Regulation, activation, and deactivation of soluble guanylate cyclase and NO-sensors

Abstract : This thesis is devoted to the regulation of soluble guanylate cyclase (sGC), the endogenous nitric oxide (NO) receptor in mammals involved in signal transduction. The enzyme is activated by the binding of NO to its heme and catalyzes the formation of cGMP from GTP. While sGC is present in many mammalian cells, the homologous bacterial domain (H-NOX) is involved in NO detection and metabolism regulation. An important objective was to find sGC inhibitors to slow down tumor progression.The screening of natural compounds from a chemical library, tested on purified sGC activity, revealed six active inhibitors (Ki = 0.2 – 1 µM). Together with two agents for photodynamic therapy (hypericin and hypocrellin) we demonstrated that these inhibitors are allosteric modulators which bind neither to the heme nor to the catalytic and activator sites, revealing a new class of pharmacological compounds targetting the NO/cGMP signaling pathway.The structural transition induced in sGC by stimulator riociguat in synergy with CO was studied by transient absorption spectroscopy to demonstrate coordination changes of the heme. Two different activation states of sGC with CO 6c-heme and 5c-heme exist simultaneously in the presence of the stimulator which induces the breaking of the heme Fe-His bond, as does the sGC natural effector NO. In addition, the effect of isoliquiritigenin, which is sold as a sGC activator, was shown to be actually an inhibitor of sGC.The dynamics of the ligands CO, NO and O2 were measured over 12 orders of magnitude in time in wild type and mutant of a bacterial NO transporter (AXCP). The single mutation Leu16Ala increased 108-fold the CO affinity, ~106-fold the NO affinity and makes this protein reactive to O2. In the case of CO and NO, whose affinities for L16A-AXCP are the largest ever measured, the bimolecular rebinding was absolutely not detectable. Molecular dynamic simulations demonstrated that dissociated CO is constrained to stay within 4 Å from Fe2+ by Ala16, contrarily to wild-type Leu16.The dynamics of O2 in Tt-H-NOX proteins measured by transient absorption spectroscopy confirmed the hypothesis that Tt-H-NOX may not be a NO-sensor stricto sensu but a redox sensor. The properties of the Tt-H-NOX protein are not compatible with the role a mere NO-carrier.
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Olga Petrova. Regulation, activation, and deactivation of soluble guanylate cyclase and NO-sensors. Biochemistry, Molecular Biology. Université Paris-Saclay, 2017. English. ⟨NNT : 2017SACLX113⟩. ⟨tel-01713240⟩

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