Postdoctoral Researcher
Presenting Author LSUHSC New Orleans - Dept. of Pharmacology
The cytosolic face of the endoplasmic reticulum (ER) membrane contains a variety of enzymes involved in oxidative metabolism. Among these, NADPH-cytochrome P450 reductase (POR) and cytochrome b5 (CYB5) provide reducing electrons necessary for cytochrome P450 (P450) and heme oxygenase-1 (HO-1) activity. HO-1 catalyzes the first and rate-limiting step in free heme catabolism while the P450s are responsible for the metabolism of endogenous and exogenous compounds, including a majority of prescribed drugs. P450s have been shown to form heteromeric and homomeric complexes with other P450s and these complexes have been associated with alterations in P450 activity. Additionally, the P450 catalytic cycle requires 2 electrons—while POR is able to provide both, CYB5 can provide the second electron in some cases. However, binding of POR and CYB5 to the same P450 has been reported to be mutually exclusive, raising the question of how CYB5 can stimulate P450 activity if POR must dissociate prior to CYB5 binding.
In order to shed light on this question, we set out to collect more information on the protein-protein interaction network that the ER-membrane proteins participate in. Using bioluminescence resonance energy transfer (BRET), we are able to detect interactions between a pair of proteins and, further, to measure the stability of that pair in the presence of a third protein. To detect protein-protein interactions, HEK293T cells were co-transfected with vectors encoding two potentially interacting proteins tagged with either Renilla luciferase (Rluc) or GFP2. While human CYP1A2 showed evidence of a specific complex, with analysis of BRET data showing the presence of homodimers, human CYP1A1 did not form a specific homomeric complex. CYP1A1 and CYP1A2 also differed in how they interacted with other ER membrane proteins. Both proteins showed evidence of heteromeric complex formation with HO-1, but CYP1A1 only did so in the presence of excess POR. POR and CYB5 do not appear to interact with each other and in the presence of CYP1A2, HO-1, and several other P450s, a complex between POR and CYB5 was not detected. However, a complex including the two proteins was detected in the presence of CYP1A1 which indicated the presence of a ternary CYB5-POR-CYP1A1 complex. The presence of a ternary complex could explain the stimulation of P450 activity by CYB5, though the specific mechanism remains to be determined.
Supported by NIH R01 GM123253 amp;amp; P42 ES013648
