Molecular and Cellular Effects of Human Mutations in Cytochrome P450 Reductase

人类细胞色素 P450 还原酶突变的分子和细胞效应

• 批准号: 7798646
• 负责人: BETTIE SUE SILER MASTERS
• 金额: $ 55.57万
• 依托单位: UNIVERSITY OF TEXAS HLTH SCIENCE CENTER
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-05-21 至 2012-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7798646
• 关键词: Address; Adenoviruses; Adult; Affect; Animal Model; Antley-Bixler syndrome; Bacterial Model; Behavior; Binding; Biological Assay; Categories; Cell model; Cell physiology; Cells; Characteristics; Colon Carcinoma; Complement component C1s; Cytochrome P450; Cytochromes b5; DNA Microarray Chip; Defect; Dependence; Development; Electron Transport; Endoplasmic Reticulum; Enzymatic Biochemistry; Enzymes; Event; Film; Flavins; Fluorescence; Fluorescence Spectroscopy; Gene Expression; Gene Expression Profiling; Gene Mutation; Genetic Polymorphism; Heme; High Pressure Liquid Chromatography; Human; Human Cell Line; Intestines; Kinetics; Knock-out; Knowledge; Lead; Liver; Mammalian Cell; Measures; Mediating; Messenger RNA; Metabolic; Metabolic Biotransformation; Metabolism; Methods; Modeling; Molecular; Molecular Analysis; Molecular Profiling; Morphology; Mus; Mutate; Mutation; NADP; NADPH-Ferrihemoprotein Reductase; Oxidation-Reduction; Oxygenases; POR gene; Phenotype; Population; Property; Proteins; Public Health; Recombinants; Research; Residual state; Signal Transduction; Solutions; Spectrum Analysis; Structure; Techniques; Temperature; Testing; Therapeutic; Therapeutic Agents; Thermodynamics; Time; Tissues; Titrations; Transfection; Transgenic Animals; Untranslated Regions; Variant; Work; X ray diffraction analysis; X-Ray Crystallography; X-Ray Diffraction; Xenobiotics; base; bone; bone cell; cofactor; cytochrome c; design; falls; intestinal epithelium; knock-down; mutant; osteosarcoma; oxidation; physical property; prenatal; public health relevance; reconstitution; research study; response; skeletal; small hairpin RNA; young adult

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.

描述（由申请人提供）：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）介导的外源性和内源性代谢的具体影响仍未解决。最近发现的四十种天然存在的人类 CYPOR 变体代表了广泛的残留活性，已根据表型相关性和初步酶学进行了分类。该研究计划旨在在分子和细胞水平上解决自然发生的 POR 突变和多态性变异如何影响 CYPOR 在支持细胞过程中的结构排列和催化功能。具体目标 1 - POR 变体的分子分析将检验以下假设：特定残基突变时会产生具有改变的电子传输和/或氧化/还原伙伴相互作用特性的蛋白质。为了实现这一目标，将利用溶液（光谱、动力学和热力学）和晶体学（X 射线衍射）技术。初步证据表明，从治疗的角度来看，特定的缺陷可能是可以解决的。此外，CYPOR 缺陷以及随后的 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，具体目标 2 - CYPOR 缺陷下游事件的细胞分析将利用受不同程度 CYPOR 缺陷影响的组织的细胞模型。为了实现这一目标，将有缺陷的 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，以测量对外源性/内源性挑战的直接代谢（CYP 介导的活性测定）和下游反应（功能测定和基因表达谱）。公共卫生相关性：这项拟议工作与公共卫生的相关性在于，它将调查在人群中发现的 CYPOR 多种基因突变，这些突变可能会影响产前发育期间以及年轻人和成年人对环境和治疗药物的反应。