Fabry disease is an X-linked lysosomal storage disease that results from a deficiency in the enzyme α-galactosidase (α-GAL). α-GAL hydrolyzes α-galactosides, and patients with Fabry disease suffer from an accumulation of these undegraded substrates. Human α-GAL naturally occurs as a homodimer, as determined through SEC and crystallographic analysis. This means its quaternary structure consists of two identical α-GAL subunits that are associated together into a single unit. Other species, such as rice, produce a monomeric form of α-GAL, consisting of only a single subunit. If α-GAL is functional as both a homodimer and monomer, then how does homodimerization affect the activity of human α-GAL? This can be answered through two model systems. First, a monomeric form of human α-GAL can be produced, testing the activity of human α-GAL in a monomeric state. A variant of α-GAL was engineered (called α-GALF273G/W277G) that appeared promising. Secondly, another system can be produced capable of stabilizing one active site of the dimer and testing the other active site for activity. Another lysosomal enzyme, α-N-acetylgalactosaminidase (α-NAGAL), shares 46% amino acid sequence identity and share 11 of 13 active site residues. Previously, an α-GAL variant (called α-GALE203S/L206A) was produced, that maintained the antigenicity of α-GAL, but had acquired the enzymatic specificity of α-N-acetylgalactosaminidase (α-NAGAL). A heterodimeric form of α-GAL can be produced combining one subunit of α-GAL with the engineered variant. The engineered site can be stabilized, while the wild-type site can be tested for activity. SEC analysis suggests α-GALF273G/W277G is a monomer, and its kinetic properties are reported. Evidence shows monomeric α-GAL could be useful as an improved enzyme replacement therapy. Western blotting and activity assays suggest the presence of the α-GAL/ α-GALE203S/L206A heterodimer.

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A new approach to fulminic acid cycloadditions has been developed. At reduced temperatures, fulminic acid is generated in situ and undergoes 1, 3-diploar cycloaddition reactions with dipolarophiles to form isoxazolines and/or its dimers. This procedure represents a novel, safe general method for the one-step generation of fulminic acid, which complements existing potentially explosive protocols.

We have synthesized a series of dimers of (+)-(7R,11R)-huprine Y and evaluated their activity against Trypanosoma brucei, Plasmodium falciparum, rat myoblast L6 cells and human acetylcholinesterase (hAChE), and their brain permeability. Most dimers have more potent and selective trypanocidal activity than huprine Y and are brain permeable, but they are devoid of antimalarial activity and remain active against hAChE. Lead optimization will focus on identifying compounds with a more favourable trypanocidal/anticholinesterase activity ratio.

Yes / Background: The receptor for activated C-kinase 1 (RACK1) is a conserved protein belonging to the WD40 repeat family of proteins. It folds into a beta propeller with seven blades which allow interactions with many proteins. Thus it can serve as a scaffolding protein and have roles in several cellular processes. Results: We identified the product of the Dictyostelium discoideum gpbB gene as the Dictyostelium RACK1 homolog. The protein is mainly cytosolic but can also associate with cellular membranes. DdRACK1 binds to phosphoinositides (PIPs) in protein-lipid overlay and liposome-binding assays. The basis of this activity resides in a basic region located in the extended loop between blades 6 and 7 as revealed by mutational analysis. Similar to RACK1 proteins from other organisms DdRACK1 interacts with G protein subunits alpha, beta and gamma as shown by yeast two-hybrid, pulldown, and immunoprecipitation assays. Unlike the Saccharomyces cerevisiae and Cryptococcus neoformans RACK1 proteins it does not appear to take over Gβ function in D. discoideum as developmental and other defects were not rescued in Gβ null mutants overexpressing GFP-DdRACK1. Overexpression of GFP-tagged DdRACK1 and a mutant version (DdRACK1mut) which carried a charge-reversal mutation in the basic region in wild type cells led to changes during growth and development. Conclusion: DdRACK1 interacts with heterotrimeric G proteins and can through these interactions impact on processes specifically regulated by these proteins. / This work was supported by the DFG and SFB670. TYR acknowledges support from the Professorinnen Program of the University of Cologne.

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of the thesis Glutamate is a main excitatory neurotransmitter in the brain of mammals, which activates both ionotropic and metabotropic glutamate receptors. Ionotropic receptors are responsible for fast synaptic transmission leading to membrane depolarization and Ca2+ influx into the cell. On the other hand mGlu receptors play an important role in regulation of the transmission via heterotrimeric G-proteins and activation of various signaling pathways. Postsynaptically localized group I mGlu receptors (mGluR1, 5) together with ionotropic NMDA and AMPA receptors share common large receptor signaling complexes, or signalosome facilitating glutamate signal transductions. Individual mGluR1 splice variants are differently associated with signalosome including scaffold proteins like PSD-95 which organize postsynaptic density (PSD). Heterodimerization of different mGluR1 splice variants is a focal point of my thesis together with investigation of recently discovered protein IL1RAPL1 (interleukin-1 receptor accessory protein-like 1) and its role in organization of postsynaptic signalosome. Using biochemical, immunocytochemical and functional assays we showed heterodimers of mGluR1a/1b were expressed on the plasma membrane and that heterodimers are fully functional in the recombinant system. Next we showed...

La vasopressine (AVP) et la corticolibérine (CRF) agissent de manière synergique lors de la réponse aux stimuli stressants. Elles régulent de manière concertée la sécrétion d'adrénocorticotrophine hypophysaire et la libération de catécholamines surrénalienne. Dans ces deux structures, les isoformes de récepteurs présents sont les récepteurs V1B et CRF1. Nous avons démontré que deux mécanismes moléculaires sous-tendent la synergie fonctionnelle de l'AVP et du CRF : un croisement des voies de seconds messagers propres à chacun des récepteurs d'une part, et une modification de leurs propriétés pharmacologiques résultant de leur interaction (hétérodimérisation) d'autre part. Pour valider ce dernier mécanisme, nous avons recherché des formes naturelles ou mutées de récepteurs à l'AVP et au CRF conservant leurs propriétés de couplage aux protéines G, mais incapables d'hétérodimériser, et avons analysé les conséquences de cette rupture d'hétérodimérisation sur leur aptitude à agir en synergie. Grâce à une approche de mutagénèse dirigée, nous avons commencé à résoudre la question des portions de récepteurs engagées dans l'hétérodimérisation. Les résultats obtenus apportent les premières évidences permettant de comprendre la synergie AVP/CRF au niveau moléculaire, et particulièrement le rôle de l'hétérodimérisation. L'hétérodimère V1B/CRF1 pourrait être impliqué dans le stress et ses états pathologiques que sont l'anxiété et la dépression. Nous montrons que les récepteurs V1B et CRF1 sont co-exprimés dans les neurones de certaines structures cérébrales régulant ces phénomènes comportementaux. Démontrer l'existence de l'hétérodimère V1B/CRF1 dans des tissus natifs sera la prochaine étape de ce travail. Si elle est validée, le complexe V1B/CRF1 pourra être considéré comme une cible pharmacologique de première importance dans le traitement de l'anxiété et de la dépression. Travail soutenu par l'Institut de Recherches SERVIER et la Fondation pour la Recherche Médicale. / Vasopressin (AVP) and Corticotropin-Releasing Factor (CRF) are involved in the stress response, mainly by regulating ACTH secretion from the pituitary and by increasing catecholamine and corticosteroids secretion from the adrenal medulla. In these two structures, AVP and CRF have been shown to act in synergism via V1B and CRF1 receptors. Recently, our group demonstrated that such synergism operates via both second messenger crosstalk and putative mechanism involving receptors heterodimerization. To further validate this last original mechanism, we monitored the influence of receptor heterodimerization selectivity and of receptor heterodimerization disruption on functional synergism. We also deciphered receptor dimers interface by synthesizing receptor mutants that do not heterodimerize anymore.These results give clues to the comprehension of AVP/CRF synergism at the molecular level and trigger the potential role of receptors heterodimerization in stress-related behaviours. Indeed both V1B and CRF1 are also co-expressed in neurons of relevant brain area. Establishing the physical association of V1B/CRF1 as heterodimers in native tissue, the next step of our project, would be of considerable importance.Work supported by SERVIER (France) an d the FRM.