CYTOCHROME P450 GENE REGULATION IN LUNG

肺部细胞色素 P450 基因调控

• 批准号: 6351531
• 负责人: Garold S Yost
• 金额: $ 28.03万
• 依托单位: UNIVERSITY OF UTAH
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-02-01 至 2004-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6351531
• 关键词: affinity chromatography; cytochrome P450; gene environment interaction; gene expression; genetic mapping; genetic regulation; molecular cloning; nucleic acid sequence; pathologic process; respiratory disorder; transcription factor

Humans are exposed to a large variety of pneumotoxic or carcinogenic chemicals that exert their toxicities after bioactivation of the parent compound by enzymes in the cytochrome P450 supergene family. Lung diseases cause significant morbidity and mortality, and specific P450 enzymes are at least partially responsible for many of these diseases. Our laboratory has demonstrated that pulmonary cytochrome P450 enzymes bioactive one important pneumotoxin, 3-methylindole, to highly reactive electrophilic intermediates in specific lung cells of animals and man. Selective toxicity to lung tissues is primarily related to the selective expression of P450 genes in bronchiolar or alveolar epithelial cells, without concomitant expression in liver or other tissues. Thus, the expression of P450 genes in certain cells leads to the selective damage of these cells by reactive intermediates produced by the enzymes. It is also likely that lung-specific expression of certain members of the P450 gene superfamily by different individuals leads to differential human susceptibilities to pneumotoxicants, but the biochemical mechanisms responsible for the selective expression of these genes in lung cells are not known. The hypothesis of this research is that the selective expression of certain cytochrome P450 genes in human lung epithelial cells is driven by unique, previously uncharacterized transcription factors; these factors act in concert to regulate the expression of two important P450 genes, CYP2F1 and CYP4B1. The major goal of this research is to precisely determine the factors that regulate the expression of P450 genes in lung cells. This goal will be realized through the following objectives: 1) clone the human pulmonary P450 genes CYP2F1 and CYP4B1, characterize the 5' regulatory regions of the genes, and screen the sequences for known cis-regulatory domains; 2) utilize deletion analysis with luciferase constructs in human bronchial epithelial lung cells and in vitro DNA binding assays to analyze and identify unique, new cis-regulatory domains; and 3) employ DNA affinity chromatography techniques and the yeast one-hybrid system to characterize the trans-regulatory proteins that regulate expression of these genes. These studies will provide vital, unique knowledge concerning the specific mechanisms that control expression of P450 genes in lung cells and will most likely have significant impact on human lung diseases that are related to bioactivation of toxicants by cytochrome P450 enzymes.

人类暴露于大量种类的具有肺毒性或致癌性的化学物质，这些化学物质在母体化合物被细胞色素P450超基因家族中的酶生物活化后发挥其毒性。 肺部疾病导致显著的发病率和死亡率，并且特异性P450酶至少部分地负责许多这些疾病。 我们的实验室已经证明，肺细胞色素P450酶的生物活性的一个重要的肺毒素，3-甲基吲哚，高活性的亲电中间体在特定的肺细胞的动物和人。肺组织的选择性毒性主要是与P450基因的选择性表达在细支气管或肺泡上皮细胞，没有伴随的表达在肝脏或其他组织。 因此，P450基因在某些细胞中的表达导致这些细胞被酶产生的活性中间体选择性损伤。 不同个体的P450基因超家族的某些成员的肺特异性表达也可能导致人类对肺毒物的不同敏感性，但这些基因在肺细胞中选择性表达的生化机制尚不清楚。 这项研究的假设是，某些细胞色素P450基因在人肺上皮细胞中的选择性表达是由独特的，以前未表征的转录因子驱动的;这些因子共同作用，调节两个重要的P450基因，CYP 2F 1和CYP 4 B1的表达。 本研究的主要目的是精确地确定调节肺细胞中P450基因表达的因素。 这一目标将通过以下目标实现：1）克隆人肺P450基因CYP 2F 1和CYP 4 B1，表征基因的5'调控区，并筛选已知顺式调控结构域的序列：2）利用荧光素酶构建体在人支气管上皮肺细胞中的缺失分析和体外DNA结合分析来分析和鉴定独特的、新的顺式调控结构域; 3）利用DNA亲和层析技术和酵母单杂交系统对调控这些基因表达的反式调节蛋白进行鉴定。 这些研究将提供关于控制肺细胞中P450基因表达的特定机制的重要的、独特的知识，并且很可能对与细胞色素P450酶对毒物的生物活化相关的人类肺部疾病产生重大影响。