CYTOCHROME P-450 POLYMORPHISM

本应用程序的一个主要目的是了解 IIC类细胞色素P450酶的多底物特异性编码 在他们的一级和三级结构中。大熊猫的遗传多样性 细胞色素P450单加氧酶提供对脂质的关键防御 易溶、有毒的外来化合物(外来生物)以及重要的作用 在内源性底物的代谢中，如类固醇。肝性最强 P450表现出独特的代谢能力，可以代谢几种不同的 外源生物。遗传学研究表明P450IIC5在肝脏中是独一无二的 P450，包括ITS中氨基酸序列高度相似的那些 孕酮21-羟基化的催化作用。然而，就像其他几个人一样 肝脏P450，它代谢致癌物质，如2-乙酰氨基荧烯和 苯并(A)芘。为了识别主要结构的片段 它们决定了这种多底物的特异性，我们将识别片段 P450IIC5的氨基酸序列，当它被替换到其他 IIC P450类，可产生有效的孕酮21-羟基酶。 将测试相互作用的结构以确定底物是否 其他酶的特性被授予IIC5。我们将首先 表达来自嵌合cDNA的杂交酶以定位这些片段，以及 然后使用寡核苷酸定向突变使单个 替换。这种方法将定义线性序列的区域， 测定底物的选择性。为了了解这些细分市场是如何 交互时，我们将获得有关拓扑组织的信息 通过定位单抗识别的表位来分类IIC酶 之前准备好了IIC P450类。氨基酸序列的差异 破坏结合的基因将被映射到嵌合酶中。我们期待着 鉴定远端并置形成的复杂表位 氨基酸序列在三维结构中的片段 酵素。表位定位研究和鉴定结果 底物选择性的决定因素将由计算机模拟在 基于同源酶P450 cam的三维结构。这 该模型应适用于其他微生物体P450。另一个主要目标是 自身免疫中P450酶如何成为自身抗原的鉴定 慢性活动性肝炎。我们之前已经证明了一种多态的， 人类P450，IID6，被与此相关的自身抗体识别 疾病。我们将自身抗体识别的表位(S)映射到P450 IID6，以确定与潜在外源抗原的相似性。 一种特定的IID6等位基因与先天性心脏病发生的潜在关联 在这种人类肝病中，P4501ID6的自身抗体也将是 调查过了。该基因的缺陷等位基因发生的频率很高。 如果建立了关联，它将更清楚地标识 IID6与自身免疫性疾病的关系及其特征 等位基因可以提供有关自身抗体如何产生的机械性信息 P450 IID6。这些研究还可以确定导致突变的等位基因 人类药物代谢能力的缺陷。

A major aim of the present application is to understand how the multi-substrate specificity of Class IIC cytochrome P450 enzymes is encoded in their primary and tertiary structures. The genetic diversity of the cytochrome P450 monooxygenases provides a critical defense against lipid soluble, toxic foreign compounds (xenobiotics) as well as an essential role in the metabolism of endogenous substrates such as steroids. Most hepatic P450s exhibit a distinct capacity to metabolize several different xenobiotics. Genetic studies indicate that P450IIC5 is unique among hepatic P450s, including those with highly similar amino acid sequences, in its catalysis of the 21-hydroxylation of progesterone. Yet, like several other hepatic P450s, it metabolizes carcinogens such as 2-acetylaminofluorene and benzo(a)pyrene. In order to identify segments of the primary structures which determine this multi-substrate specificity, we will identify segments of the amino acid sequence of P450IIC5, which when substituted into other class IIC P450s, generates efficient progesterone 21-hydroxylases. Reciprocal constructs will be tested to identify whether the substrate specificities of the other enzymes are conferred to IIC5. We will first express hybrid enzymes from chimeric cDNAs to localize these segments, and then employ oligonucleotide-directed mutagenesis to make single substitutions. This approach will define regions of linear sequence that determine substrate selectivity. In order to understand how these segments interact, we will obtain information on the topological organization of class IIC enzymes by mapping epitopes recognized by monoclonal antibodies prepared previously to class IIC P450s. Differences of amino acid sequence which disrupt binding will be mapped in chimeric enzymes. We anticipate identifying complex epitopes formed from the juxtaposition of distal segments of the amino acid sequence in the 3-dimensional structure of the enzyme. The results of epitope mapping studies and of identification of determinants of substrate selectivity will be modeled by computer on the basis of the 3-dimensional structure of a homologous enzyme, P450 cam. This model should be applicable to other microsomal P450s. Another major aim is the identification of how P450 enzymes become autoantigens in autoimmune chronic active hepatitis. We have previously shown that a polymorphic, human P450, IID6, is recognized by autoantibodies associated with this disease. We will map the epitope(s) recognized by autoantibodies to P450 IID6 in order to identify similarities with potential exogenous antigens. The potential association of a specific IID6 allele with the generation of the autoantibodies to P4501ID6 in this human liver disease will also be investigated. Defective alleles of this gene occur with a high frequency. If an association is established, it would more clearly identify the relation of IID6 to the autoimmune disease, and the characterization of the allele could provide mechanistic information on how autoantibodies arise to P450 IID6. These studies could also identify mutant alleles that contribute to deficiencies of human drug-metabolizing capacity.