The long-term objective of this proposal is to better understand how the proteins of the P450 monooxygenase system are organized in the endoplasmic reticulum, the role of P450-P450 interactions on the function of these enzymes, and how these interactions may make individuals susceptible to alkylbenzene-induced toxicity by alterations in P450-mediated hydrocarbon metabolism. Alkylbenzenes are produced in extensive quantities throughout the world, with simple aromatic hydrocarbons being major components of gasoline and used in a wide variety of consumer products. The P450 system is responsible for both aliphatic and aromatic hydroxylation of the aromatic hydrocarbons, with several forms, CYP1A2, CYP2B4, and CYP2E1, being implicated in hydrocarbon metabolism. This process requires a functional interaction between P450 and the flavoprotein NADPH-cytochrome P450 reductase. However, total P450 levels exceed those of reductase by a ratio of 20:1. These conditions raise basic questions as to how the enzymes of this microsomal electron transport chain are organized. During the previous grant period we demonstrated that P450s interact through the formation of heteromeric P450 complexes. We have identified complexes between CYP2B4-CYP1A2 as well as CYP1A2-CYP2E1. These interactions were shown to have a profound effect on xenobiotic metabolism, largely due to an alteration in the manner in which NADPH-cytochrome P450 reductase transfers electrons to P450s in the heteromeric complex. Interestingly, we did not observe an effect on P450 function from CYP2B4-CYP2E1 complexes. The proposed studies are designed to extend our investigation and address questions related to the organization of reductase and P450, their interactions within the endoplasmic reticulum, and how these interactions affect xenobiotic metabolism, including the metabolism of alkylbenzenes. We plan to continue our characterization of these interactions, examining (1) the functional consequences of P450-P450 interactions, (2) the structural basis for these interactions by identifying the region(s) responsible for P450-P450 complex formation, and (3) the organizational consequences to P450-P450 complex formation (i.e. how do such interactions affect their regional distribution in the endoplasmic reticulum). These studies will increase our understanding of how the P450 electron transport chain is organized, and will provide new important information on the role of the P450 system in the bioactivation of aromatic hydrocarbons and the generation of reactive oxygen - a process that can have a significant influence on chemical toxicity.

Public Health Relevance

The purpose of this proposal is to examine the organization of the enzymes of the P450 system in membranes within the cell. How the P450s are organized will govern not only their effectiveness in removing foreign compounds from the body, but also their ability to form reactive compounds that can lead to toxicity, cancer, and aging.

Agency
National Institute of Health (NIH)
Institute
National Institute of Environmental Health Sciences (NIEHS)
Type
Research Project (R01)
Project #
5R01ES004344-19
Application #
8215695
Study Section
Xenobiotic and Nutrient Disposition and Action Study Section (XNDA)
Program Officer
Carlin, Danielle J
Project Start
1988-01-15
Project End
2014-01-31
Budget Start
2012-02-01
Budget End
2013-01-31
Support Year
19
Fiscal Year
2012
Total Cost
$380,951
Indirect Cost
$111,372
Name
Louisiana State Univ Hsc New Orleans
Department
Pharmacology
Type
Schools of Medicine
DUNS #
782627814
City
New Orleans
State
LA
Country
United States
Zip Code
70112
Brignac-Huber, Lauren M; Park, Ji Won; Reed, James R et al. (2016) Cytochrome P450 Organization and Function Are Modulated by Endoplasmic Reticulum Phospholipid Heterogeneity. Drug Metab Dispos 44:1859-1866
Reed, James R; Backes, Wayne L (2016) The functional effects of physical interactions involving cytochromes P450: putative mechanisms of action and the extent of these effects in biological membranes. Drug Metab Rev 48:453-69
Scott, Emily E; Wolf, C Roland; Otyepka, Michal et al. (2016) The Role of Protein-Protein and Protein-Membrane Interactions on P450 Function. Drug Metab Dispos 44:576-90
Reed, James R; dela Cruz, Albert Leo N; Lomnicki, Slawo M et al. (2015) Inhibition of cytochrome P450 2B4 by environmentally persistent free radical-containing particulate matter. Biochem Pharmacol 95:126-32
Reed, James R; dela Cruz, Albert Leo N; Lomnicki, Slawo M et al. (2015) Environmentally persistent free radical-containing particulate matter competitively inhibits metabolism by cytochrome P450 1A2. Toxicol Appl Pharmacol 289:223-30
Park, Ji Won; Reed, James R; Backes, Wayne L (2015) The Localization of Cytochrome P450s CYP1A1 and CYP1A2 into Different Lipid Microdomains Is Governed by Their N-terminal and Internal Protein Regions. J Biol Chem 290:29449-60
Park, Ji Won; Reed, James R; Brignac-Huber, Lauren M et al. (2014) Cytochrome P450 system proteins reside in different regions of the endoplasmic reticulum. Biochem J 464:241-9
Reed, James R; Cawley, George F; Ardoin, Taylor G et al. (2014) Environmentally persistent free radicals inhibit cytochrome P450 activity in rat liver microsomes. Toxicol Appl Pharmacol 277:200-9
Johnson, Eric F; Connick, J Patrick; Reed, James R et al. (2014) Correlating structure and function of drug-metabolizing enzymes: progress and ongoing challenges. Drug Metab Dispos 42:9-22
Reed, James R; Cawley, George F; Backes, Wayne L (2013) Interactions between cytochromes P450 2B4 (CYP2B4) and 1A2 (CYP1A2) lead to alterations in toluene disposition and P450 uncoupling. Biochemistry 52:4003-13

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