Molecular and Cellular Effects of Human Mutations in Cytochrome P450 Reductase

描述(申请人提供)：NADPH-细胞色素P450氧化还原酶(CYPOR，由POR基因编码)缺陷已与Antley-Bixler综合征(ABS)的骨骼和类固醇生成异常相关，尽管其对细胞色素P450(CYP)介导的异生和内生代谢的特定影响仍未解决。最近发现的40个自然产生的人类CYPOR变异体代表了广泛的残留活性，已经根据表型相关性和初步的酶学进行了分类。这项研究计划旨在从分子和细胞水平上研究自然发生的POR突变和多态变异如何影响CYPOR在支持细胞过程中的结构安排和催化功能。特定目标1-POR变体的分子分析将检验这样的假设，即特定残基在突变时，产生具有改变的电子传递和/或氧化/还原伙伴相互作用性质的蛋白质。为了实现这一目标，将同时使用溶液(光谱、动力学和热力学)和结晶学(X射线衍射)技术。初步证据表明，从治疗的角度来看，具体的缺陷是可以解决的。此外，CYPOR缺乏，以及随后的CYP代谢物失衡，预计会导致基因表达谱改变，从而导致发育缺陷。因此，《特定目的2--CYPOR缺乏症下游事件的细胞分析》将利用受不同程度的CYPOR缺乏症影响的组织的细胞模型。为了达到这一目的，我们将把有缺陷的POR基因引入原代(从PORlox/lox小鼠组织中分离)和转化的人类(肝、肠和骨)细胞模型中，以测量对异种生物/内生生物挑战的直接代谢(CYP介导的活性分析)和下游反应(功能分析和基因表达谱)。公共卫生相关性：这项拟议工作与公共卫生的相关性在于，它将调查在人类群体中发现的CYPOR的多种基因突变，这些突变可能会影响产前发育期间以及在年轻人和成年人中对环境和治疗药物的反应。

DESCRIPTION (provided by applicant): NADPH-cytochrome P450 oxidoreductase (CYPOR, encoded by the POR gene) deficiency has been correlated with the skeletal and steroidogenic anomalies of Antley-Bixler syndrome (ABS), although its specific effects on cytochrome P450 (CYP)-mediated xenobiotic and endobiotic metabolism remain unresolved. Forty naturally occurring human CYPOR variants, recently discovered and representing a broad range of residual activities, have been categorized based on phenotypic correlation and preliminary enzymology. This research plan is designed to address, on a molecular and cellular level, how naturally occurring POR mutations and polymorphic variations affect the structural arrangement and catalytic function of CYPOR in supporting cellular processes. Specific Aim 1 - Molecular Analysis of POR Variants will examine the hypothesis that specific residues, when mutated, produce proteins with altered electron transport and/or oxidation/reduction partner interaction properties. To address this aim, both solution (spectroscopic, kinetic, and thermodynamic) and crystallographic (X-ray diffractions) techniques will be utilized. Preliminary evidence suggests that specific deficiencies may be addressable from a therapeutic perspective. Furthermore, CYPOR deficiency, as well as the subsequent imbalance of CYP metabolites, is predicted to result in altered gene expression profiles that lead to developmental defects. Therefore, Specific Aim 2 - Cellular Analysis of Downstream Events in CYPOR Deficiency will utilize cellular models of tissues affected by varying degrees of CYPOR deficiency. To address this aim, defective POR genes will be introduced into both primary (isolated from PORlox/lox mouse tissues) and transformed human (liver, intestinal, and bone) cell models in order to measure the direct metabolic (CYP-mediated activity assays) and downstream response (functional assays and gene expression profiling) to xenobiotic/endobiotic challenge. Public Health Relevance: The relevance of this proposed work to public health is that it will investigate the multiple genetic mutations in CYPOR being found in the human population, which could influence the response to environmental and therapeutic agents during prenatal development, as well as in young and adult human beings.