AROMATASE STRUCTURE FUNCTION RELATIONSHIPS AND CANCER

长期以来，雌激素一直被认为与肿瘤的发展和/或进展有关 某些形式的人类癌症，包括子宫内膜癌和 胸部。在这方面，雌激素拮抗剂以及 雌激素生物合成抑制剂在人类乳房管理中的应用 癌症已经广为人知。然而，尽管雌激素受体拮抗剂 如他莫昔芬在这种情况下是有效的，有必要 开发更有效和更特异的生物合成抑制剂 雌激素。由雄激素形成雌激素是由一种 一种称为芳香酶的酶复合体，由一种特定形式的 细胞色素P-450(P-450AROM)和黄素蛋白NADPH-细胞色素P-450 还原酶。我们之前的研究表明，一种单一形式的P- 450AROM存在于人体组织中，其表达是编码的 由单个基因决定的。本提案的目标是了解 函数与初级序列的关系，最终与三个序列的关系- 人P-450AROM的空间结构。这是特别的 出于几个原因，它具有重要意义。首先，这样的知识将 极大地促进了设计更有效和更特异的抑制剂 雌激素生物合成在乳房治疗中的应用 癌症。由于在人类中只有一种形式的P-450AROM，任何给定的 芳香酶抑制剂在抑制该酶方面应该同样有效。 无论发文地点如何表达。其次，有必要 理解复杂的多步骤反应序列的机理 由这种酶催化的。这一目标将在第一个月中得到解决 编码全长cDNA插入片段的逐个定点突变 P-450AROM，并最终通过分辨率的三维 用X射线结晶学研究P-450AROM的结构通过以下方式 定点突变，我们将改变结构域中的选定氨基酸 已被确定对P-450有重要意义的蛋白质 总体而言是催化反应。这些选定的氨基酸是一种 它们是P-450AROM独有的，并已通过计算机建模进行了识别 底物结合区在反应中可能起重要作用 芳香酶的作用机理。我们还将寻求表达P-450AROM 单细胞生物体，即大肠杆菌和酿酒酵母，如 杆状病毒感染的昆虫细胞，以产生大量的 该酶作为纯化的起点，并最终 结晶。定点突变将被用作一种手段 尝试在单细胞生物体中表达一种可溶形式的酶 如大肠杆菌和酵母菌，以促进其纯化和 结晶。这将通过密码子的顺序改变来实现 对于氨基末端的蛋氨酸，为了删除膜- 跨越区域。一旦获得了P-450AROM的最佳产量，则 这种材料将被用来生产酶的晶体，这些晶体 适用于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.