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Structural and functional studies of signaling molecules in axon guidance
Knapp, Kryštof ; Rozbeský, Daniel (advisor) ; Novák, Petr (referee)
This work aims to determine a model of the autoinhibition mechanism of MICAL proteins using biochemical, biophysical, and bioinformatical approaches. MICAL proteins are a group of flavin monooxygenases that play a key role in various cellular processes, as they facilitate the reorganization of the actin cytoskeleton. MICAL-1 has long been known for its vital role in axon guidance as an effector of repulsive signaling through oxidative destabilization of actin filaments. However, recent findings indicate that MICAL-1 can also serve as a sig- naling molecule, using localized hydrogen peroxide production to regulate other downstream effectors. Despite the consensus that MICAL-1 activity must be strictly regulated, the exact molecular mechanism of this regulation has not yet been described. In this work, we provide a novel model of MICAL-1 autoinibiton mechanism based on a comparison of steady-state kinetic experiments and molecular dynamics simulations between full-length MICAL-1 from Coturnix japonica and its truncated form lacking the C-terminal domain. In our model, we conclude that changes in MICAL-1 activity are the result of intramolecular protein interac- tions between the C-terminal and the monooxygenase domain. Furthermore, we rule out the role of MICAL-1 oligomerization in its activity...
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Inhibitory effect of 17α-ethinylestradiol on the activity of cytochrome P450 subfamilies 2B and 2C
Knapp, Kryštof ; Dračínská, Helena (advisor) ; Kukačka, Zdeněk (referee)
17α-ethinylestradiol (EE2) is a synthetic derivative of endogenous estrogen 17β-estradiol. It is widely used in pharmaceutics as a ingredient of female oral contraceptives and also in hormone replacement therapy for women with postmenopausal syndrome. Due to its high estrogenic potential together with resistance to the chemical degradation and tendency to bio- accumulate in environment it is considered as an important persistent organic pollutant. In organisms, EE2 is oxidatively metabolised by enzymes from a family of cyto- hromes P450. Some studies show that EE2 is also a potent inhibitor of some isoforms from cytochrome P450 family. In this work, we studied inhibitory effect of EE2 on two human isoforms CYP2B6, CYP2C9 and their rat orthologs CYP2B1 and 2C6, respectively. All studied isoforms exhibited reduced enzyme activity in the presence of EE2. The greatest decrease of enzyme activity was observed with CYP2B1 catalyzing pentoxyresoru n O-depentylation with value of IC50 = 9,6 µM. For hydroxylation of bupropion, selective reaction of CYP2B1 and CYP2B6, EE2 behaved as a more potent inhibitor of CYP2B6 with IC50 = 60 µM. The inhibitory effect of EE2 on rat CYP2B1 for similar reaction conditions was signi cantly lower. With 200µM concentration of EE2, CYP2B1 exhibited still more than 50 % of...
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