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

J Patrick Connick,James R Reed,George F Cawley,Wayne L Backes

doi:10.1042/bcj20200768

J Patrick Connick, James R Reed + Show 2 more

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https://doi.org/10.1042/bcj20200768

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Abstract

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 with 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 the mathematical simulation, the results could not be explained by a model where CYP1A1 and HO-1 simply compete for POR, and are consistent with the formation of a stable CYP1A1•HO-1 complex that affected the functional characteristics of both moieties.

Highlights

As members of the CYP1A subfamily, CYP1A1 and CYP1A2 have similar amino acid sequences (80% identical) and induction of both enzymes is mediated by the aryl hydrocarbon receptor [1, 2]
Whereas baseline CYP1A1 expression is low, induction of CYP1A1 activity by exposure to aryl hydrocarbon receptor agonists has been observed in many tissues [1, 57]
When analyzing physical complex formation using bioluminescence resonance energy transfer (BRET), the most striking result was with the formation of the CYP1A1HO-1 complex

Summary

Introduction

As members of the CYP1A subfamily, CYP1A1 and CYP1A2 have similar amino acid sequences (80% identical) and induction of both enzymes is mediated by the aryl hydrocarbon receptor [1, 2]. Some conserved CYP1 family-specific residues are responsible for forming a small, planar active site [3], leading to both P450s sharing many of the same substrates and inhibitors [4]. Despite their high degree of sequence similarity, differences in these proteins have been observed. As the total concentration of the expressed P450s exceed that of POR in most tissues [11,12,13], each of the P450s must effectively compete for POR or be functionally silent These interactions become more complex when considering the potential for the levels of several of these proteins to be regulated by exogenous chemicals. These characteristics raise continued questions regarding how the P450 system proteins are organized in the endoplasmic reticulum (ER)

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