Altered Human CYP3A4 Activity Caused by Antley-Bixler Syndrome-Related Variants of NADPH-Cytochrome P450 Oxidoreductase Measured in a Robust In Vitro System

Moutinho, Daniela; Marohnic, Christopher C.; Panda, Satya Prakash; Rueff, José; Masters, Bettie Sue Siler; Kranendonk, Michel

doi:10.1124/dmd.111.042820

Cited by 28 publications

(37 citation statements)

References 36 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Membrane fractions of selected CPR mut /CYP clones were prepared and characterized for CYP-(CO-difference spectrophotometry), CPR-(immune-detection by western blotting) and protein-contents (Bradford assay), using previously described methods (Duarte et al, 2007;Kranendonk et al, 2008;Moutinho et al, 2012;Palma et al, 2013;Campelo et al, 2018;Esteves et al, 2018). The intrinsic electron donation capacity of the CPR-FMN-domain mutants was assessed by DCPIP reduction.…”

Section: Enzyme Activity Evaluation Using Membrane Fractionsmentioning

confidence: 99%

The Role of the FMN-Domain of Human Cytochrome P450 Oxidoreductase in Its Promiscuous Interactions With Structurally Diverse Redox Partners

et al. 2020

Self Cite

View full text Add to dashboard Cite

NADPH cytochrome P450 oxidoreductase (CPR) is the obligatory electron supplier that sustains the activity of microsomal cytochrome P450 (CYP) enzymes. The variant nature of the isoform-specific proximal interface of microsomal CYPs indicates that CPR is capable of multiple degenerated interactions with CYPs for electron transfer, through different binding mechanisms, and which are still not well-understood. Recently, we showed that CPR dynamics allows formation of open conformations that can be sampled by its structurally diverse redox partners in a CYP-isoform dependent manner. To further investigate the role of the CPR FMN-domain in effective binding of CPR to its diverse acceptors and to clarify the underlying molecular mechanisms, five different CPR-FMN-domain random mutant libraries were created. These libraries were screened for mutants with increased activity when combined with specific CYP-isoforms. Seven CPR-FMN-domain mutants were identified, supporting a gain in activity for CYP1A2 (P117H, G144C, A229T), 2A6 (P117L/L125V, G175D, H183Y), or 3A4 (N151D). Effects were evaluated using extended enzyme kinetic analysis, cytochrome b 5 competition, ionic strength effect on CYP activity, and structural analysis. Mutated residues were located either in or adjacent to several acidic amino acid stretches-formerly indicated to be involved in CPR:CYP interactions-or close to two tyrosine residues suggested to be involved in FMN binding. Several of the identified positions co-localize with mutations found in naturally occurring CPR variants that were previously shown to cause CYP-isoform-dependent effects. The mutations do not seem to significantly alter the geometry of the FMN-domain but are likely to cause very subtle alterations leading to improved interaction with a specific CYP. Overall, these data suggest that CYPs interact with CPR using an isoform specific combination of several binding motifs of the FMN-domain.

show abstract

Section: Enzyme Activity Evaluation Using Membrane Fractionsmentioning

confidence: 99%

The Role of the FMN-Domain of Human Cytochrome P450 Oxidoreductase in Its Promiscuous Interactions With Structurally Diverse Redox Partners

et al. 2020

Self Cite

View full text Add to dashboard Cite

show abstract

“…Although in vitro assays may not simulate some aspects of the natural environment of the endoplasmic reticulum, they remain useful tools for comparison of residual activities among individual CYP enzymes. At the very least, it is important that in vitro assays mimic the membrane milieu and stoichiometry of the in vivo system as closely as possible [54]. …”

Section: Discussionmentioning

confidence: 99%

Identification of Six Novel P450 Oxidoreductase Missense Variants in Ashkenazi and Moroccan Jewish Populations

et al. 2012

View full text Add to dashboard Cite

Background The enzyme NADPH–P450 oxidoreductase (POR) is the main electron donor to all microsomal CYPs. The possible contribution of common POR variants to inter- and intra-individual variability in drug metabolism is of great pharmacogenetic interest. Aim To search for POR polymorphic alleles and estimate their frequencies in a Jewish population. Materials & methods We analyzed the POR gene in 301 Ashkenazi and Moroccan Jews. Results A total of 30 POR SNPs were identified, nine in the noncoding regions and 21 in the protein-coding regions (ten synonymous, 11 missense). Six of these missense variants are previously undescribed (S102P, V164M, V191M, D344N, E398A and D648N). Conclusion The data collected in this study on missense POR SNPs, interpreted in light of the crystallographic structure of human POR, indicate that some POR missense variants may be potential biomarkers for future POR pharmacogenetic screening.

show abstract

“…The variant A287P was reported as the most common mutation found in the Caucasian population (10,14), and these studies were verified and extended to establish genotype-phenotype correlations in a cohort of patients from 11 countries (21). We and others have pursued the characterization of A287P (10,14,22,23).…”

mentioning

confidence: 85%

“…The biplasmid system in BTC strains allows the expression of fulllength proteins at a stoichiometry that reflects that found in vivo (23). Table 4 summarizes the characterization of the different BTC1A2_POR membranes, i.e.…”

Section: A287p Variant Preparation Necessitates Modified Expressionmentioning

confidence: 99%

Instability of the Human Cytochrome P450 Reductase A287P Variant Is the Major Contributor to Its Antley-Bixler Syndrome-like Phenotype

McCammon

Panda

Xia

et al. 2016

Journal of Biological Chemistry

Self Cite

View full text Add to dashboard Cite

Human NADPH-cytochrome P450 oxidoreductase (POR) gene mutations are associated with severe skeletal deformities and disordered steroidogenesis. The human POR mutation A287P presents with disordered sexual development and skeletal malformations. Difficult recombinant expression and purification of this POR mutant suggested that the protein was less stable than WT. The activities of cytochrome P450 17A1, 19A1, and 21A2, critical in steroidogenesis, were similar using our purified, full-length, unmodified A287P or WT POR, as were those of several xenobiotic-metabolizing cytochromes P450, indicating that the A287P protein is functionally competent in vitro, despite its functionally deficient phenotypic behavior in vivo. Differential scanning calorimetry and limited trypsinolysis studies revealed a relatively unstable A287P compared with WT protein, leading to the hypothesis that the syndrome observed in vivo results from altered POR protein stability. The crystal structures of the soluble domains of WT and A287P reveal only subtle differences between them, but these differences are consistent with the differential scanning calorimetry results as well as the differential susceptibility of A287P and WT observed with trypsinolysis. The relative in vivo stabilities of WT and A287P proteins were also examined in an osteoblast cell line by treatment with cycloheximide, a protein synthesis inhibitor, showing that the level of A287P protein post-inhibition is lower than WT and suggesting that A287P may be degraded at a higher rate. Current studies demonstrate that, unlike previously described mutations, A287P causes POR deficiency disorder due to conformational instability leading to proteolytic susceptibility in vivo, rather than through an inherent flavin-binding defect.

show abstract

Altered Human CYP3A4 Activity Caused by Antley-Bixler Syndrome-Related Variants of NADPH-Cytochrome P450 Oxidoreductase Measured in a Robust In Vitro System

Cited by 28 publications

References 36 publications

The Role of the FMN-Domain of Human Cytochrome P450 Oxidoreductase in Its Promiscuous Interactions With Structurally Diverse Redox Partners

The Role of the FMN-Domain of Human Cytochrome P450 Oxidoreductase in Its Promiscuous Interactions With Structurally Diverse Redox Partners

Identification of Six Novel P450 Oxidoreductase Missense Variants in Ashkenazi and Moroccan Jewish Populations

Instability of the Human Cytochrome P450 Reductase A287P Variant Is the Major Contributor to Its Antley-Bixler Syndrome-like Phenotype

Contact Info

Product

Resources

About