AROMATASE STRUCTURE FUNCTION RELATIONSHIPS AND CANCER

芳香酶结构功能关系与癌症

• 批准号: 3195805
• 负责人: EVAN R SIMPSON
• 金额: $ 19.87万
• 依托单位: UT SOUTHWESTERN MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 1989
• 资助国家: 美国
• 起止时间: 1989-12-01 至 1994-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3195805
• 关键词: Baculoviridae; Escherichia coli; Saccharomyces cerevisiae; X ray crystallography; aromatase; complementary DNA; cytochrome P450; cytochrome oxidase; enzyme complex; enzyme structure; enzyme substrate; estrogen inhibitor; oxidoreductase inhibitor; protein engineering; protein purification; protein sequence; protein structure function; site directed mutagenesis; steroid hormone biosynthesis

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.

雌激素长期以来一直与发育和/或进展有关 某些形式的人类癌症，包括子宫内膜癌和 胸部。 在这种情况下，雌激素拮抗剂以及 雌激素生物合成抑制剂在人类乳腺治疗中的应用 癌症已被广泛认识。 然而，尽管雌激素受体拮抗剂 诸如他莫昔芬之类的药物在这种情况下是有效的，因此有必要 开发更有效和特异性的生物合成抑制剂 雌激素。 雄激素转化为雌激素的过程是由 称为芳香酶的酶复合物，包含特定形式的 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，单一形式的 P- 450AROM 存在于人体组织内，其表达被编码 由单个基因。 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 人类 P-450AROM 的空间结构。 这是有特殊意义的 其重要性有几个原因。 首先，这些知识将 极大地促进了更有效和特异性抑制剂的设计 雌激素生物合成在乳腺癌患者治疗中的应用 癌症。 由于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 无论表达的问题部位如何。 其次，需要 了解复杂的多步反应序列的机理 在这种酶的催化下。 这个目标将在第一阶段实现 例如通过全长 cDNA 插入编码的定点诱变 P-450AROM，最终由三维分辨率 通过 X 射线晶体学分析 P-450AROM 的结构。 借助于 定点诱变，我们将改变域中选定的氨基酸 已被确定对 P-450 具有重要意义的蛋白质 一般的催化反应。 这些选定的氨基酸是 是 P-450AROM 独有的，并且已通过计算机建模识别 底物结合区域在反应中可能很重要 芳香化酶的作用机制。 我们还将寻求表达 P-450AROM 单细胞生物，即大肠杆菌和酿酒酵母，如下所示 杆状病毒感染的昆虫细胞，以产生大量 酶作为纯化的起点，并最终 结晶。 定点诱变将被用作一种手段 尝试在单细胞生物中表达可溶形式的酶 例如大肠杆菌和酵母，以促进其纯化和 结晶。 这将通过顺序改变密码子来实现 对于氨基末端的蛋氨酸，为了删除膜- 跨越地区。 一旦获得 P-450AROM 的最佳产量，那么 该材料将用于生产酶晶体 适用于 X 射线光谱分析。