Molecular & Cellular Effects of Human Mutations in Cytochrome P450 Reductase

描述（由申请人提供）：细胞色素P450包括一个大家族的酶，负责药物和异生物质代谢、类固醇和胆汁酸生物合成以及脂肪酸和类花生酸羟基化等多种反应。NADPH-细胞色素P450氧化还原酶（POR）是微粒体细胞色素P450和血红素加氧酶的唯一电子提供者，血红素加氧酶分解代谢血红素降解。因为在哺乳动物系统中POR没有冗余，所以序列变体可能会根据突变的序列位置和严重程度表现出不同的多效性效应，特别是如果它影响POR与某些CYP的相互作用，而不是其他CYP。事实上，Antley-Bixler样综合征的患者，其特征是颅面畸形，过早的骨性结合和类固醇生成障碍，被证明有这样的变体。该提案将调查氧化还原酶POR的破坏如何影响各种组织，特别是肝脏和骨骼的发育和功能的问题。据推测，这些影响 部分是由直接或间接调节下游信号传导途径的代谢产物的损失介导的，并且POR和特定CYP之间相互作用的扰动可能不同，产生依赖于受影响CYP的不同表型。为了解决这些问题，提出了以下三个具体目标：具体目标一。分子表征。这一目标将表征人类中天然存在的POR变体的纯化形式。将采用各种生物化学和生物物理技术来确定POR及其变体的物理性质和蛋白质-蛋白质相互作用能力，包括光谱、动力学、表面等离子体共振、分析超离心、ELDOR和等温滴定量热法。这些研究将阐明引起这些表型变化的酶的分子特性。具体目标二。POR缺陷和突变对下游细胞事件的影响。在本实验中，我们将研究POR突变对下游细胞事件的影响。这些将在其中POR表达已经减少或缺失的骨和肝来源的细胞和组织中测定，例如，通过shRNA或来自组织特异性敲除小鼠。这些细胞事件依赖于骨或肝脏代谢产物，可能影响骨发育和肝细胞功能。具体目标三。POR变体和POR敲低的肝脏和骨骼特异性效应。将在肝脏特异性细胞色素P450还原酶敲除小鼠和新开发的（PI实验室）骨特异性敲除小鼠模型中，使用显微CT、差异染色和测定这些小鼠中的维生素D、视黄酸、胆固醇、类固醇和脂质血清水平，检查骨发育和缺损。新的人类多态性的鉴定将继续确定POR缺陷的可能下游生物标志物是一个潜在的结果。

DESCRIPTION (provided by applicant): 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.