Function and Regulation of Human Cytochrome P4502S1

性状（由申请方提供）：CYP 2S 1是最近鉴定的人细胞色素P450，在上皮组织中广泛表达。我们认为，CYP 2S 1在环境原致癌物的代谢活化以及药物和内源性化合物的代谢中起着重要作用。该提案将解决这一假设，并表征酶的调节。有三个具体目标：（i）我们已经在细菌中表达了人CYP 2S 1，并证明它代谢了几种对上皮组织有毒和/或致癌的化合物。我们还将在哺乳动物细胞中过度表达这种酶。使用这些表达系统，我们将筛选额外的底物，以及通过CYP 2S 1活化为致突变（因此可能致癌）衍生物的前致癌物。将测定代表性化合物的Km和Vmax值，并鉴别形成的代谢产物。CYP 2S 1的程度有助于对总代谢的特定底物在人体上皮组织将使用抑制抗体的酶，（ii）我们已经表明，CYP 2S 1是诱导二恶英，致癌的多环芳烃（PAH），和缺氧。我们将研究人类CYP 2S 1基因5'侧翼区潜在的异生素反应元件（XREs）或潜在的抗氧化剂反应元件（ARE）是否介导二恶英和/或多环芳烃的诱导，并解决以下假设：由于上述XREs的特定核苷酸序列，该基因在某些细胞中对多环芳烃的反应优于对二恶英的反应。我们还将分析该基因的缺氧诱导机制。（iii）我们将产生Cyp 2s 1的敲除小鼠，然后产生含有人CYP 2S 1基因（包括其侧翼调控区）的该小鼠的衍生物。这种“CYP 2S 1人源化”小鼠将用于研究人CYP 2S 1底物的代谢、该代谢的生物学后果以及外源性物质和缺氧对人CYP 2S 1基因的调节，从而补充和扩展特定目标1和2。我们的研究可能证明CYP 2S 1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群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.