scholarly journals Identification of the contact region responsible for the formation of the homomeric CYP1A2•CYP1A2 complex

2021 ◽  
Author(s):  
Aratrika Saha ◽  
J. Patrick Connick ◽  
James R. Reed ◽  
Charles S. Lott ◽  
Wayne L. Backes
Keyword(s):  
Cytochrome P450 ◽  
Complex Formation ◽  
Contact Region ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Proximal Region ◽  
Homologous Region ◽  
Cytochrome P450 Reductase ◽  
Metabolic Characteristics ◽  
Impaired Binding

Previous studies showed that cytochrome P450 1A2 (CYP1A2) forms a homomeric complex that influences its metabolic characteristics. Specifically, CYP1A2 activity exhibits a sigmoidal response as a function of NADPH-cytochrome P450 reductase (POR) concentration and is consistent with an inhibitory CYP1A2•CYP1A2 complex that is disrupted by increasing [POR] (Reed et al., (2012) Biochem. J. 446, 489-497). The goal of this study was to identify the CYP1A2 contact regions involved in homomeric complex formation. Examination of X‑ray structure of CYP1A2 implicated the proximal face in homomeric complex formation. Consequently, involvement of residues L91-K106 (P1 region) located on the proximal face of CYP1A2 was investigated. This region was replaced with the homologous region of CYP2B4 (T81-S96) and the protein was expressed in HEK293T/17 cells. Complex formation and its disruption was observed using bioluminescence resonance energy transfer (BRET). The P1‑CYP1A2 (CYP1A2 with the modified P1 region) exhibited a decreased BRET signal as compared to wild-type CYP1A2 (WT‑CYP1A2). On further examination, P1‑CYP1A2 was much less effective at disrupting the CYP1A2•CYP1A2 homomeric complex, when compared to WT‑CYP1A2, thereby demonstrating impaired binding of P1‑CYP1A2 to WT‑CYP1A2 protein. In contrast, the P1 substitution did not affect its ability to form a heteromeric complex with CYP2B4.  P1‑CYP1A2 also showed decreased activity as compared to WT‑CYP1A2, which was consistent with a decrease in the ability of P1‑CYP1A2 to associate with WT‑POR, again implicating the P1 region in POR binding. These results indicate that the contact region responsible for the CYP1A2•CYP1A2 homomeric complex resides in the proximal region of the protein.

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 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 ◽  
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 A Large-Scale Allosteric Transition in Cytochrome P450 3A4 Revealed by Luminescence Resonance Energy Transfer (LRET)

PLoS ONE ◽  
2013 ◽  
Vol 8 (12) ◽  
pp. e83898 ◽  
Author(s):  
Elena V. Sineva ◽  
Jessica A. O. Rumfeldt ◽  
James R. Halpert ◽  
Dmitri R. Davydov
Keyword(s):  
Cytochrome P450 ◽  
Energy Transfer ◽  
Large Scale ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Cytochrome P450 3A4 ◽  
Allosteric Transition
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