isoform-specific drugs may be a desirable goal to achieve therapeutic utility with a minimum of undesired side effects. Therefore, an isoform-specific analysis of the catalytic subunits of the PI3K class I (p110α, p110β, p110γ, p110δ) was undertaken to reveal their individual role(s) for MM cell survival. Initially, isoform-specific knockdown experiments in MM cell lines were performed to assess their survival and the activation states of down-stream components of the PI3K pathway. These experiments were then complemented using isoform-specific pharmacological inhibitors (BYL 719 for p110α, TGX 221 for p110β, CAY10505 for p110γ and CAL 101 for p110δ) in MM cells and primary MM cells. Cell lines with constitutively phosphorylated Akt reduced this signal after p110α knockdown or pharmacologic inhibition and these treatments also affected their survival. Conversely, neither knockdown nor drug-mediated inhibition of any of the other three p110 isoforms influenced MM cell survival. In addition, whereas most primary MM samples were sensitive against BYL-719 only a few samples displayed apoptotic effects when treated with TGX 221, CAY10505 or CAL-101. These results showed that p110α is the major contributor of PI3K-mediated cell survival, and therefore the inhibitor BYL 719 was tested in combination with clinically relevant therapeutics for MM. Such treatment led to increased rates of apoptosis in MM cell lines in comparison to the respective single drug treatments. Taken together, we assume that PI3K/p110α is a therapeutically valuable target structure for the treatment of MM that would warrant more extensive pre-clinical studies.