SPR-Based study of affinity of cytochrome P450s / redox partners interactions modulated by steroidal substrates.

Autor: Ershov PV; Federal State Budgetary Institution 'V.N. Orekhovich Research Institute of Biomedical Chemistry', 119121, Moscow, Pogodinskaya str. 10, building 8, Russia. Electronic address: lmi.orechovich@yandex.ru., Yablokov ЕO; Federal State Budgetary Institution 'V.N. Orekhovich Research Institute of Biomedical Chemistry', 119121, Moscow, Pogodinskaya str. 10, building 8, Russia., Florinskaya AV; Federal State Budgetary Institution 'V.N. Orekhovich Research Institute of Biomedical Chemistry', 119121, Moscow, Pogodinskaya str. 10, building 8, Russia., Mezentsev YV; Federal State Budgetary Institution 'V.N. Orekhovich Research Institute of Biomedical Chemistry', 119121, Moscow, Pogodinskaya str. 10, building 8, Russia., Kaluzhskiy LА; Federal State Budgetary Institution 'V.N. Orekhovich Research Institute of Biomedical Chemistry', 119121, Moscow, Pogodinskaya str. 10, building 8, Russia., Tumilovich AM; Institute of Bioorganic Chemistry National Academy of Science of Belarus, 220141, Minsk, Kuprevicha str. 5/2, Belarus., Gilep АА; Federal State Budgetary Institution 'V.N. Orekhovich Research Institute of Biomedical Chemistry', 119121, Moscow, Pogodinskaya str. 10, building 8, Russia; Institute of Bioorganic Chemistry National Academy of Science of Belarus, 220141, Minsk, Kuprevicha str. 5/2, Belarus., Usanov SA; Institute of Bioorganic Chemistry National Academy of Science of Belarus, 220141, Minsk, Kuprevicha str. 5/2, Belarus., Ivanov АS; Federal State Budgetary Institution 'V.N. Orekhovich Research Institute of Biomedical Chemistry', 119121, Moscow, Pogodinskaya str. 10, building 8, Russia.
Jazyk: angličtina
Zdroj: The Journal of steroid biochemistry and molecular biology [J Steroid Biochem Mol Biol] 2019 Mar; Vol. 187, pp. 124-129. Date of Electronic Publication: 2018 Nov 20.
DOI: 10.1016/j.jsbmb.2018.11.009
Abstrakt: The goal of this work was to test the hypothesis that the affinity of protein-protein interactions in the cytochrome P450-dependent monooxygenase system is modulated by the low-molecular-weight compounds (substrates or inhibitors). The surface plasmon resonance (SPR) based study was carried out using the recombinant protein preparations of three microsomal cytochromes P450 (CYP17A1, CYP21A2, and CYP2C19) and their redox partners: cytochrome b5 (CYB5A), NADPH - cytochrome P450 reductase (CPR), and also iron-sulfur protein adrenodoxin (Adx). As a result, we have revealed some specificity of the influence of the steroid substrates on the binding affinity of CYPs with their redox partners, namely: the lack of effect on CPR/CYPs and Adx/CYP complex formation, and a significant effect on interactions between CYB5A and steroidogenic CYPs. The equilibrium dissociation constant (Kd) value of the CYB5A/CYP17A1 complex decreased by 5 times in the presence of progesterone (P4), which was due to a 10 times increase in the association rate constant (k on ). In this case, a twofold increase in the dissociation rate constant (k off ) value of CYB5A/CYP17A1 complex formation was observed. It was also demonstrated that the affinity of CYB5A/CYP17A1 interaction increased in the presence of two other steroidal substrates 17α-hydroxyprogesterone and pregnenolone and that effect was comparable with P4. In contrast, only the twofold decrease in the affinity of CYB5A/CYP21A2 interaction in the presence of P4 was caused by a slight increase in the k off value (the k on value of the complex did not change). This indicates a different format of the steroidal substrates effects expressed in a change in the stability of the CYB5A/CYPs complexes. Thus, it was found that P4 modulated the both kinetic and equilibrium constants of CYB5A/CYP17A1 and CYB5/CYP21A2 complex formation and complexes, while not affecting the CYB5A/CYP2C19 interaction (2C19 is the cytochrome P450 isoenzyme possessing broad substrate specificity), thereby indicating a specific influence of steroidal substrates on interactions involving steroidogenic CYPs. Our results are consistent with current understanding of the role of CYB5A as a regulator of cytochrome P450 activity in P450-dependent monooxygenase system.
(Copyright © 2018 Elsevier Ltd. All rights reserved.)
Databáze: MEDLINE
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