The cytochrome P450s comprise a large family of enzymes responsible for such diverse reactions as drug and xenobiotic metabolism, steroid and bile acid biosynthesis, and fatty acid and eicosanoid hydroxylation. NADPH-cytochrome P450 oxidoreductase (POR) is the sole provider of electrons to the microsomal cytochromes P450 and to heme oxygenase, which catabolizes heme degradation. Because there is no redundancy for POR in mammalian systems, sequence variants might be expected to exhibit varied and pleiotropic effects depending on the sequence location and severity of the mutation, particularly if it impacts POR interactions with some CYPs, but not others. Indeed, patients with Antley-Bixler-like syndrome, which is characterized by craniofacial dysmorphism, premature synostoses, and disordered steroidogenesis, were shown to have such variants. This proposal will investigate the question of how disruption of the redox enzyme POR affects development and function of various tissues, particularly liver and bone. It is hypothesized that these effects are mediated in part by loss of CYP metabolites that regulate downstream signaling pathways directly or indirectly, and that perturbation of the interaction between POR and particular CYPs may vary, producing different phenotypes dependent upon the affected CYPs. To address these questions, three Specific Aims are proposed as follows:
Specific Aim I. Molecular Characterization.
This aim will characterize the purified forms of naturally occurring POR variants in humans. A variety of biochemical and biophysical techniques will be employed to determine the physical properties and protein-protein interaction capabilities of POR and its variants, including spectral, kinetic, surface plasmon resonance, analytical ultracentrifugation, ELDOR and isothermal titration calorimetry. These studies will elucidate the molecular properties of the enzymes that cause these phenotypic changes.
Specific Aim II. Effect of POR Deficiency and Mutations in Downstream Cellular Events. In this experimental aim, we will examine the effects of POR mutations on downstream cellular events. These will be determined in bone- and liver-derived cells and tissues in which POR expression has been diminished or deleted, for example, by shRNA or from tissue- specific knockout mice. These cellular events, dependent on bone or liver metabolites, may affect bone development and hepatocyte function.
Specific Aim III. Liver- and Bone-Specific Effects of POR Variants and POR Knock-down. Bone development and defects will be examined in liver-specific cytochrome P450 reductase knockout mice and a newly developed (in the PI's laboratory) bone-specific knockout mouse model using micro CT, differential staining and determination of vitamin D, retinoic acid, cholesterol, steroid, and lipid serum levels in these mice. Identification of new human polymorphisms will continue to determine possible downstream biomarkers of POR deficiency is a potential outcome.
: The multifaceted approach taken by this proposal addresses the consequences of human cytochrome P450 reductase (POR) deficiency in human drug metabolism and bone development. Understanding these processes is crucial for tailoring drug regimens and other possible therapeutic interventions. By searching for new genetic mutations in this enzyme, identification of relevant biomarkers for POR deficiency is also a goal.
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