Cyclic-3',5 '-adenosine monophosphate (cAMP) is an ancient signaling molecule that is found in a variety of species from prokaryotes to eukaryotes and translates extra-cellular stimuli into tightly controlled intra-cellular responses. The two major mammalian cAMP sensors are protein kinase A (PKA), for the phosphorylation of the downstream effectors, and the exchange protein directly activated by cAMP (Epac ), for the guanine nucleotide exchange in the small GTPase Rap proteins. In this study, we investigated the intra-molecular cAMP dependent allosteric network of Epac cyclic nucleotide binding domain (CBD) via solution NMR spectroscopy. Epac proteins have been shown to employ an auto-inhibition strategy in the control of the equilibrium between the active and the inactive states. In the absence of cAMP, the periphery of the Rap recognition site is masked via an ionic interface provided by the N-terminus of the CBD. Binding of cAMP at the distal Phosphate Binding Cassette (PBC), results in weakening of this interface. Here we show that the cAMP binding signal is propagated to the sites important in Epac activation, i.e. the ionic interface, via two key allosteric spots within the CBD. We have also determined the dynamics as a key carrier of cAMP effects to the region forming the ionic interface (ionic latch). Hence entropic enhancements emerged as a key effector in the cAMP mediated ionic latch weakening. We have also provided initial evidence of a negative allosteric contribution from the C-terminal Hinge-Lid region (CHLR) on the cAMP induced Epac activation. In addition to these findings, we also observed critical differences in the mode of cAMP recognition and inter-subdomain communication between the Epac and PKA. A detailed understanding of these two ubiquitous systems, will aid in the development of agonists and antagonists that are relevant in the drug lead development for related diseases, such as Alzheimer's and diabetes. / Thesis / Master of Science (MSc)

Thesis (Ph.D.)--Hong Kong University of Science and Technology, 2006. / Includes bibliographical references. Also available in electronic version.

Faculty of Science School of Pathology(Molecular Medicine and Haematology). 9707563v tridium@acenet.co.za / The erythrocyte carries gases between the cells and the lungs, and has to distort to negotiate narrow splenic sinuses and capillaries. This distortion necessitates a high surface area to volume ratio that is maintained by the erythrocyte membrane skeleton, a network of proteins including spectrin and protein 4.1. The skeleton anchors the lipid bilayer through attachment to integral membrane proteins, notably the anion exchange protein, band 3. Abnormalities of the erythrocyte membrane proteins cause loss of cell elasticity and ultimately the erythrocytes become prematurely trapped in the spleen where they are phagocytosed, resulting in haemolytic anaemia. Three mutations causing band 3-deficient hereditary spherocytosis (HS), a haemolytic anaemia characterised by spherical erythrocytes, were located using restriction enzyme analysis and DNA sequencing. Proband A (Black) is heterozygous for band 3 Pinhal (R490H) and has mild clinical symptoms. Proband B and his mother (Caucasian) are heterozygous for band 3 Bicetre (R490C) and have severe anaemia requiring transfusions and splenectomy, respectively. These results confirm codon 490 as a hotspot for mutations and indicate the effect of different amino acid substitutions in the same position on clinical severity. Proband C (Black) is homozygous for a novel mutation (E508K) for which her parents are heterozygous. The proband is severely affected and transfusion- dependent whereas her father has moderate anaemia and her mother is asymptomatic. It is speculated that a secondary factor modulates their clinical symptoms. All of these mutations occur in a CpG dinucleotide, a common source of DNA mutations, and are located within the highly conserved exon 13, which encodes the third to fifth α-helices and the second extracellular loop of the transmembrane region of band 3. The mutations are likely to alter the conformation of band 3, impairing its insertion into the erythrocyte membrane. No causative mutations were located in another 12 band 3-deficient HS kindred using restriction enzymes and single strand conformation polymorphism analysis. Ten protein 4.1-deficient patients with hereditary elliptocytosis, a haemolytic anaemia characterised by elliptical erythrocytes, were also studied. Immunoblot analyses ruled out abnormally sized protein 4.1 and three known DNA mutations were excluded using restriction enzyme analysis. Further studies are required to elucidate the cause of the haemolytic anaemia in these kindred. This study advanced our knowledge of the molecular basis of HS in South African kindred and highlighted the susceptibility of CpG dinucleotides to mutations.

Yes / Pharmaceutical manipulation of cAMP levels exerts beneficial effects through the regulation of the exchange protein activated by cAMP (EPAC) and protein kinase A (PKA) signalling routes. Recent attention has turned to the specific regulation of EPAC isoforms (EPAC1 and EPAC2) as a more targeted approach to cAMP-based therapies. For example, EPAC2-selective agonists could promote insulin secretion from pancreatic β cells, whereas EPAC1-selective agonists may be useful in the treatment of vascular inflammation. By contrast, EPAC1 and EPAC2 antagonists could both be useful in the treatment of heart failure. Here we discuss whether the best way forward is to design EPAC-selective agonists or antagonists and the current strategies being used to develop isoform-selective, small-molecule regulators of EPAC1 and EPAC2 activity.

Chronic postoperative pain impacts millions of individuals worldwide that undergo a variety of surgical procedures. Opioids remain the mainstay analgesics of acute and perioperative pain; however, prolonged opioid therapy may lead to life-threating adverse effects, tolerance, dependence, and addiction. Therefore, unraveling the cellular mechanisms that drive persistent pain states and opposing endogenous analgesia provided by opioid receptor signaling, may lead to novel analgesics. Evidence suggests that tissue injury leads to increased sensitization of the spinal cord nociceptive neurons which increases susceptibility to chronic pain via an N-methyl-D-aspartate (NMDA) receptor activation of calcium-sensitive adenylyl cyclase isoform 1 (AC1). This phenomenon, named latent pain sensitization (LS), is mediated by a compensatory response of endogenous inhibitory systems. In this dissertation, we test the hypothesis that surgical insult promotes prolonged activation of kappa opioid receptors (KOR) which mask LS via attenuation of pro-nociceptive AC1 signaling pathways in both male and female animals. We employed a murine model of chronic postoperative pain that promotes LS in the spinal cord and closely resembles the phenotypic features of postoperative pain in human subjects. When behavioral signs of hyperalgesia resolved, we targeted spinal opioid receptor systems and pronociceptive modulators with intrathecal delivery of selective pharmacological antagonists and assessed behavioral signs of hyperalgesia and spinal nociceptive sensitization. We propose that LS is kept in remission by a long-lasting compensatory response of tonic endogenous KOR signaling that hinders a pronociceptive LS pathway that includes not only AC1 but also two downstream targets: protein kinase A (PKA) and exchange protein activated by cAMP (Epac1/2) - in a sex-dependent manner. Our results propose new therapeutic targets for the management of persistent postoperative pain and underscore the importance of tailoring sex-specific pain management strategies.

<p>Exchange proteins directly activated by cAMP (EPAC) are guanine nucleotide exchange factors for the small GTPases, Rap1 and Rap2. The central regulatory module of EPAC is a cAMP binding domain (CBD), which in the absence of cAMP provides auto-inhibition of the catalytic guanine nucleotide exchange activity. Binding of the allosteric effector, cAMP, removes the auto-inhibition exerted by the CBD of EPAC. Herein, we investigate through NMR spectroscopy the structural and dynamical basis of auto-inhibition and cAMP-dependent allosteric activation in the CBD of EPAC. Specifically, the work described in this dissertation proposes novel methods that utilize NMR chemical shifts to define the network of residues that mediates long-range intra-molecular signalling, <em>i.e.</em> the chemical shift covariance analysis (CHESCA) and the chemical shift projection analysis (CHESPA). Using CHESCA as explained in Chapter 2, we identified an allosteric network that bridges the sites of cAMP-binding and cAMP-dependent structural changes to those of cAMP-dependent dynamical changes, which are critical for the release of auto-inhibition. The CHESCA results therefore rationalize how cAMP leads to activation through modulation of both structure and dynamics. In order to dissect the determinants of auto-inhibition in the absence of cAMP, several mutations along the signaling pathways identified by CHESCA were implemented and their effect on the auto-inhibitory conformational equilibrium of the apo-CBD was assessed through CHESPA, as outlined in Chapters 3 and 4. Overall, we have shown how CHESCA and CHESPA provide unprecedented insight into the allosteric networks underlying auto-inhibition and cAMP dependent activation in the CBD of EPAC. In addition, the methods employed here to map EPAC allostery are likely to be generally applicable to other systems.</p> / Doctor of Philosophy (PhD)

Gegenstand dieser Arbeit ist die Entwicklung funktionalisierter Cellulose-Tiefenfilterschichten. Dazu werden kommerzielle Kationenaustauscher in einer Kugelmühle nass zerkleinert und bei der Herstellung in die Tiefenfilterschichten eingebettet. Die mechanischen Eigenschaften und das Filtrationsverhalten dieser Schichten sind vergleichbar mit kommerziellen Produkten. Der maximale, technisch einsetzbare Masseanteil an Ionenaustauscher beträgt dabei ca. 40 %. In Durchströmungsversuchen wird die Funktion der eigesetzten Partikel innerhalb der Cellulose-Matrix untersucht. Die Adsorptionskinetik kann mit Hilfe eines in der Arbeit weiterentwickelten Modells nach Langmuir beschrieben werden. Für die Austauschkinetik wird auf der Basis dieses Modells eine abweichende Gesetzmäßigkeit ermittelt. Zudem wird eine Anlagen-Auslegung zur maximalen Kapazitätsausnutzung für einen Adsorptionsprozess vorgestellt.