No / Vascular relaxation induced by 3′,5′-cyclic adenosine monophosphate (cAMP) is both endothelium-dependent and endothelium-independent, although the underlying signaling pathways are not fully understood. Aiming to uncover potential mechanisms, we performed contraction–relaxation experiments on endothelium-denuded and intact rat aorta rings and measured NO levels in isolated human endothelial cells using single cell fluorescence imaging. The vasorelaxant effect of forskolin, an adenylyl cyclase activator, was decreased after selective inhibitor of protein kinase A (PKA), a cAMP-activated kinase, or L-NAME, an endothelial nitric oxide synthase (eNOS) inhibitor, only in intact aortic rings. Both selective activation of PKA with 6-Bnz-cAMP and exchange protein directly activated by cAMP (Epac) with 8-pCPT-2′–O-Me-cAMP significantly relaxed phenylephrine-induced contractions. The vasorelaxant effect of the Epac activator, but not that of the PKA activator, was reduced by endothelium removal. Forskolin, dibutyryl cAMP (a cAMP analogue), 6-Bnz-cAMP and 8-pCPT-2′–O-Me-cAMP increased NO levels in endothelial cells and the forskolin effect was significantly inhibited by inactivation of both Epac and PKA, and eNOS inhibition. Our results indicate that the endothelium-dependent component of forskolin/cAMP-induced vasorelaxation is partially mediated by an increase in endothelial NO release due to an enhanced eNOS activity through PKA and Epac activation in endothelial cells. / This work was supported by grants from the Ministerio de Ciencia e Innovación, Spain (SAF2010-22051) and Xunta de Galicia, Spain (INCITE08PXIB203092PR)

No description available.

No description available.

Cyclic AMP (cAMP) is a second messenger that translates extracellular signals into tightly regulated biological responses. The cAMP binding domain (CBD) is a conserved regulatory switch that binds to cAMP and allosterically controls multiple cellular functions. All CBDs share a common architecture comprised of α- and β-subdomains. cAMP binds to the phosphate binding cassette (PBC) nested within the β-subdomain. In mammals the main cAMP receptors are protein kinase A (PKA), guanine exchange factors (EPAC) and ion channel proteins, including both the hyperpolarization-activated cyclic nucleotide-dependent channels (HCN channels) and the cyclic nucleotide-gated channels (CNG channels). Impaired activities of these proteins are associated with diabetes, cardiovascular diseases, cancer and Alzheimer's disease. Therefore, these proteins represent promising therapeutical targets. However, the mechanism of their cAMP-dependent allosteric control is not completely understood. In the present thesis we have studied the allosteric mechanism of activation in PKA and EPAC using an NMR-based approach and we have proposed a model explaining how cAMP allosterically controls the activity of PKA and EPAC. Binding of cAMP to the Regulatory (R) subunit of PKA facilitates the release of the Catalytic (C) subunit. According to our model, binding of cAMP triggers long range perturbations that propagate from the PBC to the R:C interface through both direct and indirect pathways. The indirect pathway involves two key relay sites located at the C-terminus of β2 (1163) and at the N-terminus of β3 (D170). D170 functions as an electrostatic switch that mediates the communication between the PBC and the helical subdomain, whereas 1163 controls the global unfolding. Hence, removal of cAMP uncouples the α- and β-subdomains by breaking the circuitry of cooperative interactions radiating from the PBC. The proposed model was further validated by the cAMP agonist Sp-cAMPS and the cAMP antagonist Rp-cAMPS. It was observed that Rp-cAMPS, in which the equatorial exocylic oxygen is replaced by sulphur, does not activate a necessary indirect allosteric pathway, while its diastereoisomer (Sp-cAMPS) with opposite phosphorus chirality behaves similarly to cAMP activating all allosteric pathways. Our data also showed that the cAMP-antagonist stabilizes a ternary inhibitory complex between the effector ligand and both the regulatory and the catalytic subunits of PKA. At this point it is still not understood how the proposed model of cAMP allostery is conserved in other cAMP binding proteins such as EPAC. EPAC is a multidomain guanine nucleotide exchange factor specific for small GTP-binding proteins and is directly activated by cAMP. We have probed how cAMP docks into the EPAC1 CBD and how its signal allosterically propagates from the cAMP binding site to the helical subdomain, which mediates the inhibitory interactions between the regulatory and catalytic regions of EPAC. Our comparative NMR investigation of cAMP signalling in PKA and EPAC revealed key functionally significant differences between these two systems that will facilitate the design of EPAC-selective therapeutics. / Thesis / Doctor of Philosophy (PhD)

Thesis (Ph. D.)--University of Washington, 1997. / Vita. Includes bibliographical references (leaves [115]-133).

All organisms must sense and respond to their environment in order to survive. The processes that allow a living cell to sense changes in its environment, and respond appropriately are collectively referred to as ‘signal transduction’. Cyclic AMP is a ubiquitously used second messenger molecule that plays diverse roles from hormone signalling in mammalian cells to catabolite repression in enteric bacteria. In several bacterial pathogens such as Pseudomonas aeruginosa, cAMP has also been found to mediate pathogenesis, usually by regulating the production of several virulence factors aiding in colonisation of the host. Cyclic AMP signalling has been suggested to regulate the virulence of the obligate intracellular Mycobacterium tuberculosis. Mycobacteria, including M. tuberculosis, code for a large number of adenylyl cyclases, enzymes that synthesise cAMP. Of the 16 putative adenylyl cyclases encoded by M. tuberculosis H37Rv, 10 have received extensive biochemical attention. A knockout of one of these cyclases, Rv0386, resulted in compromised virulence of M. tuberculosis. Ten proteins predicted to bind cAMP and mediate its cellular roles have also been identified in M. tuberculosis. Among these are the cAMP-regulated transcription factor, CRPMt, and cAMP-regulated protein acetyl transferase, KATmt. Comparatively little information is available, however, regarding the roles of cAMP-degrading machinery in mycobacteria. Two phosphodiesterases, with modest activity against cAMP in vitro, have been identified from M. tuberculosis, and are encoded by the Rv0805 and Rv2795c loci. Of these, Rv2795c has orthologs in all sequenced mycobacterial genomes. However, Rv0805-like proteins are coded only by slow growing mycobacteria such as the M. tuberculosis-complex, M. marinum and M. leprae, several of which are human or animal pathogens. Rv0805 belongs to the metallophosphoesterase superfamily of proteins, consisting of metal-dependent phosphoesterases with substrates ranging from large polymers like nucleic acids to small molecules like cAMP and glycerophospholipids. Like other metallophosphoesterases, Rv0805 displays promiscuous substrate utilisation and efficient hydrolysis of 2’3’-cAMP in vitro. Rv0805 also hydrolyses 3’5’-cAMP in vitro. Overexpression of Rv0805 is reported to lead to reduction in intracellular cAMP levels in M. smegmatis and M. tuberculosis, suggesting that it is capable of hydrolysing cAMP in the bacterial cell as well. The structure of Rv0805 revealed a sandwich-like α/β fold, typical of metallophosphoesterases, along with a relatively flexible C-terminal domain of unknown function. Despite extensive biochemical and structural information on Rv0805 however, its roles in mycobacteria remain unknown. In this study, the cellular roles of Rv0805 are explored and using information from biochemical and structural analyses, novel activities and interactions of Rv0805 have been identified. Rv0805, when expressed in M. smegmatis, led to a reduction in intracellular cAMP, as previously reported. However, the extent of reduction was modest (~30 %) and limited to the exponential phase of growth when both Rv0805 and intracellular cAMP are at their highest levels. Overexpression of Rv0805 also resulted in hypersensitivity to cell wall perturbants like crystal violet and sodium dodecyl sulphate (SDS) indicative of a change in the properties of the cell envelope of M. smegmatis. Importantly, these effects were independent of cAMP-hydrolysis by Rv0805, as overexpression of catalytically inactive Rv0805N97A also elicited similar changes. Unexpectedly, Rv0805 was localised to the cell envelope, both in M. tuberculosis as well as in M. smegmatis. The ability of Rv0805 to localise to the cell envelope was dependent on it C-terminus, as truncation of Rv0805 in this region (Rv0805Δ10, Rv0805Δ20 and Rv0805Δ40) resulted in progressively greater enrichment in the cytosol of M. smegmatis. Overexpression of Rv0805Δ40, which was localised almost completely to the cytosol, did not result in hypersensitivity to SDS, suggesting that cell envelope localisation, rather than cAMP-hydrolysis, was crucial for the cell envelope modifying roles of Rv0805. A possible mechanism behind the cell envelope-related effects of Rv0805 overexpression was the ability of the protein to interact with the cell wall of mycobacteria in a C-terminus-dependent manner. Purified Rv0805, but not Rv0805Δ40, could associate with crude mycobacterial cell wall as well as purified cell wall core polymer (mycolyl-arabinogalactan-peptidoglycan complex) in vitro. In addition to the C-terminus, the architecture of the active site was also crucial for this interaction as mutations in the active site that compromised metal-binding also resulted in poor interaction with the cell wall. Most significant among these residues was His207, which when mutated to Ala almost completely abrogated interaction with the cell wall in vitro. Further, Rv0805H207A was unable to localise to the cell envelope when expressed in M. smegmatis, even in the presence of the C-terminus, highlighting the importance of this residue in maintaining the structural integrity of Rv0805, and demonstrating that the structure of the C-terminus, rather than its sequence alone, played a role in cell envelope localisation and interaction. In order to verify that the observed sensitivity of Rv0805-overexpressing M. smegmatis to cell wall perturbants was due to a change in cell envelope properties atomic force microscopy was employed. Two distinct modes of operation were used to analyse surface and bulk properties of the mycobacterial cell envelope. These were tapping mode phase imaging, and contact mode force spectroscopy. Using tapping mode phase imaging, it was found that the cell surface of M. smegmatis was inherently heterogeneous in its mechanical properties. Further, contact mode force-spectroscopy revealed that the cell envelope of M. smegmatis in cross-section had at least three layers of varying stiffness. Typically, a middle layer of high stiffness was observed, sandwiched between two lower stiffness layers. This organisation is reminiscent of the current model of the mycobacterial cell envelope, possessing a central polysaccharide rich layer and outer and inner lipid rich layers. Treatment of wild type M. smegmatis with cell wall-perturbing antibiotics isoniazid and ethambutol resulted in markedly altered phase images, as well as significantly lower stiffness of the bacterial cell envelopes, validating that the methodology employed could indeed be used to assess cell wall perturbation in mycobacteria. Further, M. smegmatis harbouring deletions in cell envelope biosynthesis related genes, MSMEG_4722 and aftC, showed significantly lower cell wall stiffness than wild type M. smegmatis, providing evidence that genetic perturbation of the cell wall of mycobacteria could also be studied using atomic force microscopy. While phase imaging revealed similar surface properties of Rv0805-overexpressing and control M. smegmatis, force spectroscopy revealed significantly lower cell envelope stiffness, particularly of the middle layer, of the former. Cell envelope stiffness was, however, unaffected by expression of Rv0805Δ40 in M. smegmatis, providing direct evidence for C-terminus-dependent cell envelope perturbation upon Rv0805 overexpression. Additionally, overexpression of Rv0805N97A, but not Rv0805H207A led to reduced stiffness of the cell envelope of M. smegmatis, demonstrating that the cell wall remodelling activity of Rv0805 was independent of cAMP-hydrolysis, but dependent on cellular localisation and cell wall interaction. Like in M. smegmatis, overexpression of Rv0805 also led to lower cAMP levels in M. tuberculosis. Using a microarray-based transcriptomics approach, pathways affected by Rv0805 overexpression were identified. Rv0805 overexpression elicited a transcriptional response, leading to the down-regulation of a number of virulence associated genes such as whiB7, eis, prpC and prpD. Importantly, Rv0805-overexpression associated gene expression changes did not include genes regulated by CRPMt, the primary cAMP-regulated transcription factor in M. tuberculosis. Further, Rv0805N97A overexpression in M. tuberculosis led to similar changes in gene expression as overexpression of the wild type protein. These observations reiterated that, at least upon overexpression, the effects of Rv0805 were largely independent of cAMP-hydrolysis. Using overexpression in M. smegmatis and M. tuberculosis, cAMP-hydrolysis independent roles of Rv0805 in mycobacteria were identified. To further validate these observations, a knockout strain of the Rv0805 gene was generated in M. bovis BCG, a well-established model to study M. tuberculosis. Curiously, deletion of Rv0805 did not lead to a change in intracellular cAMP levels, demonstrating that cAMP-hydrolysis by Rv0805 may not contribute to the modulation of mycobacterial cAMP levels under standard laboratory growth conditions. Rv0805 deletion led to altered colony morphology and possible reduction in cell wall thickness, reaffirming the roles of this phosphodiesterase in regulating cell envelope physiology of mycobacteria. Additionally, Rv0805 deletion also resulted in compromised growth of M. bovis BCG in fatty acid-deficient media, implicating Rv0805 as a possible regulator of carbon metabolism. In summary, this thesis explores novel links between Rv0805 and the mycobacterial cell wall and elucidates the critical importance of the C-terminus domain of this metallophosphodiesterase in modulating its cellular localisation to, and interaction with, the mycobacterial cell envelope. En route to understanding the effects of Rv0805 overexpression on the cell wall of M. smegmatis, an atomic force microscopy-based methodology to assess perturbation of the cell envelope of mycobacteria was also developed. Finally, using a combination of biochemical and genetic analyses, cellular roles of Rv0805, independent of cAMP-hydrolysis, were identified in slow-growing mycobacteria. This study therefore provides direct evidence against the sole role of this mycobacterial phosphodiesterase as a regulator of intracellular cAMP levels, and opens up new avenues to understanding the cellular functions of Rv0805 and indeed other members of the metallophosphoesterase superfamily.

L'acide ascorbic acid (AA) a été considéré, pendant longtempss, comme une molecule devantêtre absorbée dans la nutrition, et prévenant le scorbut. Notre hypothèse, fondé sur desrésulats de notre groupe, suggèrent de nouvelles fonctions.Parmi celles-ci, nous nous sommes posé la question de l'AA molècule de signalling, durantl'embryogenèse et chez l'adulte, commme l'acide rétinoique (principe actif de la vitamine A)l'est. A cet effet, nous avons utilisé deux modèles cellulaires : des cellules souchesembryonnaires murines et des lignées de cellules souches/progénétrices adultes. Nous avonsainsi montré que l'AA stimule la différentiation de ces cellules en cellules musculairessquelettiques et en osteoblastes et inhibe l'adipogenèse et la neurogenèse. Cet effet passe parle transporteur de l'AA SVCT2 et implique la voie p38/MAPK. D'autre part, nous avonsdemontré que l'AA agit en compétition avec le RA, sur la neurogenèse et la myogenèse.Enfin, dans des cellules mésenchymateuses adultes, nous avons montré que l'AA inhibel'adipogenèse et stimule l'ostéogenèse. Cette action, comme chez l'embryon implique SVCT2et une modulation du pool du cAMP.En conclusion, l'AA pousse les cellules à se différencier en cellule musculaire squelettique eten ostéoblste et inhibie l'adipogenèse et la neurogenèse. / AA has been considered for a long time as a molecule involved in nutrition, to prevent scurvy. Our hypothesis is that AA could also be involved in development during embryogenesis, as well as in cell differentiation in adults. The aim of this study is to evaluate the potential implication of AA in cell differentiation, especially of mesenchyme cells, and to propose potential pathways that could be involved in these processes. Using murine ESCs we observed that AA markedly enhance the differentiation of ESCs toward muscle cells. Furthermore, we demonstrated that induction of myocytes by AA involves p38MAPK pathway and p-CREB. Moreover, we demonstrated that AA acts in mirror with retinoic acid. ESCs treated with RA mainly differentiate into neuronal cells, but AA compete, in a dosage dependent way to this differentiation. AA induces differentiation of ESCs into cardiac myocytes and could probably acts through p38MAPK pathway. Regarding adipocyte we revealed that SVCT2 expression significantly decreased as preadipocytes cells differentiate to adipocytes. This data suggests that mature adipocytes could not receive signals from AA. In addition, our results show that the expression of SVCT2 is increased in cells treated with AA and without IBMX. Moreover, we demonstrated that AA evolves in decreasing of cells containing lipids. Finally, we demonstrated that AA is not only involved in muscle differentiation of mesenchyme but is also involved in adipose tissue as a negative inducer. In conclusion, AA drives differentiation of ESCs toward muscle cells and osteoblast, incompetition with RA, and has a negative effect on adipogenesis and neurogenesis differentiation.

Thesis advisor: Charles S. Hoffman / In this thesis, I describe the development of a screening platform for detecting PDE8A inhibitors using the cAMP-dependent glucose sensing pathway of the fission yeast Schizosaccharomyces pombe, which led us to discover several PDE8A selective inhibitors. In this system, the only PDE of the fission yeast is replaced with mammalian PDE8A1 in strains that have been engineered such that PDE inhibition is required to allow cell growth. Using this system, I screened 56 compounds obtained from PDE4 and PDE7 high throughput screens (HTSs) and identified a PDE4-PDE8 dual specificity inhibitor. Using this as a positive control, I developed a robust high-throughput screen (HTS) for PDE8A inhibitors and screened 240,267 compounds at the Harvard Medical School ICCB Screening Facility. Approximately 0.2 % of the screened compounds were potential PDE8A inhibitors with 0.03% displaying significant potency. Secondary assays of 367 of the most effective compounds against strains expressing PDE8A (both full length and catalytic domain), PDE4A and PDE7A or PDE7B led to the selection of structurally diverse compounds for further testing. To profile the selectivity of twenty-eight of these compounds, dose response assays were conducted using 16 yeast strains that express different PDE isoforms (representing all PDE families with the exception of the PDE6 family). These assays identified compounds with different patterns of inhibition, including structurally-distinct PDE8A-specific inhibitors. By evaluating the effects of these compounds for steroid production in mouse Leydig cells, biologically active compounds that can elevate steroid production were identified. / Thesis (PhD) — Boston College, 2011. / Submitted to: Boston College. Graduate School of Arts and Sciences. / Discipline: Biology.

Einleitung: / Introduction:

Thesis (Ph.D.)--University of Toledo, 2006. / "In partial fulfillment of the requirements for the degree of Doctor of Philosophy in Biomedical Sciences." Major advisor: Brian G. Rowan. Includes abstract. Document formatted into pages: iv, 204 p. Title from title page of PDF document. Title at ETD Web site: Molecular mechanisms of protein kinase a signaling pathway on estrogen receptor action in breast cancer . Bibliography: pages 59-65, 100-104, 137-150, 167-202.