The D3 dopamine receptor (D3R) belongs to D2-class of dopamine receptors (DARs) and is involved in emotion, movement, and reward. D3R dysfunction has been reported in some neuropsychiatric disorders such as addiction, cognitive deficits, depression, schizophrenia, and Parkinson’s disease. Genetic studies have shown two polymorphic variants of the D3R gene resulting from substitution of serine to glycine at position nine of the amino terminus. Isoform 5 of adenylyl cyclase (AC5) is one of the nine transmembrane bound ACs in the brain and myocardium. Previous studies in rats have shown that AC5 is expressed in the striatum, nucleus accumbens and olfactory tubercle and at lower levels in islands of Calleja, where the D3R is also expressed. Previous studies showed that although D2R and D4R inhibit ACs activity in different cell types, inhibition of ACs by D3R is weak and often undetectable. It has been shown that D3R selectively inhibits AC5 activity in human embryonic kidney 293 (HEK293) cells co-transfected with D3R and AC5. Co-expression of D3R and AC5 in brain regions which are major coordinators of normal and pathological movement, and the selective inhibition of AC5 activity by D3R raise the possibility of a functional link between AC5 and D3R in the modulation of signal transduction and trafficking. I hypothesized that AC5 plays a unique role in modulation of D3R trafficking and signaling pathways through interaction between D3R and AC5. Herein, I demonstrated an interaction between D3R and AC5 in vivo and in vitro using reciprocal co-immunoprecipitation/immunoblotting (co-IP/IB) assays. Interestingly, DA may facilitate the formation of protein complex between D3R and AC5 in vitro. Radio ligand binding assays revealed that heterodimerization of D3R polymorphic variants with AC5 does not change ligand binding affinity and expression of the D3R. Furthermore, taking advantages of GloSensor assays, selective inhibition of AC5 activity by D3Ser9 and D3Gly9 has been shown following activation by DA and quinpirole. Using ELISA studies showed that AC5 promotes cell surface expression and total expression of D3Ser9 and D3Gly9. Moreover, ELISA results suggested that AC5 facilitates DA-induced D3Ser9 endocytosis in dynamin and β-arrestin 2 dependent process, while having no effect on D3Gly9 polymorphic variant. The results also revealed that AC5 attenuates heterologous (PKC-induced) internalization of D3Ser9, while it does not have any effect on D3Gly9 heterologous internalization. My results also displayed a complex formation between D3R, AC5 and, β-arrestin 2 under basal and DA stimulation conditions, which emphasize the role of β-arrestin 2 in D3R signal transduction. Overall, a new regulatory mechanism for D3R has been suggested. My results suggested that complex formation between both D3R polymorphic variants with AC5 can regulate signaling and trafficking properties of D3R without changing the binding affinity of the receptor. These data will be meaningful for understanding of diseases and developing treatment strategies.

The soluble form of ADCYs, ADCY10, is ubiquitously expressed in the cytoplasm and distinct organelles including cell nucleus. In contrast to its membrane-associated isoforms (ADCY1-9) which are stimulated by G-protein-coupled receptors, ADCY10 is activated by bicarbonate (HCO3-) and can form cAMP in nearly all cell compartments. ADCY10 is involved in a variety of physiological as well as pathological processes including cell cycle control in tumor cells. However, the underlying mechanism is still unclear. Here the role of ADCY10 in cell cycle control and cell proliferation is studied in endothelial cells from human umbilical veins (HUVECs). The current study reveals that ADCY10 and α-Tubulin translocate and colocalize during mitosis suggesting a role of ADCY10 in cell division. In addition, FACS analysis demonstrated that ADCY10 plays a role in cell proliferation by modulating cell cycle control. Inhibition of ADCY10 by 0 mM HCO3- or 10 μM KH7 (specific ADCY10 inhibitor) induced cell accumulation in G2 phase rather than M phase determined by decreased mitotic indicator cyclin B1 level. Thus, ADCY10 inhibition leads to decreased cell proliferation. The known cAMP effectors, Epac and PKA, were assessed as possible downstream targets of ADCY10 in cell proliferation. It was shown that ADCY10 and Epac induce cell proliferation via ERK1/2-MAPK pathway. Inhibition of Epac was associated with suppressed cell proliferation. However, an arrest of cell cycle after Epac inhibition was observed in G0/G1 phases rather than S or G2/M phases. Thus, Epac inhibition causes a different arrest of cell cycle compared to ADCY10 inhibition. Regarding PKA, it was demonstrated that deficiency of PKA might play a role in either activation or inhibition of cell proliferation. However, direct inhibition of PKA by PKI and H-89 did not lead to cell accumulation in G2. This effect might be associated with broadened roles of PKA in different pathways. In contrast, direct stimulation of PKA under ADCY10 inhibition revealed that PKA is a downstream molecule of ADCY10 as a regulator of cell cycle transition from G2 to mitotic phase. However, the underlying pathway remains to be investigated. The cell cycle transition of G2/M phase is regulated by an auto-amplification loop of cyclin B1/CDK1, which is controlled by the kinase WEE1 and the phosphatase PP2A. WEE1 content was regulated via ADCY10 but was independent of PKA or Epac. Direct inhibition of PP2A showed a suppression of cell proliferation and induced cell cycle arrest in G2. These results were in accordance with those observed after the ADCY10. Furthermore, inhibition of ADCY10 had no effect on PP2A expression level but rather affected PP2A activity and was independent of Epac and PKA. Therefore, this data provides evidence that ADCY10 controls cell proliferation and cell cycle regulation via PP2A. Taken together, ADCY10 coordinates the cell cycle progression in a complex framework. Downstream of ADCY10, Epac promotes G1/S transition, whereas PKA mediates cell cycle transition of G2/M.

The deposition of β-amyloid peptides and of α-synuclein proteins is a neuropathological hallmark in the brains of Alzheimer’s disease (AD) and Parkinson’s disease (PD) subjects, respectively. However, there is accumulative evidence that both proteins are not exclusive for their clinical entity but instead co-exist and interact with each other. Here, we investigated the presence of a newly identified, pyroglutamate79-modified α-synuclein variant (pGlu79-aSyn)—along with the enzyme matrix metalloproteinase-3 (MMP-3) and glutaminyl cyclase (QC) implicated in its formation—in AD and in the transgenic Tg2576 AD mouse model. In the human brain, pGlu79-aSyn was detected in cortical pyramidal neurons, with more distinct labeling in AD compared to control brain tissue. Using immunohistochemical double and triple labelings and confocal laser scanning microscopy, we demonstrate an association of pGlu79-aSyn, MMP-3 and QC with β-amyloid plaques. In addition, pGlu79-aSyn and QC were present in amyloid plaque-associated reactive astrocytes that were also immunoreactive for the chaperone heat shock protein 27 (HSP27). Our data are consistent for the transgenic mouse model and the human clinical condition. We conclude that pGlu79-aSyn can be generated extracellularly or within reactive astrocytes, accumulates in proximity to β-amyloid plaques and induces an astrocytic protein unfolding mechanism involving HSP27.

Lebewesen nutzen nukleotid-basierte sekundäre Botenstoffe um extra- und intrazelluläre Signale zur Induktion einer entsprechenden Zellantwort weiterzuleiten. Das zyklische Dinukleotid c-di-AMP steuert verschiedene physiologische Prozesse und ist für viele Bakterien unter bestimmten Bedingungen essentiell. Dieses Signalmolekül muss präzise reguliert werden, da seine Akkumulation oft toxisch ist. Diadenylatzyklasen mit einer DAC-Domäne synthetisieren c-di-AMP, welches von Phosphodiesterasen (PDE) mit DHH/DHHA1- oder HD-Domänen abgebaut wird. Streptomyceten sind im Boden lebende, Gram-positive Actinobakterien mit einem komplexen Lebenszyklus, während welchem sie vielzählige sekundäre Metabolite, inklusive Antibiotika, produzieren. Die Regulierung des zellulären c-di-AMP und seine Bedeutung in der Streptomyceten-Biologie waren zu Beginn dieser Studie weitgehend unbekannt. Zur c-di-AMP-Synthese nutzen Streptomyces die DAC DisA, besitzen aber keine der typischen PDEs sowie die meisten der bekannten c-di-AMP-bindenden Effektoren. Diese Arbeit zeigt, dass DisA die wichtigste c-di-AMP-Synthetase in Streptomyces venezuelae ist. AtaC wurde als eine PDE identifiziert, welche eine neue Klasse von c-di-AMP PDEs begründet. Während eine ataC-Deletion zu Störungen in Differenzierung und Wachstum in S. Venezuelae führt, führt die Inaktivierung von disA zur Sensitivität gegenüber erhöhten Konzentrationen von monovalenten Kationen im Medium. CpeA und CpeD wurden als erste c-di-AMP-bindende Proteine im Streptomyces Signalnetzwerk charakterisiert. Die entsprechenden Gene sind in Operons mit putativen Kation/Proton-Antiportern cpeB bzw. cpeE kodiert und die jeweiligen Genprodukte interagieren c-di-AMP-abhängig in vivo. Obwohl Deletion von cpe und Überexpression von cpeABC in S. venezuelae keine Phänotypen zeigten, verbesserte die CpeABC-Expression in Escherichia coli das Wachstum in Kalium-supplementierten Medien, was auf eine Funktion von cpe in der Regulation von Kalium hindeutet. / Nucleotide second messengers are used by all forms of life to transduce extra and intracellular signals and translate them into a physiological cell response. The cyclic dinucleotide c-di-AMP is a signaling molecule involved in diverse functions in bacterial physiology and is essential for many bacteria under certain growth conditions. However, this second messenger has to be tightly regulated since increased levels of c-di-AMP can be toxic. In many bacteria diadenylate cyclases with a conserved DAC domain synthesize c-di-AMP and phosphodiesterases (PDEs) with a DHH/DHHA1 or HD domain degrade it. Streptomyces spp. are soil-inhabiting gram-positive Actinobacteria characterized by a sophisticated developmental life cycle during which they produce various secondary metabolites, including antibiotics. The regulation and role of c-di-AMP is not well understood in Streptomyces biology. For c-di-AMP synthesis, streptomycetes utilize the DAC DisA but do not encode any canonical PDE and most of the known effector proteins for c-di-AMP signal transduction are absent. In this work, I demonstrated that DisA is the primary c-di-AMP synthetase in Streptomyces venezuelae and characterized AtaC as the founding member of a novel class of c-di-AMP-specific PDEs. In S. venezuelae, deletion of ataC interferes with development and growth, whereas disA inactivation affects bacterial survival under high ion osmotic stress conditions. Further, I identified CpeA and CpeD as the first c-di-AMP-binding proteins in Streptomyces. The respective genes are encoded in operons with the predicted cation/proton antiporters cpeB and cpeE, respectively, and the gene products interact in vivo in a c-di-AMP-dependent manner. Although neither cpe deletion nor overexpression of cpeABC produced a phenotype in S. venezuelae, expression of cpeABC in Escherichia coli improved growth in liquid media supplemented with potassium, suggesting that Cpe transporters are involved in potassium homeostasis.

No description available.

No description available.

Parkinson’s disease (PD) is a progressive neurodegenerative disorder that is neuropathologically characterized by degeneration of dopaminergic neurons of the substantia nigra (SN) and formation of Lewy bodies and Lewy neurites composed of aggregated α-synuclein. Proteolysis of α-synuclein by matrix metalloproteinases was shown to facilitate its aggregation and to affect cell viability. One of the proteolysed fragments, Gln79-α-synuclein, possesses a glutamine residue at its N-terminus. We argue that glutaminyl cyclase (QC) may catalyze the pyroglutamate (pGlu)79-α-synuclein formation and, thereby, contribute to enhanced aggregation and compromised degradation of α-synuclein in human synucleinopathies. Here, the kinetic characteristics of Gln79-α-synuclein conversion into the pGlu-form by QC are shown using enzymatic assays and mass spectrometry. Thioflavin T assays and electron microscopy demonstrated a decreased potential of pGlu79-α-synuclein to form fibrils. However, size exclusion chromatography and cell viability assays revealed an increased propensity of pGlu79- α-synuclein to form oligomeric aggregates with high neurotoxicity. In brains of wild-type mice, QC and α-synuclein were co-expressed by dopaminergic SN neurons. Using a specific antibody against the pGlu-modified neo-epitope of α-synuclein, pGlu79-α-synuclein aggregates were detected in association with QC in brains of two transgenic mouse lines with human α-synuclein overexpression. In human brain samples of PD and dementia with Lewy body subjects, pGlu79-α-synuclein was shown to be present in SN neurons, in a number of Lewy bodies and in dystrophic neurites. Importantly, there was a spatial co-occurrence of pGlu79-α-synuclein with the enzyme QC in the human SN complex and a defined association of QC with neuropathological structures. We conclude that QC catalyzes the formation of oligomer-prone pGlu79-α-synuclein in human synucleinopathies, which may—in analogy to pGlu-Aβ peptides in Alzheimer’s disease—act as a seed for pathogenic protein aggregation.

Die Biosynthese von Chlorophyll ist komplex und umfasst mehr als ein Dutzend enzymatische Schritte. Es ist nur allzu selbstverständlich, dass eine Deregulation der Chlorophyllsynthese globale Effekte auf die Zelle hat. Um diese Konsequenzen näher zu beleuchten, wurden Arabidopsis thaliana Pflanzen mit chemisch induzierter Deaktivierung von zwei Chlorophyllbiosynthesegenen (CHLH bzw. CHL27) erzeugt sowie photoautotophe Zellsuspensionskulturen von Arabidopsis thaliana hinsichtlich kurzzeitig induzierter Signalprozesse untersucht. Die Resultate verdeutlichen, dass durch Fehlregulationen innerhalb der Chlorophyllbiosynthese erzeugte reaktive Sauerstoffspezies die Transkriptionskontrolle kernkodierter Gene beeinflussen. Die Untersuchung eines enzymatischen Schrittes der Chlorophyllbiosynthese trat in dieser Arbeit in den Hauptfokus: Die Bildung des fünften, isozyklischen Ringes im Chlorophyllmolekül, katalysiert durch das bisher unzureichend erforschte Enzym Mg-Protoporphyrin-IX-monomethylester-Cyclase (Cyclase). Anhand von transgenen Cyanobakterien und Pflanzen sollte das noch unbekannte Gen ycf54 hinsichtlich seiner physiologischen Funktion in dem Cyclase-Enzymschritt analysiert werden. Das Fehlen von Ycf54 in Synechocystis sp. PCC6803 bzw. des homologen LCAA-Proteins in Nicotiana tabacum und Arabidopsis thaliana führt zu starken Cyclase-Substrat-Akkumulationen, verringerten Chlorophyllgehalten und reduzierten Ycf59- bzw. CHL27-Proteingehalten. Ein Mangel von Ycf54/LCAA beeinträchtigt daher die Funktionalität des Cyclase-Komplexes und scheint sich zudem interessanterweise auch auf die Stabilität photosynthetischer Antennenkomplexe auszuwirken. Mittels Pulldown-Assays konnte für Arabidopsis thaliana die direkte physikalische Interaktion zwischen LCAA und CHL27 bestätigt werden. Darüber hinaus sind erste Hinweise für die Ferredoxin-NADP-Reduktase als potenziellen Interaktionspartner gezeigt. / Synthesis of chlorophyll is a complex metabolic process and encompasses more than a dozen enzymatic reactions. It is self-evident that a deregulation of chlorophyll biosynthesis evokes global cellular impacts. To elucidate these consequences Arabidopsis thaliana plants with chemically inducible deactivation of two chlorophyll biosynthesis genes (CHLH and CHL27, respectively) were generated and photoautotrophic cell suspension cultures of Arabidopsis thaliana were used for short induced signal processes. The results illustrate that reactive oxygen species provoked by a deregulated chlorophyll synthesis affect the control of transcription of nuclear genes. The investigation of one enzymatic step of chlorophyll biosynthesis was placed as main focus: The formation of the isocyclic ring of the chlorophyll molecule catalyzed by the Mg protoporphyrin IX monomethyl ester cyclase (short: cyclase), an enzyme which is not fully investigated so far. The still unknown hypothetical chloroplast open reading frame (ycf) ycf54 should be analyzed concerning it’s physiological function in the enzymatic step of the cyclase using transgenic cyanobacteria and plants. Lack of Ycf54 in Synechocystis sp. PCC6803 and the homologous LCAA protein in Nicotiana tabacum and Arabidopsis thaliana, respectively, leads to chlorophyll deficiency, a strong accumulation of the cyclase substrate and reduced protein contents of Ycf59 and CHL27, respectively. A deficit of Ycf54/LCAA impairs the functionality of the cyclase complex and also might compromise the stability of photosynthetic antenna complexes. Using pull-down assays a direct physical interaction between LCAA and CHL27 could be confirmed. Additionally, first evidences for ferredoxin NADP reductase as a potential interaction partner was given.

<p>The duodenal epithelium secretes bicarbonate ions and this is regarded as the primary defence mechanism against the acid discharged from the stomach. For an efficient protection, the duodenum must also function as a sensory organ identifying luminal factors. Enteroendocrine cells are well-established intestinal “taste” cells that express signaling peptides such as orexins and guanylins. Luminal factors affect the release of these peptides, which may modulate the activity of nearby epithelial and neural cells.</p><p>The present thesis considers the effects of orexins and guanylins on duodenal bicarbonate secretion. The duodenal secretory response to the peptides was examined in anaesthetised rats <i>in situ</i> and the effects of orexin-A on intracellular calcium signaling by human as well as rat duodenal enterocytes were studied <i>in vitro</i>.</p><p>Orexin-A, guanylin and uroguanylin were all stimulants of bicarbonate secretion. The stimulatory effect of orexin-A was inhibited by the OX₁-receptor selective antagonist SB-334867. The muscarinic antagonist atropine on the other hand, did not affect the orexin-A-induced secretion, excluding involvement of muscarinic receptors. Orexin-A induced calcium signaling in isolated duodenocytes suggesting a direct effect at these cells. Interestingly, orexin-induced secretion and calcium signaling as well as mucosal orexin-receptor mRNA and OX₁-receptor protein levels were all substantially downregulated in overnight fasted rats compared with animals with continuous access to food. Further, secretion induced by Orexin-A was shown to be dependent on an extended period of glucose priming.</p><p>The uroguanylin-induced bicarbonate secretion was reduced by atropine suggesting involvement of muscarinic receptors. The melatonin receptor antagonist luzindole attenuated the secretory response to intra-arterially administered guanylins but had no effect on secretion when the guanylins were given luminally. </p><p>In conclusion, the results suggest that orexin-A as well as guanylins may participate in the regulation of duodenal bicarbonate secretion. Further, the duodenal orexin system is dependent on the feeding status of the animals.</p>

Adenylate cyclase toxin (CyaA) produced by the causative agent of whooping cough Bordetella pertussis, is a key virulence factor important for colonization of the host. CyaA targets preferentially myeloid phagocytes expressing CD11b/CD18 integrin. By elevating cytosolic cAMP in the host cells, CyaA interferes with their phagocytic, chemotactic and oxidative burst capacities. Furthermore, CyaA modulates the secretion of cytokines and the maturation state in LPS-stimulated dendritic cells (DC) by affecting the expression of costimulatory molecules. In this study, we investigated the effects of CyaA on the capacity of murine bone-marrow DC to prime CD4+ and CD8+ T cells in response to ovalbumin epitopes delivered by the CyaA-AC- toxoid, as a model antigen. Further, we examined the possible impact of CyaA on the antigen uptake and processing for MHC class I and II-restricted presentation by DC, as we previously observed a decreased T cell stimulatory capacity of CyaA-treated DC in response to soluble ovalbumin. We found out that the high levels of cAMP generated by CyaA in LPS-stimulated DC account for the decreased presentation of ovalbumin epitopes carried by CyaA-AC- toxoid on MHC class I and II molecules, thereby impairing the CD8+ and CD4+ T cell responses. Whereas CyaA did not influence the...