Signaltransduktion in membran-gebundenen Adenylatcyclasen

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Dokumentart: PhDThesis
Date: 2020-08-06
Language: German
Faculty: 7 Mathematisch-Naturwissenschaftliche Fakultät
Department: Pharmazie
Advisor: Schultz, Joachim E. (Prof. Dr.)
Day of Oral Examination: 2020-07-03
DDC Classifikation: 500 - Natural sciences and mathematics
Keywords: Cyclo-AMP , Rezeptor , Adenylatcyclase , Membran-Anker-Sequenz
Other Keywords: Sinorhizobium meliloti
Cyclic AMP
Membrane anchor
Adenylate cyclase
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Class III adenylate cyclases are important transmitters for external signals into the intracellular second messenger cAMP. According to sequence similarities, they are classified into four distinct classes, IIIa-d. Among them, the classes IIIa and IIIb stand out the most not only for being present in both prokaryotic and eukaryotic cells but also for carrying a large hexahelical membrane anchor with a potential regulatory function. Previous work on membrane-bound adenylate cyclases has indicated the ability to perceive extracellular signals through the similarly structured quorum sensors CqsS and LqsS from V. harveyi and L. pneumophila. However, a possible regulatory mechanism by the membrane anchors has not been defined yet. One of 26 cyclases from S. meliloti, CyaCSm, shows a noteworthy similarity to succinate:quinone oxidoreductases regarding its membrane anchor. It is a monomer consisting of one 6TM domain, a ferredoxin domain, and a catalytic domain. According to UV-Vis measurements, four highly conserved histidine residues are binding two heme B molecules as a prosthetic group in the first four α-helices of the membrane anchor. Hence, specifically replacing these residues by alanine demonstrated that these histidines are responsible for binding of heme B. Subsequently, in vivo and in vitro measurements of these mutants indicated a significant decrease in AC activity. The hypothesis of AC regulation by the quinone Q0 and its reduced counterpart Q0H2 has been confirmed in this work. Experiments have shown a nearly threefold increase in cyclase activity under reduced (addition of Q0H2) to oxidized (addition of Q0) conditions. The formation of cAMP in mammalian cells is mainly regulated by GPCRs through the binding of the dissociated G protein subunit Gsα to the intracellular catalytic dimer. So far, direct regulation by an extracellular ligand for these adenylate cyclases has not been shown. First in vitro experiments on the effect of several hormones and neurotransmitters on the human adenylate cyclase isoform 5 showed no significant changes in the amount of produced cAMP after activation with Gsα. Only the addition of heat-inactivated human serum resulted in a significant inhibition of the Gsα-stimulated cyclase. This observation not only indicated the structural ability of the catalytic domains to recognize external signals, but also emphasizes the existence of a ligand in blood serum. The identification of ligands provides the basis for future research.

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