Role of a hydrophobic polypeptide in the N-terminal region of NADPH-cytochrome P-450 reductase in complex formation with P-450lm

Black, Shaun D.; French, John S.; Williams, Charles H.; Coon, Minor J.

doi:10.1016/0006-291x(79)91238-5

Cited by 108 publications

(36 citation statements)

References 22 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…The discrepancy is probably due to the use of truncated POR proteins in their studies. It has been observed that the N-terminal sequence of POR is important for the electron transfer function (Black et al, 1979;Bonina et al, 2005). Although the study by Black et al (1979) suggested that the membrane anchor of POR is required for interaction and reduction of cytochrome P450 but not cytochrome c, it is not clear whether the reported activity decrease in cytochrome c reduction could be due to a combined effect of C569Y change and the N-terminal truncation in the POR protein.…”

Section: Discussionmentioning

confidence: 72%

Genetic Variation of Human Cytochrome P450 Reductase as a Potential Biomarker for Mitomycin C-Induced Cytotoxicity

Wang¹,

Han²,

He³

et al. 2006

Drug Metab Dispos

View full text Add to dashboard Cite

ABSTRACT:The importance of genetic variation in clinical response to various drugs is now well recognized. Identification of genetic biomarkers that can predict efficacy and toxicity of chemotherapeutic drugs in cancer patients holds great promise in treatment improvement and cost reduction. Mitomycin C (MMC) is a common anticancer drug used for the treatment of numerous types of tumors. Metabolismmediated activation, by either one-electron or two-electron reduction, plays a critical role in the chemotherapeutic action of MMC. NADPH-cytochrome P450 (oxido)reductase (POR) is a major enzyme responsible for MMC activation through the one-electron reductive pathway, which leads to the production of semiquinone anion radicals and subsequent DNA damage in the cells. Recently, a total of six naturally occurring human POR variants with single amino acid changes (Y181D, A287P, R457H, V492E, C569Y, and V608F) have been identified. Although the catalytic efficiency of these variants in reduction of cytochrome c was reported to be altered, their capability in activating MMC, a direct substrate of POR, has not been examined. In the present study, we demonstrated that except for the C569Y variant, MMC-induced toxicity assayed as cell viability and proliferative capability was significantly decreased in the Flp-In Chinese hamster ovary cells stably expressing all the other POR variants in comparison with the cells expressing wild-type human POR. Cells expressing the V608F and Y181D variants had a complete loss of the capability to activate MMC. Our finding suggests that these functional POR genetic variations may serve as a potential biomarker to predict the chemotherapeutic response to MMC.

show abstract

Section: Discussionmentioning

confidence: 72%

Genetic Variation of Human Cytochrome P450 Reductase as a Potential Biomarker for Mitomycin C-Induced Cytotoxicity

Wang¹,

Han²,

He³

et al. 2006

Drug Metab Dispos

View full text Add to dashboard Cite

show abstract

“…In the absence of the N terminus, the two proteins may be unable to directly interact through the N terminus, or because the truncated protein is unable to insert itself into the lipid milieu and hence is incapable of interacting. Previous studies have shown that CPR and b5, lacking their hydrophobic membrane binding N termini and C termini, respectively, were unable to provide electrons to P450s, indicating the relevance of nonspecific hydrophobic interactions in P450-CPR and P450-b5 interactions (Black et al, 1979;Lee-Robichaud et al, 1997;Chudaev et al, 2001;Mulrooney et al, 2004). Other studies of P450-P450 and P450-redox interactions studies have suggested the importance of electrostatic forces (Shimizu et al, 1991;Shen and Strobel, 1993;Omata et al, 1994;Bridges et al, 1998;Chudaev et al, 2001;Mulrooney et al, 2004;Kelley et al, 2005).…”

Section: Discussionmentioning

confidence: 99%

CYP2C9-CYP3A4 Protein-Protein Interactions: Role of the Hydrophobic N Terminus

Subramanian

Tam²,

Zheng³

et al. 2010

Drug Metab Dispos

View full text Add to dashboard Cite

ABSTRACT:Cytochromes P450 (P450s) interact with redox transfer proteins, including P450 reductase (CPR) and cytochrome b 5 (b5), all being membrane-bound. In multiple in vitro systems, P450-P450 interactions also have been observed, resulting in alterations in enzymatic activity. The current work investigated the effects and mechanisms of interaction between CYP2C9 and CYP3A4 in a reconstituted system. CYP2C9-mediated metabolism of S-naproxen and S-flurbiprofen was inhibited up to 80% by coincubation with CYP3A4, although K m values were unchanged. Increasing CYP3A4 concentrations increased the degree of inhibition, whereas increasing CPR concentrations resulted in less inhibition. Addition of b5 only marginally affected the magnitude of inhibition. In contrast, CYP2C9 did not alter the CYP3A4-mediated metabolism of testosterone. The potential role of the hydrophobic N terminus on these interactions was assessed by incubating truncated CYP2C9 with full-length CYP3A4, and vice versa. In both cases, the inhibition was fully abolished, indicating an important role for hydrophobic forces in CYP2C9-CYP3A4 interactions. Finally, a CYP2C9/CYP3A4 heteromer complex was isolated by coimmunoprecipitation techniques, confirming the physical interaction of the proteins. These results show that the N-terminal membrane binding domains of CYP2C9 and CYP3A4 are involved in heteromer complex formation and that at least one consequence is a reduction in CYP2C9 activity.Cytochromes P450 (P450s) are the primary oxidative enzymes responsible for the metabolism of xenobiotics by humans and animals (Guengerich, 2006). P450s can oxidize structurally diverse substrates ranging from small hydrocarbons to large molecules such as cyclosporine. P450s function by transferring electrons via a P450 catalytic pathway, wherein the first electron is transferred to the P450 via cytochrome NADPH P450 reductase (CPR) and the second electron is transferred via either CPR or cytochrome b 5 (b5) (Guengerich, 2002). Inefficiencies in the catalytic pathway, in part, govern the rate of transformation. P450s and CPR are bound to the cell membrane via a hydrophobic N terminus, whereas b5 is bound via its C terminus, each terminal being composed of 30 to 60 amino acids (Szczesna-Skorupa and Kemper, 2008). This hydrophobic region also contributes to protein aggregation, resulting in the formation of high molecular weight polymers in membranes and reconstituted systems (SzczesnaSkorupa and Kemper, 2008). Considering that P450s and CPR, and P450s and b5 interact within the cell membrane, P450-P450 interactions also appear conceivable. Indeed, in vitro evidence of P450-P450 interactions have been observed in several studies, including interactions of human CYP2C9-CYP2C19, rabbit CYP1A2-CYP2B4, rabbit CYP1A2-CYP2E1, human CYP3A4-CYP1A1, human CYP3A4-CYP1A2, and human CYP2C9-CYP2D6 (Cawley et al., 1995;Yamazaki et al., 1997;Backes et al., 1998;Backes and Cawley, 1999;Kelley et al., 2005Kelley et al., , 2006Subramanian et al., 2009). Substrate-dependent activation o...

show abstract

“…dmd.aspetjournals.org structs, are generally compromised in the ability to interact productively (Black et al, 1979). For productive alignment of the two proteins, insertion of both proteins in an appropriate lipid microenvironment is considered essential (Hlavica et al, 2003).…”

Section: Michaelis-menten Kinetic Parameters For Purified Cypor Variantsmentioning

confidence: 99%

Human Cytochrome P450 Oxidoreductase Deficiency Caused by the Y181D Mutation: Molecular Consequences and Rescue of Defect

Marohnic

Panda²,

McCammon

et al. 2009

Drug Metab Dispos

View full text Add to dashboard Cite

Role of a hydrophobic polypeptide in the N-terminal region of NADPH-cytochrome P-450 reductase in complex formation with P-450lm

Cited by 108 publications

References 22 publications

Genetic Variation of Human Cytochrome P450 Reductase as a Potential Biomarker for Mitomycin C-Induced Cytotoxicity

Genetic Variation of Human Cytochrome P450 Reductase as a Potential Biomarker for Mitomycin C-Induced Cytotoxicity

CYP2C9-CYP3A4 Protein-Protein Interactions: Role of the Hydrophobic N Terminus

Human Cytochrome P450 Oxidoreductase Deficiency Caused by the Y181D Mutation: Molecular Consequences and Rescue of Defect

Contact Info

Product

Resources

About