RelA is a guanosine tetraphosphate synthetase which catalyzes the production of (p)ppGpp during the stringent response in Escherichia coli. RelA consists of an N-terminus, which is responsible for the catalytic activity, and a C-terminus, which is thought to be involved in the regulation of RelA activity. Furthermore, the C-terminus has dimerization and ribosome binding ability. ‘RelA-2, which is a fragment of C-terminus, is a major domain responsible for dimerization and ribosome binding. In this study, it was demonstrated that combination of two mutations (C612G, D637R) in ‘RelA-2 significantly reduced the dimerization. This dimerization-defective mutant still bound to ribosomes both in vivo and in vitro, indicating that dimerization is not required for its ribosome binding and that the dimerizaton domain is separated from its ribosome binding domain. The overexpression of the dimerization-defective mutant in amino acid starved cells inhibited chromosome-encoded wild type RelA activity. As a result, the starved cells did not show a stringent response. This finding does not support the oligomerization model proposed by Gropp group. Previous studies in this laboratory have shown, and were confirmed here, that the overexpressed ‘RelA-3, another fragment of C-terminus, which is devoid of dimerization and ribosome binding ability, did not inhibit the RelA activity when cells are under amino acid starvation. This evidence supports the hypothesis that ribosome binding is somehow involved in the regulation of RelA activity. It was demonstrated in this study that RelA was localized to the 50S subunit in vivo by Western Blot analysis. This result confirmed a previous study showing that the 50S subunit had the enzymatic activity in vitro, but not the 30S subunit. However, an in vitro study using pure 50S and 30S ribosomal subunits for the binding experiments indicated that RelA mainly bound to the 30S subunit and weakly to the 50S subunit. A model has been proposed to explain the possible mechanism of ribosome association for RelA. The involvement of L11 and EF-G in the regulation of RelA activity was also investigated. Three residues (C38, G131, and G137) in L11 have been identified to be crucial for the regulation of RelA activity. Three residues (T89, L438, and G628) in EF-G have been identified to be involved in the regulation of RelA activity. These preliminary studies implicate that the regulation of RelA inside amino acid-starved E. coli is complex.

Diss. (sammanfattning) Umeå : Univ., 2008. / Härtill 3 uppsatser.

Für die Ätiopathogenese der Atherosklerose spielen eine Vielzahl von Mediatoren eine Rolle. Dabei werden durch das Endothel sowohl protektive als auch schädliche Mediatoren sezerniert. High Density Lipoproteine (HDL) stellen einen bedeutenden protektiven Marker für das kardiovaskuläre Risiko dar, u.a. durch die Aktivierung der endothelialen NO-Synthase (eNOS). HDL besteht zu 50 % aus Proteinen und zu 50 % aus Lipiden. Welche Komponenten des HDL für die eNOS Aktivierung verantwortlich sind, ist nicht bekannt gewesen. Im ersten Abschnitt dieser Promotionsarbeit konnte erfolgreich gezeigt werden, dass die Lysophospholipide, Sphingosin-1-Phosphat (S1P) und Sphinsosylphosphorylcholin (SPC), die strukturelle Bestandteile der Lipidfraktion von HDL darstellen, für einen Teil der HDL induzierten eNOS Aktivierung durch Stimulation des S1P3-Rezeptors verantwortlich sind. Diese eNOS Aktivierung wird durch den intrazellulären Einstrom von Calcium und durch die Aktivierung der Akt-Kinase induziert. Im zweiten Abschnitt dieser Promotionsarbeit konnte nachgewiesen werden, dass das oral verfügbare Lysophospholipid-basierte Medikament, FTY720, das ein strukturelles Analogon des S1P ist, den HDL induzierten Signaltransduktionsweg der eNOS Aktivierung in gleicher Weise induziert. Im dritten Abschnitt dieser Promotionsarbeit konnte ein neuartiges endothelabhängig sezerniertes gemischtes Dinukleosidpolyphosphat, Uridin-Adenosin-Tetraphosphat (Up4A), identifiziert werden. Up4A ist ein Agonist an den P2X- und P2Y-Purinrezeptoren. Up4A induziert bei Applikation in eine isoliert perfundierte Rattenniere hauptsächlich über die Aktivierung des P2X1-Rezeptors und des P2Y2/P2Y4-Rezeptors eine starke Vasokonstriktion im renalen Perfusionsgebiet mit einhergehender Erhöhung des mittleren renalen Perfusionsdrucks. Die direkte Infusion von Up4A in vivo in eine WKY-Ratte führt zu einer signifikanten Erhöhung des mittleren arteriellen Blutdrucks. / In the pathogenesis of atherosclerosis many mediators are included. Therefore the endothelium plays a crucial part by secreting protective but also deleterious factors. High density lipoproteins are an established protective factor in the risk profile of cardiovascular events especially by activating the endothelial NO synthase (eNOS). HDL is composed of 50 % proteins and 50 % lipids. Which component of HDL is responsible for the eNOS activation was not known. In the first part of this dissertation it could be shown, that the lysophospholipids, sphingosine-1-phosphate (S1P) and sphingosylphosphorylcholine (SPC), which are structural compounds of the lipid fraction of HDL, are responsible for a significant part of the HDL induced eNOS activation by stimulating the specific S1P3 receptor. In the signal transduction mechanism the activation of Akt kinase and an influx of calcium is involved. In the second part of this dissertation it could be shown, that the orally active lysophospholipide based drug FTY720, which is a structural analogue of S1P, is able to induce the same signal transduction mechanism activated by HDL including the stimulation of the S1P3 receptor. In the last part of this dissertation a new endothelium dependent vasoconstrictor, the dinucleoside polyphosphate uridine-adenosine-tetraphosphate (Up4A), could be for the first time identified. Up4A is a potent agonist of the P2X- and P2Y-purinoceptors. Via activating the P2X1 receptor and the P2Y2/P2Y4 receptor Up4A induce a strong vasoconstriction in the renal perfusion system in the model of the isolated perfused rat kidney with an adjacent increase of the mean perfusion pressure. By injection of Up4A in vivo in a Wistar Kyoto rat the mean arterial pressure also increase significantly.