AROMATASE STRUCTURE FUNCTION RELATIONSHIPS AND CANCER

长期以来，雌激素与乳腺癌的发生和/或进展有关。 某些类型的人类癌症，包括子宫内膜癌和 乳房 在本文中，雌激素拮抗剂以及 雌激素生物合成抑制剂在人乳腺治疗中的应用 癌症是公认的。 然而，尽管雌激素受体拮抗剂 例如他莫昔芬在该上下文中是有效，需要 开发更有效和特异性的生物合成抑制剂 雌激素类。 由雄激素形成雌激素是由一种 已知为芳香酶的酶复合物，其包含特定形式的 细胞色素P-450（P-450 AROM）和黄素蛋白，NADPH-细胞色素P-450 还原酶。 我们以前的研究表明，单一形式的P- 450 AROM存在于人体组织中，其表达是由 被一个基因所控制。 本提案的目的是了解 功能与一级序列以及最终与三级序列的关系 人P-450 AROM空间结构 这是特别 重要性有几个原因。 首先，这种知识将 极大地促进了设计更有效和特异性的 雌激素生物合成用于乳腺癌患者的治疗 癌 由于人体内只有一种形式的P-450 AROM，因此任何给定的 芳香酶抑制剂在抑制酶方面应该是同样有效的 而不考虑表达的发放位点。 第二，有必要 理解复杂的多步反应序列的机理 由这种酶催化。 这一目标将在第一个 例如通过全长cDNA插入片段定点诱变，所述插入片段编码 P-450 AROM，并最终由三维分辨率 用X射线晶体学方法研究了P-450 AROM的结构。 通过 定点突变，我们将改变 已鉴定为对P-450有意义蛋白质 通常是催化反应。 这些选择的氨基酸是 是P-450 AROM所独有的，并已通过计算机模拟识别 底物结合区的结构在反应中可能是重要的 芳香化酶作用机制 我们还将寻求在以下方面表达P-450 AROM 单细胞生物，即E. coli和革兰氏阳性菌S.啤酒，如在 杆状病毒感染的昆虫细胞，以产生大量的 酶作为纯化的起始点，并最终 晶化 定点诱变将被用作 在单细胞生物体中表达可溶形式的酶的尝试 如大肠大肠杆菌和酵母菌以便于其纯化 晶化 这将通过密码子的顺序改变来实现 对于氨基末端的甲硫氨酸，为了删除膜- 跨越区域。 一旦获得了P-450 AROM的最佳产率，则 该材料将用于生产酶的晶体， 适合于X射线光谱分析。

Estrogens have long been implicated in the development and/or progression of certain forms of human cancer, including those of the endometrium and breast. In this context, the usefulness of estrogen antagonists as well as inhibitors of estrogen biosynthesis in the management of human breast cancer is well recognized. However, although estrogen receptor antagonists such as tamoxifin are efficacious in this context, there is a need to develop more effective and specific inhibitors of the biosynthesis of estrogens. The formation of estrogens from androgens is catalyzed by an enzyme complex known as aromatase which comprises a specific form of cytochrome P-450 (P-450AROM) and a flavoprotein, NADPH-cytochrome P-450 reductase. Our previous studies are indicative that a single form of P- 450AROM is present within human tissues and that its expression is encoded by a single gene. The objective of the present proposal is to understand the relationship of function to primary sequence and ultimately to three- dimensional structure of human P-450AROM. This is of particular significance for several reasons. In the first place, such knowledge will greatly facilitate the design of more effective and specific inhibitors of estrogen biosynthesis for use in the management of patients with breast cancer. Since there is only one form of P-450AROM in humans, any given aromatase inhibitor should be equally efficacious in inhibiting the enzyme regardless of the issue site of expression. Secondly, there is the need to understand the mechanism of the complex multi-step reaction sequence catalyzed by this enzyme. This goal will be addressed in the first instance by site-directed mutagenesis of a full-length cDNA insert encoding P-450AROM, and ultimately by the resolution of the three-dimensional structure of P-450AROM by means of X-ray crystallography. By means of site-directed mutagenesis, we will alter selected amino acids in domains of the protein which have been identified as being significant to P-450 catalyzed reactions in general. These selected amino acids are one which are unique to P-450AROM and which have been identified by computer-modeling of the substrate binding region as potentially important in the reaction mechanism of aromatase. We will also seek to express P-450AROM in unicellular organisms, namely E. coli and S. cerevisiae, as will as in baculovirus-infected insect cells, in order to produce large quantities of the enzyme as a starting point for purification and ultimately crystallization. Site-directed mutagenesis will be used as a means to attempt to express a soluble form of the enzyme in unicellular organisms such as E. coli and yeast in order to facilitate its purification and crystallization. This will be achieved by sequential alteration of condons for methionines at the amino-terminus, in order to delete the membrane- spanning region. Once optimum yields of P-450AROM have been obtained, then this material will be used to produce crystals of the enzyme which are suitable for analysis by X-ray spectrometry.