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

Abstract

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 owing to their common electron donor, they generally have been studied separately. As the expressions 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. The POR•CYP1A2 complex was readily disrupted by the addition of HO-1, whereas the POR•HO-1 complex was not significantly affected by the addition of CYP1A2. Interestingly, enzyme activities did not follow this pattern. BRET data suggested substantial inhibition of CYP1A2-mediated 7-ethoxyresorufin de-ethylation in the presence of HO-1, whereas 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 a simple competition for POR. 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 owing to their common electron donor, they generally have been studied separately. As the expressions 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. The POR•CYP1A2 complex was readily disrupted by the addition of HO-1, whereas the POR•HO-1 complex was not significantly affected by the addition of CYP1A2. Interestingly, enzyme activities did not follow this pattern. BRET data suggested substantial inhibition of CYP1A2-mediated 7-ethoxyresorufin de-ethylation in the presence of HO-1, whereas 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 a simple competition for POR. Cytochrome P450 (EC 1.14.14.1) is a heme-containing, membrane-bound protein that catalyzes the oxygenation of a wide variety of exogenous and endogenous compounds (1Guengerich F.P. Cytochrome P450 and chemical toxicology.Chem. Res. Toxicol. 2008; 21: 70-83Crossref PubMed Scopus (1059) Google Scholar, 2Rendic S. Guengerich F.P. Update information on drug metabolism systems--2009, part II: summary of information on the effects of diseases and environmental factors on human cytochrome P450 (CYP) enzymes and transporters.Curr. Drug Metab. 2010; 11: 4-84Crossref PubMed Scopus (80) Google Scholar). Generally, metabolism by P450 converts lipid-soluble substrates to more water-soluble products and thereby facilitates their elimination. However, in many instances, metabolism by P450 can generate reactive intermediates that lead to toxicity, mutagenesis, and/or carcinogenesis (2Rendic S. Guengerich F.P. Update information on drug metabolism systems--2009, part II: summary of information on the effects of diseases and environmental factors on human cytochrome P450 (CYP) enzymes and transporters.Curr. Drug Metab. 2010; 11: 4-84Crossref PubMed Scopus (80) Google Scholar, 3Rendic S. Di Carlo F.J. Human cytochrome P450 enzymes: a status report summarizing their reactions, substrates, inducers, and inhibitors.Drug Metab. Rev. 1997; 29: 413-580Crossref PubMed Scopus (1057) Google Scholar). Additionally, the standard monooxygenase reaction can be “uncoupled,” leading to generation of superoxide and H2O2 (4Loida P.J. Sligar S.G. Molecular recognition in cytochrome P-450: mechanism for the control of uncoupling reactions.Biochemistry. 1993; 32: 11530-11538Crossref PubMed Scopus (209) Google Scholar). Heme oxygenase 1 (EC 1.14.14.18; HO-1) is responsible for the first step of heme degradation, converting the substrate to biliverdin (5Kikuchi G. Yoshida T. Noguchi M. Heme oxygenase and heme degradation.Biochem. Biophys. Res. Commun. 2005; 338: 558-567Crossref PubMed Scopus (315) Google Scholar, 6Abraham N.G. Kappas A. Pharmacological and clinical aspects of heme oxygenase.Pharmacol. Rev. 2008; 60: 79-127Crossref PubMed Scopus (861) Google Scholar). This enzyme protects cells from oxidative damage and is induced by agents that are related to oxidative stress, including many xenobiotics, such as aspirin, statins, niacin, etc. (7Abraham N.G. Junge J.M. Drummond G.S. Translational significance of heme oxygenase in obesity and metabolic syndrome.Trends Pharmacol. Sci. 2016; 37: 17-36Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar, 8Hsu M. Muchova L. Morioka I. Wong R.J. Schroder H. Stevenson D.K. Tissue-specific effects of statins on the expression of heme oxygenase-1 in vivo.Biochem. Biophys. Res. Commun. 2006; 343: 738-744Crossref PubMed Scopus (79) Google Scholar, 9Loboda A. Jazwa A. Grochot-Przeczek A. Rutkowski A.J. Cisowski J. Agarwal A. Jozkowicz A. Dulak J. Heme oxygenase-1 and the vascular bed: from molecular mechanisms to therapeutic opportunities.Antioxid. Redox Signal. 2008; 10: 1767-1812Crossref PubMed Scopus (194) Google Scholar). HO-1 levels are also influenced by disease states including inflammation, diabetes, hepatic injury, infectious disease, and cancer (6Abraham N.G. Kappas A. Pharmacological and clinical aspects of heme oxygenase.Pharmacol. Rev. 2008; 60: 79-127Crossref PubMed Scopus (861) Google Scholar, 7Abraham N.G. Junge J.M. Drummond G.S. Translational significance of heme oxygenase in obesity and metabolic syndrome.Trends Pharmacol. Sci. 2016; 37: 17-36Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar, 8Hsu M. Muchova L. Morioka I. Wong R.J. Schroder H. Stevenson D.K. Tissue-specific effects of statins on the expression of heme oxygenase-1 in vivo.Biochem. Biophys. Res. Commun. 2006; 343: 738-744Crossref PubMed Scopus (79) Google Scholar). A feature common to these conditions is oxidative stress, where HO-1 induction leads to the elimination of pro-oxidants (heme) and the generation of antioxidants (e.g., bilirubin and CO). There is evidence that HO-1 and some P450s are capable of forming homomeric complexes that can affect function. HO-1 (10Marohnic C.C. Huber III, W.J. Patrick C.J. Reed J.R. McCammon K. Panda S.P. Martasek P. Backes W.L. Masters B.S. Mutations of human cytochrome P450 reductase differentially modulate heme oxygenase-1 activity and oligomerization.Arch. Biochem. Biophys. 2011; 513: 42-50Crossref PubMed Scopus (17) Google Scholar, 11Hwang H.W. Lee J.R. Chou K.Y. Suen C.S. Hwang M.J. Chen C. Shieh R.C. Chau L.Y. Oligomerization is crucial for the stability and function of heme oxygenase-1 in the endoplasmic reticulum.J. Biol. Chem. 2009; 284: 22672-22679Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar), CYP1A2 (12Reed J.R. Connick J.P. Cheng D. Cawley G.F. Backes W.L. Effect of homomeric P450•P450 complexes on P450 function.Biochem. J. 2012; 446: 489-497Crossref PubMed Scopus (23) Google Scholar), CYP2C2 (13Hu G. Johnson E.F. Kemper B. 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Heme oxygenase and P450s not function but with NADPH-cytochrome P450 reductase Masters B.S. evidence for of heme oxygenase with the electron Biol. Chem. Full Text PDF PubMed Google Scholar), in is in of NADPH-cytochrome P450 H. Cytochrome Google Scholar). Although the HO-1 and P450 systems are interconnected by their common electron donor, the systems generally have been studied separately. The of both physical and P450•P450 interactions been whereas interactions between HO-1 and the P450s have evidence that P450s and HO-1 can affect function A. of heme oxygenase-1 the of CYP1A1, CYP1A2, and catalytic activity in Metab. Dispos. 2012; PubMed Scopus Google that the inhibition of CYP1A1, CYP1A2, and in was at by induction of HO-1, but the mechanism for these effects was not The of P450•P450 interactions and the effects the interactions on enzyme function interactions between HO-1 and P450s also influence catalytic The goal of this study was to examine the for interactions between the HO-1 and P450 on CYP1A2. The results that the presence of HO-1 a on P450 function that cannot be explained by a simple competition between HO-1 and P450 for The results also that HO-1 and CYP1A2 form a stable complex both in systems and in natural that the in both CYP1A2 and HO-1 function is the of this the that P450•P450 interactions have been with changes in protein the for physical interactions between HO-1 and P450s was In to interactions between P450s and HO-1 for the was with protein P450s and HO-1. HO-1 a the protein was with P450s and on the This was to of the with the of the to with the endoplasmic cells with the proteins at a to that the bioluminescence resonance energy transfer for for to protein expression to for the of these BRET where HO-1 was with CYP1A1, CYP2D6, and CYP1A2, with with and on these the HO-1–CYP1A2 reaction was for These results that interactions between HO-1 and P450s are for the P450 enzyme then focused on CYP1A2 of the BRET with HO-1 in the and the of CYP1A2 to form complexes with P450s W.L. C.J. Cawley G.F. P450 enzymes in of a complex with a for NADPH-cytochrome P450 37: PubMed Scopus Google Scholar, Reed J.R. Backes W.L. of on the interactions between and 2005; PubMed Scopus Google Scholar, Cheng D. Backes W.L. complex between CYP2E1 and evidence for the of 2006; PubMed Scopus (34) Google Scholar, J.R. M. Backes W.L. interactions between cytochromes P450 and both enzymes to in the same evidence for physical complex Biol. Chem. 2010; Full Text Full Text PDF PubMed Scopus Google Scholar, J.P. Reed J.R. Backes W.L. of interactions CYP1A2, and NADPH-cytochrome P450 of protein Metab. Dispos. 46: PubMed Scopus Google Scholar, N.Y. B. D.R. a systems to cytochrome P450 interactions of CYP2E1 with P450 and their on J. PubMed Scopus Google Scholar, J.R. Cawley G.F. Backes W.L. between cytochromes P450 and lead to in and P450 PubMed Scopus Google Scholar). The goal of this study was to CYP1A2 and HO-1 formed a BRET-detectable complex and to the complex was stable in the presence of POR. This was by complex for of the interactions and in the presence and of the for and cells at a of These data follow that to a function. The BRET with the of proteins with proteins and as of protein complex to the levels to that in not be to in protein expression a in was protein expression for of the was between and the protein expression of the The first goal was to that HO-1 and CYP1A2 formed a stable complex and to the complex be disrupted by the presence of POR. that the BRET a that was with the of a of to the cells of this that the complex was that the of protein expression was in both the and presence of POR. The of complex was also by the BRET as a function of protein expression these complexes not and the of protein a with a the be J.R. A. A for protein bioluminescence resonance energy 2006; PubMed Scopus Google Scholar). These results also that a of the BRET is to changes in levels of these results indicate that HO-1 and CYP1A2 form a complex cells and that the complex is stable in the presence of POR. the of HO-1 on complex was As expected, CYP1A2 and formed BRET complex of HO-1 the BRET to a in levels of the proteins in the BRET both in the and presence of HO-1 that the in BRET was not to a in protein expression but to of the POR•CYP1A2 The of the POR•CYP1A2 complex is with HO-1 to all proteins are In the the of CYP1A2 on the POR•HO-1 BRET was the complex between HO-1 and was of CYP1A2 did not significantly affect of the POR•HO-1 these results are with a complex where with its HO-1 CYP1A2 is to form homomeric complexes in was by both BRET and chemical (12Reed J.R. Connick J.P. Cheng D. Cawley G.F. Backes W.L. Effect of homomeric P450•P450 complexes on P450 function.Biochem. J. 2012; 446: 489-497Crossref PubMed Scopus (23) Google Scholar). As this homomeric BRET complex is to be disrupted by the of the of HO-1 to the BRET was The results that HO-1 can the BRET The presence of a stable complex leads to the of this complex have on P450 and HO-1 CYP1A2-mediated 7-ethoxyresorufin was as a function of where the proteins In the of HO-1, CYP1A2 a as a function of This and is with the of CYP1A2 to form a homomeric complex and (12Reed J.R. Connick J.P. Cheng D. Cawley G.F. Backes W.L. Effect of homomeric P450•P450 complexes on P450 function.Biochem. J. 2012; 446: 489-497Crossref PubMed Scopus (23) Google Interestingly, in the presence of HO-1, to be stimulated at subsaturating but inhibited at a that was HO-1 was in the In the the of CYP1A2 on HO-1–mediated heme metabolism was As W.J. C.C. M. J. Reed J.R. Masters Backes W.L. of membrane-bound human heme oxygenase-1 activity a Metab. Dispos. 2009; 37: PubMed Scopus Google Scholar, III, W.J. Backes W.L. and of human heme the presence of leads to for cytochrome P450 2007; 46: PubMed Scopus (25) Google Scholar), in the HO-1 and between the proteins was a of However, addition of CYP1A2 inhibition of HO-1 activities at subsaturating POR. The from to in the presence of CYP1A2. The P450s and HO-1 a necessary in POR. P450s are by a variety of and xenobiotic agents (1Guengerich F.P. Cytochrome P450 and chemical toxicology.Chem. Res. Toxicol. 2008; 21: 70-83Crossref PubMed Scopus (1059) Google Scholar, 2Rendic S. Guengerich F.P. Update information on drug metabolism systems--2009, part II: summary of information on the effects of diseases and environmental factors on human cytochrome P450 (CYP) enzymes and transporters.Curr. Drug Metab. 2010; 11: 4-84Crossref PubMed Scopus (80) Google Scholar). HO-1 also is by endogenous and compounds as as by oxidative J. Heme and of a protein in J. Biol. PubMed Scopus Google Scholar). is at levels are the leading to conditions where they for P. J. J. of cytochrome P450 expression with the expression of of cytochrome P450 in human Metab. Dispos. 2016; PubMed Scopus Google Scholar, B. J. A. of hepatic cytochrome P450 enzymes and their a Metab. Dispos. PubMed Scopus Google the can be by HO-1. In the HO-1 levels are of both and HO-1 can the levels of both proteins J. Heme from to therapeutic Rev. 2006; PubMed Scopus Google Scholar). These factors to the for HO-1, and to on the levels of of the induction states these interactions in of enzyme The is where HO-1 and CYP1A2 not form a and the proteins for on their that HO-1 for III, W.J. Backes W.L. and of human heme the presence of leads to for cytochrome P450 2007; 46: PubMed Scopus (25) Google Scholar, III, W.J. B. Backes W.L. of human heme oxygenase-1 a complex with cytochrome P450 2009; PubMed Scopus Google whereas the of CYP1A2 for is W.L. C.J. Cawley G.F. P450 enzymes in of a complex with a for NADPH-cytochrome P450 37: PubMed Scopus Google Scholar, Reed J.R. Backes W.L. of on the interactions between and 2005; PubMed Scopus Google Scholar, Cheng D. Backes W.L. complex between CYP2E1 and evidence for the of 2006; PubMed Scopus (34) Google Scholar). These data to that all proteins are HO-1 CYP1A2 for the and that CYP1A2 activity be inhibited by the addition of HO-1, is with the BRET and However, the BRET results also that a complex is the that these proteins in the as A is that the complex but of this complex not the of to with POR. to this complex be the BRET complex be significantly disrupted by the presence of HO-1, and the POR•HO-1 complex be affected by CYP1A2. The BRET results and are with this The BRET of physical complex are with of a complex that is capable of forming a POR•HO-1 This is by the inhibition of POR•CYP1A2 by HO-1 and the of CYP1A2 to the POR•HO-1 on BRET a substantial inhibition of CYP1A2-mediated activity be in the presence of HO-1. However, was and was CYP1A2-mediated activity actually in the presence of HO-1 BRET data of the POR•CYP1A2 CYP1A2-mediated was inhibited at CYP1A2 and in a simple CYP1A2 activities as a function of the was more at (12Reed J.R. Connick J.P. Cheng D. Cawley G.F. Backes W.L. Effect of homomeric P450•P450 complexes on P450 function.Biochem. J. 2012; 446: 489-497Crossref PubMed Scopus (23) Google Scholar, J.R. Backes W.L. of P450 P450 complexes and their on P450 2012; PubMed Scopus Google Scholar). In a that CYP1A2 a homomeric complex that is (12Reed J.R. Connick J.P. Cheng D. Cawley G.F. Backes W.L. Effect of homomeric P450•P450 complexes on P450 function.Biochem. J. 2012; 446: 489-497Crossref PubMed Scopus (23) Google Scholar). of this complex and the of a POR•CYP1A2 In a the of HO-1 the generation of POR•CYP1A2 at Although part of the be by the of not with the of the POR•CYP1A2 complex by HO-1 In HO-1 is and the be by the where the HO-1 POR. inhibition of was of a in the of of the CYP1A2 also was to affect HO-1–mediated heme metabolism on the BRET HO-1 activities to be affected by the presence of CYP1A2, at subsaturating Although the was not affected at HO-1–mediated was inhibited by CYP1A2 at subsaturating POR. This inhibition of HO-1–mediated heme metabolism at subsaturating be of the complex the catalytic of the These results provide information the interactions between the HO-1 and P450 systems but also many complex between HO-1 and P450s is not all P450s to form such complexes the BRET and the as the of the proteins to proteins is Interestingly, this complex is not significantly affected by the addition of that both proteins are they not as homomeric complexes but as a the POR•CYP1A2 BRET complex is affected by the POR•HO-1 complex is not affected by CYP1A2. the activities of both CYP1A2-mediated and HO-1–mediated heme metabolism are influenced by the presence of the protein in that cannot be explained by a simple competition for POR. The data at this multiple effects on the of the enzymes in the systems. A more analysis be to the and from The to generate BRET and the from is a that been by a results in but and Human cells from was from was from and was from was from and cytochrome was from HO-1, CYP1A2, and proteins to their generate the BRET expression for CYP1A2 and was from expression with to that the the The the and for CYP1A2 and and for POR. These products then the and for the and for was by the of enzymes The multiple of the are of the This information is in This was to that the protein to with the of the of the proteins to the to the the BRET for a to between the of the and the for the generate for the expression of CYP1A2, HO-1, and was to a to the protein was the from and from of and by of the BRET and their of the and HO-1, and was the the The BRET from in a of the and HO-1, and was the the The BRET from for human and from CO). The that as a of human was a of Masters C.C. Panda S.P. Martasek P. Masters B.S. in the and of human cytochrome P450 Biol. Chem. 2006; Full Text Full Text PDF PubMed Scopus Google Scholar). Human HO-1 was from the as III, W.J. Backes W.L. and of human heme the presence of leads to for cytochrome P450 2007; 46: PubMed Scopus (25) Google Scholar). the of HO-1 to with the P450s and M. 1 the 2012; PubMed Scopus Google Scholar, T. M. and of heme oxygenase Biophys. Res. Commun. PubMed Scopus Google Scholar), to with This the and with and and that the In to a for HO-1 the was from the the with a of the The was then by the HO-1 a BRET cells first with 1 to of at of the and The goal for these was to generate a of cells the same protein at a of to that the BRET complexes and not to changes in protein expression this the same protein expression all the results In many necessary to levels of protein at of protein cells to expression of the was with the a protein, the protein and cells with in 1 of and in of BRET of cells was in A was for BRET This was to the for of a for 1 to for at and for at The of to the addition of to a of was as the BRET and by with the of The BRET was then with the BRET from cells the protein to (10Marohnic C.C. Huber III, W.J. Patrick C.J. Reed J.R. McCammon K. Panda S.P. Martasek P. Backes W.L. Masters B.S. Mutations of human cytochrome P450 reductase differentially modulate heme oxygenase-1 activity and oligomerization.Arch. Biochem. Biophys. 2011; 513: 42-50Crossref PubMed Scopus (17) Google Scholar, J.R. Connick J.P. Cheng D. Cawley G.F. Backes W.L. Effect of homomeric P450•P450 complexes on P450 function.Biochem. J. 2012; 446: 489-497Crossref PubMed Scopus (23) Google Scholar). expression levels of and proteins by with cells a expression was by on a of the in expression was in the the of of the protein was by the addition of to a of and was for 1 The and for then to that of the protein is to have a expression that the by the of the expression In the data in the and presence of the systems as W.J. C.C. M. J. Reed J.R. Masters Backes W.L. of membrane-bound human heme oxygenase-1 activity a Metab. Dispos. 2009; 37: PubMed Scopus Google Scholar). A was in with and This was then at in a for the CYP1A2, HO-1, both with of and for at in enzyme was as as and the of to the enzyme was at Cheng D. Backes W.L. complex between CYP2E1 and evidence for the of 2006; PubMed Scopus (34) Google Scholar). HO-1 a including a of biliverdin converts the biliverdin by HO-1–mediated heme systems and for at the addition of reaction in The was at and to provide biliverdin was to a of in to the of H2O2 on reaction W.J. C.C. M. J. Reed J.R. Masters Backes W.L. of membrane-bound human heme oxygenase-1 activity a Metab. Dispos. 2009; 37: PubMed Scopus Google Scholar). activity to at for to of the reaction by the addition of to a of reaction was at in in with a of A was to bilirubin a the in at and The was for and the from the from the of the activities the of M. and of heme in and of protein and PubMed Google Scholar, Kappas A. induction of hepatic heme with evidence that cytochrome is not for this enzyme Sci. S. A. PubMed Scopus Google Scholar). the of CYP1A2 activity in 7-ethoxyresorufin as a CYP1A2 converts the for at systems A was to a of at the with the addition of to a of in at a to for of activities a standard from of data to the for in of the inhibition of by Biophys. PubMed Scopus Google Scholar). of was between a simple in all conditions for the and a for the to data a and J. PubMed Scopus Google Scholar). significantly the with was at are as the data are the The that they have of with the of this to of for the NADPH-cytochrome P450 reductase expression and of for the HO-1 also to for with protein A of this study was in a by J. Patrick Connick cytochromes heme and NADPH-cytochrome P450 reductase form multiple complexes that influence protein also to the of of the of in of J. P. C. and G. C. the J. P. C. the of the J. and L. B. the its and and the L. B. the This was by from the of and the of The is the of the and not the of the of

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