Function and Regulation of Human Cytochrome P4502S1

描述（由申请人提供）：CYP2S1是最近发现的人类细胞色素P450，在上皮组织中广泛表达。我们认为CYP2S1在环境前致癌物的代谢激活、药物和内源性化合物的代谢中发挥重要作用。该提案将解决这一假设，并表征酶的调节。有三个具体目的：(i)我们已经在细菌中表达了人类CYP2S1，并证明它代谢几种对上皮组织有毒和/或致癌的化合物。我们也将在哺乳动物细胞中过度表达这种酶。使用这些表达系统，我们将筛选其他底物，以及被CYP2S1激活为致突变（因此可能致癌）衍生物的致癌原。将确定代表性化合物的Km和Vmax值，并确定形成的代谢物。CYP2S1对人类上皮组织中特定底物的总代谢的贡献程度将通过酶的抑制抗体来确定，（ii）我们已经证明CYP2S1是由二恶英、致癌的多环芳烃（PAHs）和缺氧诱导的。我们将研究人类CYP2S1基因5'侧翼区域的潜在异种反应元件（XREs）或潜在抗氧化反应元件（ARE）是否介导二恶英和/或多环芳烃的诱导，并解决由于上述XREs的特定核苷酸序列，基因在某些细胞中对多环芳烃的反应比对二恶英的反应更好的假设。我们还将分析该基因缺氧诱导的机制，（iii）我们将产生Cyp2s1基因敲除小鼠，然后产生含有人类Cyp2s1基因的小鼠衍生物，包括其侧翼调控区域。这只“CYP2S1人源化”小鼠将被用于研究人类CYP2S1底物的代谢，这种代谢的生物学后果，以及异种生物和缺氧对人类CYP2S1基因的调节，从而补充和扩展特定目的1和2。我们的研究可能会证明CYP2S1在致癌物、药物和内源性化合物代谢中的重要作用，并可能最终为减少环境致癌物的有害影响和某些药物对人类的不良影响提供机会。

DESCRIPTION (provided by applicant): CYP2S1 is a recently identified human cytochrome P450, expressed extensively in epithelial tissues. We propose that CYP2S1 plays a significant role in the metabolic activation of environmental procarcinogens, and the metabolism of pharmaceuticals and endogenous compounds. The proposal will address this hypothesis and characterize regulation of the enzyme. There are three specific aims: (i) We have expressed human CYP2S1 in bacteria, and demonstrated that it metabolizes several compounds that are toxic and/or carcinogenic to epithelial tissues. We will also over-express the enzyme in mammalian cells. Using these expression systems, we will screen for additional substrates, and also for procarcinogens that are activated to mutagenic (and therefore probably carcinogenic) derivatives by CYP2S1. The Km and Vmax values will be determined for representative compounds, and the metabolites that are formed will be identified. The degree to which CYP2S1 contributes towards the total metabolism of particular substrates in human epithelial tissues will be determined using an inhibitory antibody to the enzyme, (ii) We have shown that CYP2S1 is inducible by dioxin, carcinogenic polycyclic aromatic hydrocarbons (PAHs), and hypoxia. We will investigate whether the potential Xenobiotic Responsive Elements (XREs), or the potential Antioxidant Response Element (ARE) in the 5' flanking region of the human CYP2S1 gene mediate induction by dioxin and/or PAHs, and address the hypothesis that due to the particular nucleotide sequences of the above XREs, the gene responds better to PAHs than to dioxin in certain cells. We will also analyze the mechanism of hypoxic induction of the gene, (iii) We will generate a knockout mouse for Cyp2s1, and then generate a derivative of this mouse containing the human CYP2S1 gene, including its flanking regulatory regions. This "CYP2S1-humanized" mouse will be used to study the metabolism of substrates of human CYP2S1, the biological consequences of this metabolism, and the regulation of the human CYP2S1 gene by xenobiotics and hypoxia, thus complementing and extending specific aims 1 and 2. Our studies may demonstrate important roles for CYP2S1 in the metabolism of carcinogens, Pharmaceuticals and endogenous compounds, and may ultimately provide opportunities for reducing the deleterious effects of environmental carcinogens and the adverse effects of certain Pharmaceuticals in the human population.