Tesi sul tema "PDE2A"

Background and Aims : cAMP/PKA signaling is compartmentalized within the cell, with tight spatial and temporal control of signal propagation underpinning specificity of response. The cAMP-degrading enzymes, phosphodiesterases (PDEs), are key regulators of this process. They are situated in specific subcellular domains within which they control local cAMP levels. Several components of the cAMP/PKA cascade have been identified in different mitochondrial compartments, including isoform 2 of PDE2A and multiple A-kinase anchoring proteins, suggesting the presence of multiple cAMP/PKA signaling domains within the organelle. The aims of this project were to examine the role of PDE2A in cAMP/PKA signaling compartmentalization and its biological function, with a particular focus on its role at the mitochondria. Methods : Real-time PCR and fluorescence imaging were used to assess PDE2A expression and localization in different cell lines and tissues. Co-immunoprecipitation (Co-IP) was used to validate PDE2A2 interactors. Co-IP, electron microscopy and live-cell imaging were used to explore the role of PDE2A2 at the mitochondria. Fluorescence Resonance Energy Transfer (FRET) was used to investigate the involvement of PDE2A on endoplasmic reticulum (ER) Ca²⁺ handling and local cAMP/PKA dynamics. Results : PDE2A2 localizes to the outer mitochondria membrane and intermembrane space and it interacts with several mitochondria and ER proteins, including components of the MINOS (mitochondrial inner membrane organizing system) complex and Caveolins. Functionally, PDE2A2 is involved in mitochondria degradation by mitophagy while PDE2A1 seems to be involved in the regulation of Ca²⁺ release from the ER. Phosphatases are responsible for the differential PKA dynamics between cytosol and ER. Conclusions : PDE2A isoforms localize to different compartments and are involved in distinct cellular functions. The data presented in this thesis demonstrate that PDE2A2 activity at the mitochondria controls mitochondria clearance by mitophagy. This mechanism is dysregulated in multiple pathological conditions, making PDE2A2 a potential therapeutic target.

Le Syndrome de l’X Fragile (SXF) est la forme la plus fréquente de déficience intellectuelle héréditaire. Le phénotype des patients SXF est complexe. En effet, ils présentent aussi des traits autistiques, de l’hyperactivité et un déficit de l’attention. Au niveau du cerveau, le phénotype majeur est la présence d’épines dendritiques plus nombreuses, plus longues et plus fines. Ces anomalies morphologiques sont associées à des formes altérées de plasticité synaptique chez le modèle murin de SXF, la souris Fmr1-KO. Le SXF est causé par l’expansion (plus de 200 fois) du triplet nucléotidique CGG dans le 5’UTR du gène Fragile X Mental Retardation 1 (FMR1). L’hyperméthylation de cette région du gène FMR1 et, surtout de son promoteur, induit la suppression de l’expression de FMR1 et donc l’absence de la protéine FMRP codé par ce dernier. Une expansion entre 50 et 200 fois du triplet est considérée comme une prémutation et est associée à deux autres maladies : le syndrome du tremblement-ataxie lié au X Fragile (FXTAS), une maladie neurodégénérative, et l’insuffisance ovarienne liée à la prémutation de l’X Fragile (FXPOI). FMRP se lie à l’ARNm et est impliquée dans la régulation de plusieurs étapes du métabolisme de l’ARN, en particulier dans la modulation de la traduction de plusieurs protéines synaptiques. Aucune thérapie n’est aujourd’hui disponible pour traiter les patients atteints de SXF. J’ai montré que FMRP module l’expression de la Phosphodiesterase 2A (PDE2A) au niveau synaptique. PDE2A est une enzyme impliquée dans la dégradation de l’AMPc et du GMPc, deux seconds messagers qui jouent un rôle critique dans le développement et la différenciation des neurones. Quand FMRP est absente, le niveau et l’activité de PDE2A sont augmentés et, en conséquence, les niveaux d’AMPc et de GMPc sont réduits dans le cortex et l’hippocampe. J’ai montré que le traitement avec un inhibiteur spécifique de la PDE2A, le BAY 60-7550, rétablit les déficits sociaux chez les souris Fmr1-KO jeunes et adolescentes, la morphologie anormale des épines dendritiques et la Long Term Depression mGluR-dépendante exagérée. Afin de valider par une approche génétique que la réduction du niveau (et de l’activité) de la PDE2A pourrait avoir un rôle thérapeutique pour le SXF, j’ai croisé la lignée Pde2a+/- avec les souris Fmr1-KO et analysé le phénotype comportemental, la morphologie neuronale et les voies de signalisation liées à l’AMPc et au GMPc dans l’hippocampe et le cortex des souris générées par ce croisement. En effet, j’ai pu montrer que les animaux Fmr1-KOxPde2a+/- ont un phénotype sociocognitif normal comparé aux les animaux Fmr1-KO et Pde2a+/-. En effet, les souris Pde2a+/- ont un comportement très similaire à celui des animaux Fmr1-KO même si les altérations moléculaires dans le cerveau apparaissent inversées comparées à celles des souris Fmr1-KO. Dans leur ensemble, mes résultats montrent l’importance des voies AMPc et GMPc qui dépendent de PDE2A dans la pathophysiologie de FXS et dans le neurodéveloppement. En effet, j’ai identifié un nouveau modèle de maladies du développement du cerveau dû à la réduction du niveau d’activité de Pde2a+/-. Dans la première partie de mon doctorat, j’ai contribué à l’identification de la morphologie des neurones corticaux obtenus à partir d’un modèle murin de FXTAS
Fragile X Syndrome (FXS) is the most common form of inherited intellectual disability (ID). In addition to ID, patients might exhibit autistic features, hyperactivity and attention-deficit disorder, language dysfunction and seizures. Approved and effective therapies are not yet available for FXS. The main hallmark of FXS in the brain is the presence of immature and denser dendritic spines. This abnormality is associated with altered synaptic plasticity in our animal model of FXS, the Fmr1-KO mice. FXS is caused by a CGG repeat expansion with a number of repeats higher than 200 in the 5’UTR of the Fragile X Mental Retardation 1 (FMR1) gene. Methylation of this region and of its surrounding sequences, including the promoter of FMR1, results into the silencing of the gene and the loss of its encoded protein, FMRP. An expansion with a number of repeats variable between 50 and 200 is considered as a premutation and is associated with two pathological conditions other than FXS: the Fragile X-associated Tremor Ataxia Syndrome (FXTAS), and the Fragile Xassociated Primary Ovarian Insufficiency (FXPOI). FMRP is an RNA-binding protein implicated in various steps of RNA metabolism. In particular, it plays a role in the translational regulation of a subset of synaptic proteins. We showed that FMRP modulates the synaptic expression of Phosphodiesterase 2A (PDE2A), an enzyme implicated in the degradation of cAMP and cGMP. In. the absence of FMRP, the levels and the activity of PDE2A are increased, and, consequently, cAMP/cGMP levels are reduced in the cortex and hippocampus of Fmr1-KO mice. The blockade of PDE2A with a specific and powerful inhibitor of PDE2A, Bay 607550, rescues various in vitro, ex vivo and in vivo FXS phenotypes. In particular, I contributed to this study by showing that the treatment with Bay 60-7550 rescues social deficits in young and adolescent Fmr1-KO mice and abnormal dendritic spine morphology in hippocampus. In order to validate this new therapeutic target for FXS, I crossed Pde2a+/- mice with Fmr1-KO mice and analyzed their behavior, neuronal morphology and signaling pathways linked to cAMP/cGMP levels in the hippocampus and cortex. I found that double mutant animals (Fmr1-KO x Pde2a+/-) have a normal social and cognitive behavior compared to both Fmr1-KO and Pde2a+/- mice. Indeed, Pde2a+/- mice display behavioral deficits similar to those characterizing the Fmr1-KO mouse, even if the molecular alterations seem to be opposite. In conclusion, my results highlight the key role of PDE2A-dependent cAMP and cGMP levels and their correlated pathways in neurodevelopment. Furthermore, in addition to my main project, in the first part of my doctoral stage, I participated to the characterization of neuronal morphology in the mice model of FXTAS showing that this neurodevelopmental marker is dependent on the level of the Fmr1 mRNA, which displays elevated levels in these mice in the presence of the Fmr1 premutation

Cyclic 3’, 5’ adenosine monophosphate (cAMP) signalling downstream of prostacyclin (PGI₂) is a key inhibitory pathway in blood platelets. This pathway is dynamically regulated by phosphodiesterase 3A (PDE3A), which hydrolyses cAMP into metabolically inactive AMP. Although PDE3A is an established drug target in anti-platelet therapies, the molecular mechanisms that underlie its function in platelets remain unclear. Therefore, the major aim of this study was to further explore PDE3A signalling in human platelets. Using a combination of cell fractionation and immunoblotting we identified two PDE3A splice variants in platelets, PDE3A1 and PDE3A2, that were differentially localised within the cell. PDE3A1 was located in the membrane fraction, whereas PDE3A2 was primarily located in the cytosolic fraction. Treatment of platelets with PGI2 induced a transient phosphorylation of PDE3A2 at Ser³¹² in a PKA dependent manner. In contrast, no phosphorylation of PDE3A1 was detected. The phosphorylation of PDE3A2 was associated with increased PDE3A enzymatic activity, which suggested that cAMP signalling activated only the cytosolic form of the enzyme. In many cells, A-kinase anchoring proteins (AKAPs) orchestrate a coordinated response between PKA and its effector proteins. The phosphorylation and activation of PDE3A2 in response to PGI2 was blunted by a cell permeable peptide inhibitor of PKA-AKAP interactions suggesting that PKA-mediated activation of PDE3A2 was dependent on an AKAP. Using a cAMP-pull down approach to enrich cAMP binding proteins combined with immunoblotting, we confirmed the presence of two AKAP7 isoforms (δ and γ) in platelets. Additionally, we found that AKAP7δ co-precipitated with PDE3A2 and possessed associated PDE3A activity. Furthermore, AKAP7 also possessed PKA activity, which was a result of its constitutive association with PKA-II. Critically, immunoprecipitated PDE3A was found to be co-associated with both PKA-II and AKAP7δ. The findings in this thesis suggest that blood platelets express multiple differentially-regulated PDE3A splice variants, of which PDE3A2 is regulated by PKA-II within a novel cytosolic AKAP7δ facilitated signalling complex. The selective inhibition of PDE3A splice variants and/or pharmacological disruption of PDE3A signalosomes may provide safer and more specific ways of controlling pathological platelet activation.

3’,5’-cyclic adenosine monophosphate (cAMP) is a near ubiquitous second messenger responsible for the regulation of a myriad of physiological processes. It is produced by the adenylyl cyclases (AC) and degraded by phosphodiesterases (PDEs). The primary effector of cAMP is protein kinase A (PKA). In order to overcome cross-contamination of separate cAMP-mediated processes within the same cell strict spatiotemporal control is required. Compartmentalisation sculpts cAMP gradients allowing targeted cAMP/PKA action with the cell. This is achieved by the tethering of unique isoforms of AC, PKA and PDEs to distinct subcellular locations. This subcellular targeting is often carried out by A kinase anchoring proteins (AKAPs) which act as docking sites for the components of the cAMP signalling machinery. A number of AKAPs have been identified as tethering components of the cAMP signalling cascade to organelles facilitating the cultivation of a discrete localised pool of cAMP. This allows for the highly specific PKA-mediated phosphorylation of target proteins. There are reports of two AKAPs localised at the mitochondria: optic atrophy 1 (OPA1) and sphingosine kinase anchoring protein (SKIP). However, despite the acknowledged presence of these two key components of the cAMP signalling cascade being present at the mitochondria little is known about the functional relevance of cAMP signalling within the mitochondria. In this study, I established PDE2 as located within the mitochondria of both primary cardiac cells and a cardiac cell line. Furthermore, the PDE2 isoform present was identified as PDE 2A2. It was then demonstrated that PDE 2A2 was part of a previously identified protein complex located within the mitochondria known as the mitochondrial inner membrane organising system (MINOS) complex. Furthermore, the potential for direct protein-protein interactions between PDE2 and MINOS constituents was examined. It was then demonstrated that when PDE2 activity/expression was reduced mitochondrial length would significantly increase and when PDE2 was overexpressed mitochondrial length would significantly decrease. Furthermore, manipulation of PDE2 expression/activity also led to significant changes in mitochondrial membrane potential (MMP). In summary, the data presented here indicate that PDE 2A2 is part of a multiprotein complex located within the mitochondria. Furthermore, disruption of PDE 2A2 within this complex leads to alterations in mitochondrial physiology.

Les nucléotides cycliques sont des seconds messagers intracellulaires possédant une grande importance dans la signalisation cellulaire du follicule ovarien. Les niveaux intracellulaires en nucléotides cycliques tel que l'adénosine monophosphate cyclique (AMPc) dépendent de leur synthèse, assurée par l'adenylyl-cyclase (AC) ainsi que leur dégradation par les phosphodiéstérases (PDE). Ces dernières appartiennent à la superfamille des métalophosphohydrolases, elles hydrolysent le groupement phosphate en 3' des nucléotides cycliques pour produire un nucléotide 5' phosphate. Dans les cellules ovariennes, plusieurs familles de PDE ont été identifiées, agissant comme modulateurs des taux intracellulaires de nucléotides cycliques. Dans le présent projet, nous nous attarderons à étudier la signalisation cellulaire chez les cellules ovariennes impliquant l'AMPc. La régulation de ses concentrations intracellulaires affecte plusieurs processus physiologiques. Le projet s'intéresse particulièrement à une famille d'enzyme de dégradation de l'AMPc, la phosphodiestérase8A (PDE8A). Nous voulons donc valider la présence fonctionnelle de la famille de PDE8A dans les cellules ovariennes ainsi que la mitochondrie afin de comprendre l'implication de cette enzyme dans la physiologie cellulaire en s'attardant à la stéroïdogenèse, étant donné que les mitochondries sont un organite cellulaire essentiel pour la stéroïdogenèse parce qu'elles représentent le site de synthèses de plusieurs hormones stéroïdiennes. En effet, des récents travaux ont montré la famille PDE8 comme un régulateur de la stéroïdogenèse dans les cellules de Leydig. Dans cette étude, nous avons montré que le transcrit de PDE8A ainsi que sa protéine étaient exprimés dans les cellules de la granulosa, les cellules du cumulus et l'ovocyte chez le porc. Ainsi, la protéine PDE8a a été détectée par western blot dans des mitochondries isolées à partir des cellules de granulosa et des complexe ovocyte-cumulus (COC). Une co-localisation entre le signal immunoréactive de la PDE8A et le marquage réalisé par mitotracker a été observée dans les cellules de granulosa et des mitochondries isolées. De plus, la présence fonctionnelle de la PDE8 mesurée en tant qu'activité AMPc-PDE sensible au PF-04957325 a été détectée dans des cellules de la granulosa et des mitochondries isolées supportant la présence fonctionnelle de la PDE8A dans les mitochondries isolées. De ce fait, cette observation soutient encore la localisation fonctionnelle mitochondriale de la PDE8A. Une association entre la mitochondrie et la PDE8A a aussi été démontrée par immunomicroscopie électronique qui a confirmé l'ancrage de la protéine sur la membrane mitochondriale externe. Pour évaluer l'implication de la mitochondrie dans la stéroïdogenèse, l'effet de PDE8A sur la production de progestérone a été mesuré dans les complexes ovocyte-cumulus en utilisant le PF-04957325 comme un inhibiteur spécifique de PDE8. Lorsque les COC sont cultivés sans FSH, un niveau basal de progestérone est mesuré. Par contre en présence de FSH une augmentation significative de sécrétion de progestérone est observée. En utilisant un inhibiteur spécifique de la PDE8 (PF-04957325) avec la FSH nous avons obtenu une augmentation encore plus importante de la sécrétion de progestérone. Ces résultats démontrent la présence fonctionnelle de PDE8A dans les cellules de la granulosa et les complexes ovocyte-cumulus. Puisque les mitochondries sont l'une des localisations de PDE8A, l'effet de l'inhibiteur spécifique de PDE8 sur la sécrétion de progestérone soutient la contribution mitochondriale de PDE8 dans la stéroïdogenèse.

Mestrado em Finanças
Os serviços públicos de abastecimento de água para consumo humano e saneamento de águas residuais, designados de serviços de águas, sendo prestados por entidades que operam em regime de monopólio natural, e nesta medida, regulados na maioria dos países, são o objeto de estudo neste trabalho. Coloca-se a questão de como podem estes serviços, de interesse económico geral, e recentemente reconhecidos pela ONU como direitos humanos, ser universalmente prestados com qualidade, continuidade e a preços acessíveis, sem que as eventuais ineficiências das entidades que os prestam penalizem significativamente o preço cobrado aos consumidores. Tendo presente o modelo de regulação económica vigente na Colômbia para os serviços de ?acueducto y alcantarillado?, internacionalmente reconhecido como uma boa prática, pretende-se com este trabalho de investigação compreender este modelo nos seus aspetos fundamentais, para posteriormente se conseguir avaliar a possibilidade de implementação no contexto português. Para além de uma análise teórica aos fatores de contexto, foi possível testar a aplicação do modelo colombiano a um conjunto de entidades gestoras que operam em Portugal e retirar algumas conclusões. Deu-se especial relevância à componente eficiência incluída nas fórmulas tarifárias uma vez que os incentivos à melhoria contínua são um dos principais aspetos a melhorar no modelo regulatório português.
Public services of water supply for human consumption and wastewater disposal, also known as water services, are provided by operators that operate on natural monopolies and are, therefore, object of regulation in most countries. This reality brings the question of how can these services of general economic interest, recently recognized by the UN as human rights, be universally provided with quality, continuity and affordable, without increasing significantly the price charged to consumers due to the operators? inefficiencies. This research intends to understand the current model of economic regulation of these services in Colombia (services of ?acueducto y alcantarillado?), internationally recognized as good practice, and to assess the possibility of its implementation in the Portuguese context. Besides the theoretical analysis of context factors, it was possible to test the applicability of the Colombian model using a set of Portuguese operators, and draw some conclusions. The emphasis was put on the efficiency component included in pricing formulas, because the incentives for continuous improvement seem to be one of the main aspects to improve in the Portuguese regulatory model.

Heart failure is a global epidemic. 50% of patients die within 5 years of diagnosis and 30–50% will be of sudden death due to an arrhythmia. New medicines for heart failure are urgently required. This thesis used explanted hearts from patients with heart failure undergoing heart transplantation to investigate the cause of dangerous ventricular arrhythmias. It was found that both noradrenaline and adrenaline through activation of β1- and β2-adrenoceptors caused arrhythmias which could be controlled by enzymes called phosphodiesterases. These enzymes provide a novel target for future medicines to reduce the risk of arrhythmias in patients with heart failure.

碩士
國立清華大學
生物科技研究所
103
Mitochondria are dynamic organelles that continuously undergo fusion and fission. Mitochondrial dynamics is important for maintaining mitochondrial integrity and cellular function. Aging is a degenerative process that exhibit physiological and mitochondrial dysfunction. Previous studies of our lab revealed mitochondrial fragmentation was present in senescent yeast cells. Resveratrol, a polyphenol mitigated mitochondrial fragmentation and superoxide level. In addition, resveratrol elevated membrane potential in the senescent yeast cells. However, the mechanism of how resveratrol-mediated mitochondria in senescent yeast cells remains unclear. We aim to elucidate the potential mechanisms. Previous studies indicated resveratrol can ameliorate aging-related metabolic phenotypes via inhibiting phosphodiesterase activity. In this thesis, I applied biotin-streptavidin system to isolate replicative senescent yeast cells to clarify the effect of phosphodiesterase, Pde2 on mitochondria. My results demonstrated that deletion of PDE2 reverted the effects of resveratrol on mitochondria dynamics and showed different transcription level of fission/ fusion genes. In the mitochondrial activity assay, resveratrol did not mitigate the superoxide level in Δpde2 senescent cells. Those results indicated that Pde2 is required for resveratrol-mediated mitochondrial dynamics and superoxide scavenging. These evidences provide hints to elucidate resveratrol signaling pathway in the regulation of mitochondrial dynamics in cellular senescence.

碩士
中山醫學大學
醫學研究所
97
Eurycoma longifolia (Tongkat Ali) is a plant with folk medicine in Indonesia, Malaysia, Thailand and Vietnam. The residences in Malaysia usually treat it as “ginseng of Malaysia”, and think it possesses multiple medical functions such as anti-hypertension and appetite stimulation. The purpose of this project was to examine the effect of E. longifolia extract on plasma NO and cGMP concentration in mice. Viagra was also used for comparison. Mice were treated by E. longifolia, Viagra, L-Arginine, Tongkat Ali plus ginseng, Dioscorea for 2 and 4 weeks. Plasma NO, cGMP level and the activity of type 5 phophodiesterase and cytochrome P450 were analyzed. Results showed that 5 treatments increased the NO and cGMP levels, and inhibited the activity of type 5 phophodiesterase. Tongkat Ali and Tongkat Ali plus ginseng treated mice at 4 weeks has significantly higher cytochrome P450 mRNA expression. These treatments did not affect the anti-oxidative defense in liver and kidney. These findings implied that Tongkat Ali could provide protective or medical functions via elevating plasma NO and cGMP concentration and decreasing PDE5 enzyme activity.

Herzinsuffizienz ist ein weltweites Gesundheitsproblem mit hoher Morbidität und Mortalität und immer noch schlechter Prognose. Ein charakteristisches Merkmal der molekularen und damit verbundenen strukturellen Veränderungen, die der terminalen Insuffizienz vorangehen ist die durch Desensitivierungsmechanismen vermittelte Abnahme des beta-adrenergen (β-AR) Signalmoleküls zyklisches Adenosinmonophosphat (cAMP) auf der einen Seite und der gleichzeitigen Zunahme des von natriuretischen Peptiden (NP) und Stickstoffmonoxid (NO) generierten zyklischen Guanosinmonophosphat (cGMP) auf der anderen Seite. Während hohe cAMP-Spiegel im Herzen als schädlich gelten, werden cGMP-abhängige Signalkaskaden vorwiegend als protektiv verstanden. Amplitude, Lokalisation und Halbwertszeit beider Signalmoleküle werden durch spezifische Enzyme, den Phosphodiesterasen (PDE) reguliert. Unter der PDE-Superfamilie wird die Isoform PDE2 als einzige von cGMP aktiviert, um dann verstärkt cAMP abzubauen und steht damit im Zentrum eines negativen Crosstalks dieser beiden Signalwege. PDE2 ist sowohl in der humanen als auch der experimentellen Herzinsuffizienz hochreguliert und scheint dort am β-AR Desensitivierungsprozess beteiligt zu sein. Im Rahmen dieser Arbeit wurde die pathophysiologische Rolle der PDE2 im Herzen näher charakterisiert. Es wird gezeigt, dass die PDE2 nicht nur in Kardiomyozyten, sondern auch in kardialen Fibroblasten exprimiert wird. In Fibroblasten inhibieren cAMP/cGMP-Signalwege die Transformation von kardialen Fibroblasten (CF) zu Myofibroblasten (MyoCF), einem zellulären Phänotyp, der unter anderem mit der persistenten Fibrotisierung des erkrankten Herzgewebes in Verbindung gebracht wird. In CF führte eine Überexpression von PDE2 zu eine starken Abnahme der basalen und β2-AR-vermittelten cAMP-Synthese und war ausreichend, um in Abwesenheit exogener, pro-fibrotischer Stimuli die Transformation zum MyoCF zu induzieren. In Übereinstimmung zeigten funktionale Analysen mit künstlich hergestelltem Bindegewebe aus PDE2-überexprimierenden CF eine deutliche Zunahme der Gewebssteifigkeit. PDE2 übte keinen Einfluss auf basale oder durch das atriale NP generiertes cGMP aus und reduzierte nur partiell die NO-induzierte cGMP-Akkumulation. Interessanterweise waren beide Stimuli in der Lage, trotz niedriger cAMP-Spiegel die PDE2-induzierte CF-Transformation zum MyoCF zu verhindern und lassen daher eine Redundanz dieser beiden sonst so gegensätzlichen Signalwege vermuten. Zur Untersuchung von PDE2 in Kardiomyozyten wurde ein transgenes (TG) Mausmodell mit spezifischer kardialer Überexpression herangezogen. Die Basalcharakterisierung zeigte eine erniedrigte Herzfrequenz (HR) mit kompensatorisch erhöhter, basaler Kontraktionskraft, sowie eine verminderte Maximalantwort bezüglich der HR nach akuter β-AR Stimulation. Auf molekularer Ebene war dieser Phänotyp mit einer verminderten Phosphorylierung verschiedener β-AR Zielstrukturen wie Troponin I, Phospholamban und Ryanodinrezeptor-2 assoziiert. Langzeitstudien belegten, dass eine Überexpression von PDE2 keine pathologischen Konsequenzen hat, sondern im Gegenteil die durchschnittliche Lebensspanne der Tiere eher verlängerte. Erste Studien im Herzinsuffizienzmodel der transversalen Aortenkonstriktion (TAC) zeigten bisher eine beständig erniedrigte HR und verminderte Wanddicken bei allerdings vergleichbarer Abnahme der kardialen Kontraktionskraft. Trotz der klaren Befunde und neuen Erkenntnisse über die vielfältige Rolle der PDE2 im Herzen lässt sich bisher noch nicht klar belegen, ob eine zusätzliche Aktivierung von myokardialen PDE2 tatsächlich im Sinne einer intrazellulären β-AR-Blockade die Progression zur Herzinsuffizienz verlangsamen oder verhindern könnte. Weitere darauf aufbauende Untersuchungen, wie z.B. eine akut induzierbare Aktivierung bzw. Deaktivierung in experimentellen Herzinsuffizienzmodellen könnten den Weg für die Entwicklung klinisch anwendbarer Ansätze zur therapeutischen Modulation dieser viel versprechenden Zielstruktur ebnen.

Protein degradation by ATP dependent proteases is a universally conserved process. Recognition of substrates by such proteases commonly occurs via direct interaction or with the aid of a regulatory adaptor protein. An example of this regulation is found in Caulobacter crescentus, where key regulatory proteins are proteolysed in a cell-cycle dependent fashion. Substrates include essential transcription factors, structural proteins, and second messenger metabolism components. In this study, we explore sequence and structural requirements for regulated adaptor mediated degradation of PdeA, an important regulator of cyclic-di-GMP levels. Robust degradation of PdeA is dependent on the response regulator CpdR in vivo and in vitro. Here, I structurally identify a novel PAS domain in PdeA that is necessary and sufficient for CpdR mediated PdeA degradation. The PAS domain was found to contain a unique dimerization element that is associated with PdeA function. I show specifically that PdeA engages ClpXP through C-terminal recognition motifs. Finally, we present evidence that PdeA contains cryptic ClpXP recognition sites that are revealed during partial processing. Due to these uncommon degradation characteristics of PdeA, unique proteolytic insights may be gained by investigating this model system.

Cystic Fibrosis (CF) is the most common lethal autosomic recessive disorder among Caucasian population. It is caused by mutations in the gene encoding the Cystic Fibrosis Transmembrane conductance Regulator (CFTR) protein, a cAMP-regulated chloride channel expressed at the apical surface of epithelial cells. The most common CF-causing mutation is a deletion of phenylalanine 508 (F508del, present in 85% of CF patients) and leads to CFTR misfolding which is recognized by the endoplasmic reticulum (ER) quality control (ERQC) resulting in ER retention and degradation. Regulation of CFTR at the plasma membrane (PM) is a complex process involving different interaction partners and signalling pathways. Among these, cAMP regulates both channel gating through a protein kinase A (PKA)-dependent process and PM stability through activation of the exchange protein directly activated by cAMP 1 (EPAC1). Here, we aim to provide new insights into three main areas of CFTR regulation namely: 1) CFTR trafficking to the PM; 2) CFTR anchoring at the PM through EPAC1 activation and 3) impact of cAMP levels in the cell homeostasis (using CFTR as a mode). Using interactomics we identified factors specifically involved in the recogniton of ER exit/retention motifs. Extended comparative analysis showed that the CFTR interactome is much more shaped by the folding status of the protein than by its subcellular location. Using protein interaction profiling and bioinformatic analysis, we identified proteins that interact differentially with CFTR under EPAC1 activation. We characterized, for the first time, the actin cytoskeleton dynamics regulators INF2 and CAPZA2 as modulators of CFTR anchoring at the PM. Finally, we dissected the impact of cAMP levels in the stress response and trafficking pathways. We show that PDE2 regulates cAMP levels within the cell, with impact in the EPAC1 pathway, and also ER stress controlling the cellular homeostasis
Programa doutoral BioSys - Sistemas Biológicos, Genômica Funcional & Integrativa (FCT / PD/ 00065 / 2012) da Faculdade de Ciências da Universidade de Lisboa