scholarly journals Heteromeric complex formation between human cytochrome P450 CYP1A1 and heme oxygenase-1

2021 ◽  
Author(s):  
J. Patrick Connick ◽  
James R. Reed ◽  
George F. Cawley ◽  
Wayne L. Backes
Keyword(s):  
Cytochrome P450 ◽  
Complex Formation ◽  
Heme Oxygenase ◽  
Protein Interactions ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Stable Complex ◽  
Heme Oxygenase 1 ◽  
Protein Protein Interactions ◽  
Human Cytochrome

P450 and heme oxygenase-1 (HO-1) receive their necessary electrons by interaction with the NADPH-cytochrome P450 reductase (POR).  As the POR concentration is limiting when compared to P450 and HO-1, they must effectively compete for POR to function.  In addition to these functionally required protein-protein interactions, HO-1 forms homomeric complexes, and several P450s have been shown to form complexes with themselves and with other P450s, raising the question, “How are the HO-1 and P450 systems organized in the endoplasmic reticulum?”  Recently, CYP1A2 was shown to associate with HO-1 affecting the function of both proteins.  The goal of this study was to determine if CYP1A1 formed complexes with HO-1 in a similar manner.  Complex formation among POR, HO-1, and CYP1A1 was measured using bioluminescence resonance energy transfer, with results showing HO-1 and CYP1A1 form a stable complex that was further stabilized in the presence of POR.  The POR•CYP1A1 complex was readily disrupted by the addition of HO-1.  CYP1A1 also was able to affect the POR•HO-1 complex, although the effect was smaller.  This interaction between CYP1A1 and HO-1 also affected function, where the presence of CYP1A1 inhibited HO-1-mediated bilirubin formation by increasing the KmPOR•HO-1 without affecting the Vmaxapp.  In like manner, HO-1 inhibited CYP1A1-mediated 7-ethoxyresorufin dealkylation by increasing the KmPOR•CYP1A1.   Based on mathematical simulation, the results could not be explained by a model where CYP1A1 and HO-1 simply compete for POR, and are consistent with formation of a stable CYP1A1•HO-1 complex that affected the functional characteristics of both moieties.

scholarly journals Heme oxygenase-1 affects cytochrome P450 function through the formation of heteromeric complexes: Interactions between CYP1A2 and heme oxygenase-1

2020 ◽  
pp. jbc.RA120.015911
Author(s):  
J. Patrick Connick ◽  
James R. Reed ◽  
George F. Cawley ◽  
Wayne L. Backes
Keyword(s):  
Cytochrome P450 ◽  
Heme Oxygenase ◽  
Cytochromes P450 ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Stable Complex ◽  
Heme Oxygenase 1 ◽  
Cytochrome P450 Reductase ◽  
Xenobiotic Exposure ◽  
Substantial Inhibition

Heme oxygenase 1 (HO‑1) and the cytochromes P450 (P450s) are endoplasmic reticulum-bound enzymes that rely on the same protein, NADPH-cytochrome P450 reductase (POR), to provide the electrons necessary for substrate metabolism.  Although the HO‑1 and P450 systems are interconnected due to their common electron donor, they generally have been studied separately.  As the expression of both HO-1 and P450s are affected by xenobiotic exposure, changes in HO‑1 expression can potentially affect P450 function, and conversely, changes in P450 expression can influence HO‑1.  The goal of this study was to examine interactions between the P450 and HO‑1 systems.  Using bioluminescence resonance energy transfer (BRET), HO‑1 formed HO‑1•P450 complexes with CYP1A2, CYP1A1, and CYP2D6, but not all P450s.  Studies then focused on the HO‑1/CYP1A2 interaction.  CYP1A2 formed a physical complex with HO-1 that was stable in the presence of POR.  As expected, both HO‑1 and CYP1A2 formed BRET-detectable complexes with POR.  Whereas the POR•CYP1A2 complex was readily disrupted by the addition of HO‑1, the POR•HO-1 complex was not significantly affected by the addition of CYP1A2.  Interestingly, enzyme activities did not follow this pattern.  Whereas BRET data suggested substantial inhibition of CYP1A2-mediated 7-ethoxyresorufin deethylation in the presence of HO-1, its activity was actually stimulated at subsaturating POR.  In contrast, HO‑1-mediated heme metabolism was inhibited at subsaturating POR.  These results indicate that HO‑1 and CYP1A2 form a stable complex and have mutual effects on the catalytic behavior of both proteins that cannot be explained by simple competition for POR.

scholarly journals Heme oxygenase-1 affects cytochrome P450 function through the formation of heteromeric complexes: Interactions between CYP1A2 and heme oxygenase-1

2020 ◽  
Author(s):  
J. Patrick Connick ◽  
James R. Reed ◽  
George F. Cawley ◽  
Wayne L. Backes
Keyword(s):  
Cytochrome P450 ◽  
Heme Oxygenase ◽  
Cytochromes P450 ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Stable Complex ◽  
Heme Oxygenase 1 ◽  
Cytochrome P450 Reductase ◽  
Xenobiotic Exposure ◽  
Substantial Inhibition

AbstractHeme oxygenase 1 (HO-1) and the cytochromes P450 (P450s) are endoplasmic reticulum-bound enzymes that rely on the same protein, NADPH-cytochrome P450 reductase (POR), to provide the electrons necessary for substrate metabolism. Although the HO-1 and P450 systems are interconnected due to their common electron donor, they generally have been studied separately. As the expression of both HO-1 and P450s are affected by xenobiotic exposure, changes in HO-1 expression can potentially affect P450 function, and conversely, changes in P450 expression can influence HO-1. The goal of this study was to examine interactions between the P450 and HO-1 systems. Using bioluminescence resonance energy transfer (BRET), HO-1 formed HO-1•P450 complexes with CYP1A2, CYP1A1, and CYP2D6, but not all P450s. Studies then focused on the HO-1/CYP1A2 interaction. CYP1A2 formed a physical complex with HO-1 that was stable in the presence of POR. As expected, both HO-1 and CYP1A2 formed BRET-detectable complexes with POR. Whereas the POR•CYP1A2 complex was readily disrupted by the addition of HO-1, the POR•HO-1 complex was not significantly affected by the addition of CYP1A2. Interestingly, enzyme activities did not follow this pattern. Whereas BRET data suggested substantial inhibition of CYP1A2-mediated 7-ethoxyresorufin deethylation in the presence of HO-1, its activity was actually stimulated at subsaturating POR. In contrast, HO-1-mediated heme metabolism was inhibited at subsaturating POR. These results indicate that HO-1 and CYP1A2 form a stable complex and have mutual effects on the catalytic behavior of both proteins that cannot be explained by simple competition for POR.

scholarly journals Measuring ligand-dependent and ligand-independent interactions between nuclear receptors and associated proteins using Bioluminescence Resonance Energy Transfer (BRET2)

2006 ◽  
Vol 4 (1) ◽  
pp. nrs.04021 ◽  
Author(s):  
Kristen L. Koterba ◽  
Brian G. Rowan
Keyword(s):  
Energy Transfer ◽  
Protein Interactions ◽  
Fusion Proteins ◽  
Fluorescent Protein ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Renilla Luciferase ◽  
Receptor Protein ◽  
Protein Protein Interactions

Bioluminescent resonance energy transfer (BRET2) is a recently developed technology for the measurement of protein-protein interactions in a live, cell-based system. BRET2 is characterized by the efficient transfer of excited energy between a bioluminescent donor molecule (Renilla luciferase) and a fluorescent acceptor molecule (a mutant of Green Fluorescent Protein (GFP2)). The BRET2 assay offers advantages over fluorescence resonance energy transfer (FRET) because it does not require an external light source thereby eliminating problems of photobleaching and autoflourescence. The absence of contamination by light results in low background that permits detection of very small changes in the BRET2 signal. BRET2 is dependent on the orientation and distance between two fusion proteins and therefore requires extensive preliminary standardization experiments to conclude a positive BRET2 signal independent of variations in protein titrations and arrangement in tertiary structures. Estrogen receptor (ER) signaling is modulated by steroid receptor coactivator 1 (SRC-1). To establish BRET2 in a ligand inducible system we used SRC-1 as the donor moiety and ER as the acceptor moiety. Expression and functionality of the fusion proteins were assessed by transient transfection in HEK-293 cells followed by Western blot analysis and measurement of ER-dependent reporter gene activity. These preliminary determinations are required prior to measuring nuclear receptor protein-protein interactions by BRET2. This article describes in detail the BRET2 methodology for measuring interaction between full-length ER and coregulator proteins in real-time, in an in vivo environment.

scholarly journals Heterodimerization Between the Lutropin and Follitropin Receptors is Associated With an Attenuation of Hormone-Dependent Signaling

Endocrinology ◽  
2013 ◽  
Vol 154 (10) ◽  
pp. 3925-3930 ◽  
Author(s):  
Xiuyan Feng ◽  
Meilin Zhang ◽  
Rongbin Guan ◽  
Deborah L. Segaloff
Keyword(s):  
Protein Interactions ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
G Protein Coupled Receptors ◽  
Fsh Receptor ◽  
Bioluminescence Resonance Energy Transfer ◽  
Protein Protein Interactions ◽  
Lh Receptor ◽  
G Protein Coupled ◽  
Stimulation Of

The LH receptor (LHR) and FSH receptor (FSHR) are each G protein-coupled receptors that play critical roles in reproductive endocrinology. Each of these receptors has previously been shown to self-associate into homodimers and oligomers shortly after their biosynthesis. As shown herein using bioluminescence resonance energy transfer to detect protein-protein interactions, our data show that the LHR and FSHR, when coexpressed in the same cells, specifically heterodimerize with each other. Further experiments confirm that at least a portion of the cellular LHR/FSHR heterodimers are present on the cell surface and are functional. We then sought to ascertain what effects, if any, heterodimerization between the LHR and FSHR might have on signaling. It was observed that when the LHR was expressed under conditions promoting the heterodimerization with FSHR, LH or human chorionic gonadotropin (hCG) stimulation of Gs was attenuated. Conversely, when the FSHR was expressed under conditions promoting heterodimerization with the LHR, FSH-stimulated Gs activation was attenuated. These results demonstrate that the coexpression of the LHR and FSHR enables heterodimerizaton between the 2 gonadotropin receptors and results in an attenuation of signaling through each receptor.

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