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Physical complexes formed by CYP1A2 and CYP2B4 are associated with altered P450 activity
Author(s) -
Connick John Patrick,
Reed James Robert,
Backes Wayne L
Publication year - 2017
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.31.1_supplement.669.8
Subject(s) - cyp1a2 , chemistry , cytochrome p450 , biochemistry , biophysics , enzyme , biology
It has been demonstrated that the presence of one P450 can affect the activity of another. Previous studies have shown, for example, that when CYP1A2, CYP2B4, and stoichiometrically‐limiting concentrations of NADPH‐cytochrome P450 reductase (CPR) were incorporated into phospholipid vesicles, CYP1A2 activity was higher and CYP2B4 activity was lower than observed in single‐P450 systems. These data are inconsistent with a model of simple competition among P450s for CPR binding but could be caused by physical interactions among the pair of P450s. The goal of this work was to determine how physical interactions between CYP1A2 and CYP2B4 might play a role in modulating the P450‐CPR interaction and P450 activity. Various pairs of P450 and CPR constructs were tagged with one each of GFP and Renilla luciferase and transfected into HEK293T cells. Complexes between these proteins were detected by measuring energy transfer between the tags. A complex between CYP1A2 and CYP2B4 was detected both in the presence and absence of cotransfected, untagged CPR. Interactions of both P450s with CPR were detected, and untagged CYP1A2 was shown to disrupt the CPR‐CYP2B4 interaction while untagged CYP2B4 had no effect on the CPR‐CYP1A2 interaction. These data are consistent with a model in which CPR preferentially binds to the CYP1A2 moiety of a CYP1A2‐CYP2B4 complex. Previous work has also shown—using BRET, crosslinking, and kinetic measurements—that CYP1A2 forms homomeric complexes associated with inhibition of CYP1A2 activity at subsaturating CPR. Here it is shown using BRET that these complexes were disrupted by the presence of either CPR or CYP2B4. The activity of GFP‐tagged CYP1A2 and CYP2B4 co‐expressed in HEK293T cells was measured and shown to be consistent with data from the prior experiments on purified proteins. These findings explain the observed effects of CYP1A2 and CYP2B4 on each other's activity. Support or Funding Information NIH ES004344 NIH ES013648 Louisiana Board of Regents