scholarly journals The effect of isatin on protein-protein interactions between cytochrome b5 and cytochromes P450

2017 ◽  
Vol 63 (2) ◽  
pp. 170-175 ◽  
Author(s):  
P.V. Ershov ◽  
E.O. Yablokov ◽  
Yu.V. Mezentsev ◽  
L.A. Kalushskiy ◽  
A.V. Florinskaya ◽  
...  
Keyword(s):  
Protein Interactions ◽  
Contact Region ◽  
Cytochromes P450 ◽  
Cytochrome B5 ◽  
Optical Biosensor ◽  
Test System ◽  
Feedback Mechanism ◽  
Dissociation Rate ◽  
Protein Protein Interactions ◽  
High Concentration

Cytochromes P450 (CYP) are involved in numerous biochemical processes including metabolism of xenobiotics, biosynthesis of cholesterol, steroid hormones etc. Since some CYP catalyze indol oxidation to isatin, we have hypothesized that isatin can regulate protein-protein interactions (PPI) between components of the CYP system thus representing a (negative?) feedback mechanism. The aim of this study was to investigate a possible effect of isatin on interaction of human CYP with cytochrome b5 (CYB5A). Using the optical biosensor test system employing surface plasmon resonance (SPR) we have investigated interaction of immobilized CYB5A with various CYP in the absence and in the presence of isatin. The SPR-based experiments have shown that a high concentration of isatin (270 mM) increases Kd values for complexes CYB5A/CYP3А5 and CYB5A/CYP3A4 (twofold and threefold, respectively), but has no influence on complex formation between CYB5A and other CYP (including indol-metabolizing CYP2C19 and CYP2E1). Isatin injection to the optical biosensor chip with the preformed molecular complex CYB5A/CYP3A4 caused a 30%-increase in its dissociation rate. Molecular docking manipulations have shown that isatin can influence interaction of CYP3А5 or CYP3A4 with CYB5A acting at the contact region of CYB5A/CYP.

SPR Analysis of Protein-Protein Interactions Involving Cytochromes P450 and Cytochrome b5 Integrated into Lipid Membrane

2020 ◽  
Vol 14 (2) ◽  
pp. 168-173
Author(s):  
L. A. Kaluzhskiy ◽  
P. V. Ershov ◽  
K. S. Kurpedinov ◽  
D. S. Sonina ◽  
E. O. Yablokov ◽  
...  
Keyword(s):  
Protein Interactions ◽  
Lipid Membrane ◽  
Cytochromes P450 ◽  
Cytochrome B5 ◽  
Protein Protein Interactions

scholarly journals Protein-protein interactions of cytochromes P450 3A4 and 3A5 with their intermediate redox partners cytochromes b5

2015 ◽  
Vol 61 (4) ◽  
pp. 468-474
Author(s):  
O.V. Gnedenko ◽  
A.S. Ivanov ◽  
E.O. Yablokov ◽  
S.A. Usanov ◽  
D.V. Mukha ◽  
...  
Keyword(s):  
Protein Interactions ◽  
Dissociation Constants ◽  
Cytochromes P450 ◽  
Cytochrome B5 ◽  
Electrochemical Analysis ◽  
Monooxygenase System ◽  
Protein Protein Interactions ◽  
Reduction Potentials ◽  
Drug Biotransformation ◽  
Redox Partners

Molecular interactions between proteins redox partners (cytochromes Р450 3А4, 3А5 and cytochrome b5) within the monooxygenase system, which is known to be involved in drug biotransformation, were investigated. Human cytochromes Р450 3A4 and 3А5 (CYP3A4 and CYP3A5) form complexes with various cytochromes b5: the microsomal (b5mc) and mitochondrial (b5om) forms of this protein, as well as with 2 “chimeric” proteins, b5(om-mc), b5(mc-om). Kinetic constants and equilibrium dissociation constants were determined by the SPR biosensor. Essential distinction between CYP3A4 and CYP3A5 was only observed upon their interactions with cytochrome b5om. Electroanalytical characteristics of electrodes with immobilized hemoproteins were obtained. The electrochemical analysis of CYP3A4, CYP3A5, b5mc, b5om, b5(om-mc), and b5(mc-om) immobilized on screen printed graphite electrodes modified with membranous matrix revealed that these proteins have very close reduction potentials -0.435  -0.350 V (vs. Ag/AgCl). Cytochrome b5mc was shown to be capable of stimulating the electrocatalytic activity of CYP3A4 in the presence of its substrate testosterone.

scholarly journals Biosensor selection of small compounds, modulating a complex formation between cytochrome P450s and NADPH-dependent P450 oxidoreductase

2020 ◽  
Vol 3 (3) ◽  
pp. e00134
Author(s):  
P.V. Ershov ◽  
E.O. Yablokov ◽  
Y.V. Mezentsev ◽  
L.A. Kaluzhskiy ◽  
A.Ya. Luschik ◽  
...  
Keyword(s):  
Complex Formation ◽  
Protein Interactions ◽  
Cytochromes P450 ◽  
Cytochrome B5 ◽  
Clinical Importance ◽  
Dissociation Rate ◽  
Monooxygenase System ◽  
P450 Monooxygenase ◽  
P450 Oxidoreductase ◽  
First Choice

The study of the effect of low-molecular-weight compounds (substrates, endogenous metabolites, drugs and xenobiotics) on the kinetic and equilibrium parameters of functionally significant binary protein-protein interactions (PPIs) is of both fundamental and clinical importance. The surface plasmon resonance (SPR) is the method of the first choice for studying PPIs. Earlier, SPR analysis revealed the modulating effect of steroidal substrates on the affinity of interactions between steroidogenic microsomal cytochromes P450 (CYP) and their redox partner (cytochrome b5). In this work, we have shown the suitability of the experimental approach for assessing the selective effect of the cofactor NADPH on the interaction between cytochromes CYP3A4 or CYP2E1 with NADPH-dependent P450 oxidoreductase (CPR). Experiments have shown that the CYP3A4/CPR complex is not modulated by NADPH, while the dissociation rate of the CYP2E1/CPR complex in the presence of NADPH significantly decreased: the koff values in the absence and presence of NADPH were (3.6 ± 0.2) • 10-3 s-1 and (3.8 ± 0.2) • 10-4 s-1, respectively. Thus, in the presence of NADPH, an increase in the affinity of CYP2E1/CPR complex formation by approximately one order of magnitude was observed, while NADPH did not affect the kon value of this complex. Co-injection of NADPH at the CYP2E1/CPR complex preformed in the absence of NADPH had minor influence on the koff values (<10%). This suggests a stabilizing role of NADPH for the CYP2E1/CPR complex formation. Thus, the use of our approach made it possible to assess the effect of the main electron supplier for the microsomal cytochrome P450 monooxygenase system on the kinetic rate constants of CYP/CPR complexes.

scholarly journals Study specificity of isatin interactions with P450 cytochromes

2018 ◽  
Vol 64 (1) ◽  
pp. 61-65 ◽  
Author(s):  
P.V. Ershov ◽  
Yu.V. Mezentsev ◽  
E.O. Yablokov ◽  
L.A. Kalushskiy ◽  
A.V. Florinskaya ◽  
...  
Keyword(s):  
Cytochrome P450 ◽  
Protein Interactions ◽  
Cytochromes P450 ◽  
Cytochrome B5 ◽  
Direct Interaction ◽  
Protein Protein Interactions ◽  
Living Organisms ◽  
Adrenodoxin Reductase ◽  
Equilibrium Dissociation ◽  
Dissociation Constant Kd

Cytochrome P450-dependent monooxygenase systems exist basically in all living organisms, where they perform various important functions. The coordinated functioning of these systems involves many proteins participating in different protein-protein interactions (PPI). Previously, we have found that the endogenous non-peptide bioregulator isatin (indoledione-2,3), synthesized from indole by means of certain cytochromes P450 (e.g. P450 2E1, P450 2C19, P450 2A6) regulates affinity of some PPI. In this work, an attempt has been undertaken to register a direct interaction of isatin with a set of different proteins related to the functioning of cytochrome P450-dependent monooxygenase: five isoforms of cytochromes P450, two isoforms of cytochrome b5, cytochrome P450 reductase, adrenodoxin, adrenodoxin reductase and ferrochelatase. The study has shown that isatin binds specifically only to cytochromes P450 with high affinity (the equilibrium dissociation constant (Kd) is about 10-8 M).

Kinetics of Protein-Protein Interactions at the Surface of an Optical Biosensor

1995 ◽  
Vol 231 (1) ◽  
pp. 210-217 ◽  
Author(s):  
P.R. Edwards ◽  
A. Gill ◽  
D.V. Pollardknight ◽  
M. Hoare ◽  
P.E. Buckle ◽  
...  
Keyword(s):  
Protein Interactions ◽  
Optical Biosensor ◽  
Protein Protein Interactions ◽  
Kinetics Of

scholarly journals Surface plasmon resonance: a useful technique for cell biologists to characterize biomolecular interactions

2013 ◽  
Vol 24 (7) ◽  
pp. 883-886 ◽  
Author(s):  
Robert V. Stahelin
Keyword(s):  
Surface Plasmon Resonance ◽  
Surface Plasmon ◽  
Protein Interactions ◽  
Plasmon Resonance ◽  
Dissociation Rate ◽  
Biomolecular Interactions ◽  
Protein Protein Interactions ◽  
Interaction Kinetics ◽  
Lipid Protein Interactions ◽  
Dissociation Rate Constants

Surface plasmon resonance (SPR) is a powerful technique for monitoring the affinity and selectivity of biomolecular interactions. SPR allows for analysis of association and dissociation rate constants and modeling of biomolecular interaction kinetics, as well as for equilibrium binding analysis and ligand specificity studies. SPR has received much use and improved precision in classifying protein–protein interactions, as well as in studying small-molecule ligand binding to receptors; however, lipid–protein interactions have been underserved in this regard. With the field of lipids perhaps the next frontier in cellular research, SPR is a highly advantageous technique for cell biologists, as newly identified proteins that associate with cellular membranes can be screened rapidly and robustly for lipid specificity and membrane affinity. This technical perspective discusses the conditions needed to achieve success with lipid–protein interactions and highlights the unique lipid–protein interaction mechanisms that have been elucidated using SPR. It is intended to provide the reader a framework for quantitative and confident conclusions from SPR analysis of lipid–protein interactions.

scholarly journals Quantifying the monomer-dimer equilibrium of tubulin with mass photometry

2020 ◽  
Author(s):  
Adam Fineberg ◽  
Thomas Surrey ◽  
Philipp Kukura
Keyword(s):  
Dissociation Constant ◽  
Protein Interactions ◽  
Building Block ◽  
Dissociation Rate ◽  
Protein Protein Interactions ◽  
Cellular Processes ◽  
Fundamental Building Block ◽  
Kinetics Of

AbstractThe αβ-tubulin heterodimer is the fundamental building block of microtubules, making it central to several cellular processes. Despite the apparent simplicity of heterodimerisation, the associated energetics and kinetics remain disputed, largely due to experimental challenges associated with quantifying affinities in the <μM range. We use mass photometry to observe tubulin monomers and heterodimers in solution simultaneously, thereby quantifying the αβ-tubulin dissociation constant (8.48±1.22 nM) and its tightening in the presence of GTP (3.69±0.65 nM), at a dissociation rate >10−2 s−1. Our results demonstrate the capabilities of mass photometry for quantifying protein-protein interactions and clarify the energetics and kinetics of tubulin heterodimerisation.Abstract Figure

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