No description available.

No description available.

No description available.

Intrinsically disordered proteins (IDPs) are an abundant class of proteins and protein regions which rapidly change between multiple structures without an equilibrium position. IDPs exist as a series of conformational ensembles of semi-stable conformations that can be adopted based on a hilly landscape of shallow free energy minima. Disordered sequences share characteristic features differentiating them from globular proteins, including low sequence complexity, low occurrence of hydrophobic residues, high polar and charged residue content, and high flexibility. IDPs are commonly involved in regulation in the cell, and frequently function as, or interact with, hub proteins in protein-protein interaction networks, making them an important class of macromolecules for understanding regulatory and other processes. Given their functional importance, these proteins are widely studied. Many analytical techniques are used, though rapid conformational sampling by IDPs makes it difficult to detect many states with NMR or other techniques. Computational approaches such as molecular dynamics are increasingly used to probe the binding and conformational sampling of these proteins, allowing for observation of factors that cannot be observed with traditional analytical methods such as NMR, such as differing conformational ensembles and the dipoles of individual residues. Here, we studied the role of electrostatic interactions in IDP protein-protein interaction using molecular dynamics simulations performed with the Drude-2019 force field (FF), a polarizable model that allows for more accurate representation of electrostatics, an important factor for highly charged systems like IDPs. For this project, a prototypical protein with disordered regions, p53, was simulated with two protein partners, the nuclear coactivator domain of the CREB binding protein (CBP), and the E3 ubiquitin-protein ligase mouse double minute 2 (MDM2). p53 is widely studied, and the p53 transactivation domain (TAD) is disordered and binds to many structurally diverse partners, making this protein domain a useful model for probing the role of electrostatic interactions formed by IDPs at protein-protein binding interfaces. We found that the Drude-2019 FF allows for simulation of the p53 TAD with Cα chemical shifts comparable to those observed with NMR, supporting that the Drude-2019 FF performs well in simulating IDPs. We observed large relative change in sidechain dipole moments when comparing the p53 TAD alone and when bound to either CBP or MDM2. We observed that aliphatic and aromatic amino acids experienced the largest relative change in sidechain dipole moments, and that there is sensitivity to binding shown in this dipole response. The largest percent changes in sidechain dipole moment were found to localize at and around the binding interface. Understanding the binding interactions of IDPs at a fundamental level, including the role of electrostatic interactions, may help with targeting IDPs or their partners for drug design. / Master of Science in Life Sciences / Many proteins adopt one main structure, and these proteins are called ordered proteins. Intrinsically disordered proteins (IDPs) are an abundant category of proteins which adopt multiple structures, and transition between these different structures is based on factors such as the environment around them, modifications, or interactions with other macromolecules. The flexible structures of IDPs allow them to bind to multiple different partners and to regulate processes in the cell. Since IDPs often regulate processes important to cell function, when these proteins are mutated, misfolded, or otherwise mis-regulated the resulting issues are associated with disease states. IDPs are widely studied with analytical techniques, but because IDPs frequently change shape it can be difficult to observe certain behaviors or certain factors with these techniques. Computational approaches, such as molecular dynamics (MD). MD is the study of molecular motion and interaction, and can allow observation of factors that would be difficult or impossible to observe otherwise, such as the varying structures of IDPs or the dipole moments of specific amino acids within the proteins. For this project we wanted to probe the role of dipole moments, which are charge-based interactions, in the binding of IDPs to protein partners, to better understand how IDPs bind to different partners. We used the p53 protein as an example of IDP binding and simulated it alone and bound to two other proteins, the CREB binding protein (CBP), and the E3 ubiquitin-protein ligase mouse double minute 2 (MDM2). We observed that our simulations were comparable to experiments done with nuclear magnetic resonance spectroscopy, which served to validate that our simulations were realistic. We observed that the dipole moments of the proteins change when simulating the proteins alone and in complex, and that the largest relative changes in dipole are observed for regions of the proteins involved in binding. Probing the role of charge-based interactions in protein-protein binding interactions for IDPs can help us to greater understand these interactions at a more fundamental level and could help with targeting IDPs or their partners for drug design or other problems.

Le développement et l’homéostasie cellulaire de la prostate impliquent le contrôle strict des voies de signalisation induites par les androgènes et les facteurs de croissance. Ces diverses voies sont profondément altérées dans le cancer de la prostate, notamment lors des stades les plus avancés. Dans ce travail, une lignée immortalisée à partir de l’épithélium de prostate humaine, la lignée RWPE-1, a été utilisée pour étudier certains signaux régulant la prolifération cellulaire, ainsi que les connexions entre les voies de signalisation correspondantes. La prolifération des cellules RWPE-1 est sous la dépendance de l’EGF (Epidermal Growth Factor) qui intervient physiologiquement dans le développement épithélial. Les récepteurs apparentés à l’EGF-R sont également impliqués dans la prolifération au cours de la progression tumorale. La prolifération des cellules RWPE-1 en réponse à l’EGF est strictement dépendante de la voie ERK1/2, qui est donc considérée comme un point d’intégration des signaux. L’utilisation d’inhibiteurs du récepteur aux androgènes a permis de montrer le rôle essentiel qu’il joue dans l’activation d’ERK1/2 en réponse à l’EGF. Le récepteur aux androgènes s’associe avec plusieurs molécules de signalisation dans les cellules RWPE-1. Je démontre ici pour la première fois une association entre le récepteur aux androgènes et la kinase Raf-1, activatrice de la voie ERK1/2. Ainsi, le récepteur aux androgènes contrôlerait directement un processus essentiel à la prolifération épithéliale selon un mode d’action non-génomique. Par ailleurs, j’ai montré que la réponse proliférative des cellules RWPE-1 à l’IL-6 requiert l’activation de la voie ERK1/2, et l’activité kinase de l’EGF-R, suggérant la transactivation de ce récepteur par l’IL-6. L’utilisation de divers inhibiteurs chimiques a permis de démontrer que les métalloprotéases de la famille ADAM (a disintegrin and metalloprotease), notamment ADAM17, sont impliquées dans ce processus. Ainsi, l’activation de protéines ADAM par l’IL-6 conduirait au clivage d’un ligand membranaire de l’EGF-R, aboutissant à l’activation de la voie ERK1/2. Ce nouveau mécanisme pourrait être impliqué dans les situations inflammatoires conduisant à une prolifération excessive de l’épithélium prostatique, prélude à la transformation tumorale. En conclusion, les voies de signalisation étudiées sont fortement connectées dans les cellules épithéliales normales. Les deux nouveaux mécanismes décrits ici aboutissent à l’activation des kinases ERK1/2, point d’intégration et de convergence des voies de signalisation dans les cellules épithéliales de prostate normale. / Prostate development and cell homeostasis involve strict control of androgen and growth factors induced signaling pathways. These signaling pathways are deeply altered in prostate cancer, especially during late stages. In this work, the RWPE-1 immortalized cell line derived from human prostate epithelium has been used to study the signaling pathways regulating cell proliferation and their crosstalk. Optimal RWPE-1 proliferation is dependent on EGF (Epidermal Growth Factor), that also controls normal epithelial development. EGF-R family is also involved in cancer cell proliferation. EGF-dependent RWPE- 1 cell proliferation relies strictly on the ERK1/2 pathway which is then seen as a signal integrating node. Specific inhibitors showed essential role of androgen receptor in EGF mediated ERK1/2 activation. Androgen receptor is associated with several signaling molecules in RWPE-1 cells. I show here for the first time the physical interaction between the androgen receptor and the ERK1/2 activating kinase Raf1. Then, the androgen receptor could directly regulate an essential pathway for epithelial cells proliferation through a non-genomic mechanism. In addition, I showed that IL-6 dependent RWPE-1 cells proliferation requires both ERK1/2 and EGF-R kinase activities, suggesting an IL-6 mediated transactivation of EGF-R. By using several inhibitors, I showed that ADAM (a disintegrin and metalloprotease) family metaloproteases, especialy ADAM17, are involved in this process. IL-6-mediated ADAM proteins activation could lead to the cleavage of a membrane bound EGF-R ligand, leading to ERK1/2 pathway activation. This new mechanism could be involved in the inflammatory situations inducing hyperproliferation of the prostate epithelium, the first step of the transformation process. To conclude, the signaling pathways I studied are strongly connected in normal epithelial cells. The two new mechanisms described in this study lead to ERK1/2 kinases activation, an integrating node of signaling pathways in normal prostate epithelial cells.

Oxygen homeostasis is essential to the life of aerobes, which is regulated in humans by Hypoxia Inducible Factor-1α (HIF-1α). Under hypoxic conditions, HIF-1α transactivates over a hundred genes related angiogenesis, erythropoiesis, etc. HIF-1α level and function is regulated by four HIF hydroxylase enzymes: three isoforms of prolyl hydroxylase domain (PHD1, PHD2 and PHD3) and factor inhibiting HIF-1α (FIH). PHD2 is the focus of this research. PHD2 is a non-heme Fe(II) 2-oxoglutarate dependent dioxygenase, which controls HIF-1α levels by hydroxylating two proline residues within the ODD domain of HIF-1α, then the hydroxylated prolines are recognized by pVHL, which targets HIF-1α for proteasomal degradation. Under hypoxic conditions PHD2 cannot hydroxylate HIF-1α and its level rises in cells. The aims of this research include understanding how PHD2 chooses its substrate, how the O2 activation occurs, and how certain transition metals inhibit PHD2. Our results revealed that electrostatics play a role in substrate selectivity of PHD2 by provoking a change in the opening and closing rate of β2β3 loop for NODD and CODD substrates. Mutational studies of second coordination sphere residues combined with kinetic studies indicated that decarboxylation of 2OG is the slow step in the chemical mechanism. The removal of a hydrogen-bond by the Thr387aAla mutation revealed a rate 15 times faster than WT-PHD2 by making O2 a better nucleophile. Our results indicate that this hydrogen bonding is essential for proper O2 activation. Previous reports show that certain metals increase HIF-1α levels by inhibiting PHD2. However there are conflicts about how this inhibition occurs, either through metal replacement from the active site or metals binding to a different site causing inhibition. Our competitive and non-competitive kinetic assays showed different inhibition profiles. Under competitive conditions Zn2+, Co2+, Mn2+, and Cu2+ can bind to the enzyme active site and lead to inhibition but under non-competitive conditions Zn2+, Co2+, and Mn2+ partially inhibit PHD2 suggesting that these metals cannot displace the Fe2+ from the active site. XAS experiments with Zn2+ and Fe3+ indicate that Zn2+ binds to the surface of PHD2 in a six-coordinate manner composed of two Cys201, 208, His205, Tyr197 and two water ligands.

HIV-NC is a multifunctional protein which plays an important role in almost every step of the retroviral life cycle. NC is essential in catalyzing stand transfers of HIV-1 reverse transcription, including the annealing of the transactivation response element (TAR) of the viral genome to the complementary TAR DNA in minus-strong-stop DNA. In this dissertation, the research starts with focus on elucidating the reaction mechanism of NC-facilitated TAR DNA/RNA annealing using single molecule spectroscopy (SMS) approaches. The results indicate that nucleation of TAR DNA/RNA annealing occurs in an encounter complex form in which one or two DNA/RNA strands in the partially open “Y” form associated with multiple NC molecules. This encounter complex leads to annealing through the 3’/5’ termini, namely “zipper” pathway and the annealing through the hairpin loop region, namely “kissing” pathway. By employing target oligonucleotides for specific TAR regions, we directly probed kinetic reversibility and the chaperone role of NC. Concentration-dependence of NC chaperoned melting and annealing of TAR hairpins was investigated and the results further support the proposed reaction mechanism. Additionally, we used a single-stranded DNA (ssDNA) as model to study ssDNA conformational change upon NC binding. Here we present observation of NC binding to d(TG)n and d(T)n, including NC effect on flexibility and conformation of these oligonucleotides chains. Our results reveal that the rigidity of ssDNA chain is dramatically reduced through interaction with NC. Meanwhile the results of NC dissociation experiments indicate the interaction of NC/ssDNA is complex and heterogeneous. Finally, we used SMS in vitro to systematically compare and contrast the RNA/protein interactions for the zinc-finger-binding-motif protein (NC) and the arginine-rich-binding-motif (ARM) protein (Tat) encoded by HIV-1. Tat and NC use different RNA binding motifs to recognize and interact with RNA hairpin, giving rise to very different changes in the RNA secondary structure upon protein binding. Competition experiments show that the presence of Tat can effectively inhibit the NC binding-induced local melting of TAR RNA hairpins. These results indicate that Tat specifically binds and stabilizes the TAR RNA hairpin structure, which likely inhibits the local melting of the hairpin induced by NC. / text

No description available.

Growth factor receptors have significant effects on various normal function of body such as cell proliferation, differentiation and apoptosis. They are also involved in neuronal function and dysfunction, cardiovascular diseases, and malignancies. Recently, multiple G protein-coupled receptors (GPCRs) have been shown to transactivate receptor tyrosine kinases (RTKs). Since both classes of receptors have complicated downstream cascades individually, understanding the signaling differences between GPCR-induced growth factor receptor transactivation and direct ligand activation is an important challenge. To clarifying this phenomenon we investigated the phosphorylation profile and downstream effectors of ligand-activated vs. transactivated PDGF?? receptors. Dopamine receptors (one of the receptors of the GPCRs family) were used to compare the PDGF?? receptor phosphorylation and activity during direct activation and transactivation. Dose-response and time-course data between these two stimuli were evaluated. Furthermore, the phosphorylation site profiles and the intracellular signaling pathways of PDGF?? receptor after direct activation and transactivation were examined. In addition, possible synergic effects between transactivation and direct activation were explored. The results of this project showed that the phosphorylation profile and downstream effectors of ligand activated receptors versus transactivated receptors are different. Our data indicated that transactivation-induced pathways are more involved in survival and proliferation effects compared to ligand activation. This research answered basic questions about transactivation phenomena and proposes that these transactivation pathways could be exploited as a therapeutic approach for neurodegenerative diseases.

Transcription factors (TFs) are ubiquitous gene expression regulators and play essential roles in almost all biological processes. This Ph.D. project is primarily focused on the functional characterisation of MYB112 - a member of the R2R3-MYB TF family from the model plant Arabidopsis thaliana. This gene was selected due to its increased expression during senescence based on previous qRT-PCR expression profiling experiments of 1880 TFs in Arabidopsis leaves at three developmental stages (15 mm leaf, 30 mm leaf and 20% yellowing leaf). MYB112 promoter GUS fusion lines were generated to further investigate the expression pattern of MYB112. Employing transgenic approaches in combination with metabolomics and transcriptomics we demonstrate that MYB112 exerts a major role in regulation of plant flavonoid metabolism. We report enhanced and impaired anthocyanin accumulation in MYB112 overexpressors and MYB112-deficient mutants, respectively. Expression profiling reveals that MYB112 acts as a positive regulator of the transcription factor PAP1 leading to increased anthocyanin biosynthesis, and as a negative regulator of MYB12 and MYB111, which both control flavonol biosynthesis. We also identify MYB112 early responsive genes using a combination of several approaches. These include gene expression profiling (Affymetrix ATH1 micro-arrays and qRT-PCR) and transactivation assays in leaf mesophyll cell protoplasts. We show that MYB112 binds to an 8-bp DNA fragment containing the core sequence (A/T/G)(A/C)CC(A/T)(A/G/T)(A/C)(T/C). By electrophoretic mobility shift assay (EMSA) and chromatin immunoprecipitation coupled to qPCR (ChIP-qPCR) we demonstrate that MYB112 binds in vitro and in vivo to MYB7 and MYB32 promoters revealing them as direct downstream target genes. MYB TFs were previously reported to play an important role in controlling flavonoid biosynthesis in plants. Many factors acting upstream of the anthocyanin biosynthesis pathway show enhanced expression levels during nitrogen limitation, or elevated sucrose content. In addition to the mentioned conditions, other environmental parameters including salinity or high light stress may trigger anthocyanin accumulation. In contrast to several other MYB TFs affecting anthocyanin biosynthesis pathway genes, MYB112 expression is not controlled by nitrogen limitation, or carbon excess, but rather is stimulated by salinity and high light stress. Thus, MYB112 constitutes a previously uncharacterised regulatory factor that modifies anthocyanin accumulation under conditions of abiotic stress. / Transkriptionsfaktoren (TFs) sind ubiquitäre Regulatoren der Genexpression und spielen eine essentielle Rolle in nahezu allen biologischen Prozessen. Diese Doktorarbeit hat vor allem die funktionelle Charakterisierung von MYB112 zum Thema, einem Mitglied der R2R3-MYB-TF-Familie aus der Modellpflanze Arabidopsis thaliana. Ausgesucht wurde das Gen aufgrund seiner erhöhten Expression in seneszenten Blättern, basierend auf vorangegangenen qRT-PCR Expressions-Profiling Experimenten für 1880 TFs in Arabidopsis Blättern aus drei Entwicklungsstadien (15 mm Blatt, 30 mm Blatt und 20 % vergilbtes Blatt). MYB112-Promotor-GUS-Fusionslinien wurden generiert um das Expressionsmuster von MYB112 detailliert zu untersuchen. Unter Zuhilfenahme transgener Ansätze in Kombination mit Metabolomics und Transcriptomics können wir zeigen, dass MYB112 eine wichtige Rolle in der Regulation des pflanzlichen Flavonoid-Metabolismus spielt. In MYB112 Überexpressoren und MYB112-defizienten Mutanten kommt es zu erhöhter bzw. verminderter Anthocyanin-Akkumulation. Expressions-Profiling zeigt, dass MYB112 einerseits als ein positiver Regulator des Transkriptionsfaktors PAP1 fungiert, was zu einer erhöhten Anthocyanin-Biosynthese führt, andererseits als negativer Regulator von MYB12 und MYB111 auftritt, welche beide die Flavonol-Biosynthese kontrollieren. Wir haben früh auf MYB112 reagierende Gene durch eine Kombination verschiedener Ansätze identifiziert. Diese umfassen Genexpressions-Profiling (Affymetrix ATH1 Microarrays und qRT-PCR) und Transaktivierungs-Experimente in Mesophyll-Protoplasten aus Blättern. Wir zeigen, dass MYB112 an eine 8-bp DNA-Fragment, welches die Kernsequenz (A/T/G)(A/C)CC(A/T)(A/G/T)(A/C)(T/C) aufweist. Mit Hilfe von Electrophoretic Mobility Shift Assay (EMSA) und Chromatin-Immunopräzipitation gekoppelt mit qPCR (ChIP-qPCR) zeigen wir, dass MYB112 in vitro und in vivo an die Promotoren von MYB7 und MYB32 bindet was sie damit als direkte Zielgene von MYB112 identifiziert. Es wurde bereits gezeigt, dass MYB TFs eine wichtige Rolle bei der Kontrolle der Flavonoid-Biosynthese in Pflanzen haben. Viele Faktoren, die oberhalb des Anthocyanin-Biosyntheseweges agieren, werden bei Stickstofflimitierung oder erhöhter Saccharose-Konzentration auch verstärkt exprimiert. Außer den erwähnten Bedingungen können auch andere Umweltparameter, wie z. B. erhöhter Salzgehalt und Starklicht zu erhöhter Expression führen. Im Gegensatz jedoch zu einigen anderen MYB TFs, die einen Einfluss auf Gene des Anthocyanin-Biosyntheseweges ausüben, ist die Expression von MYB112 nicht durch Stickstoff-Limitierung oder Kohlenstoffüberfluss kontrolliert, sondern wird durch erhöhten Salzgehalt sowie Starklicht stimuliert. Somit ist MYB112 ein neuer Regulator, der eine Anthocyanin-Akkumulation unter abiotischen Stressbedingungen kontrolliert.