Fusion Proteins As Probes of P450 Structure and Function

融合蛋白作为 P450 结构和功能的探针

• 批准号: 6611899
• 负责人: RICHARD J. AUCHUS
• 金额: $ 15.6万
• 依托单位: UT SOUTHWESTERN MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-04-15 至 2005-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6611899
• 关键词: chimeric proteins; crystallization; cytochrome P450; enzyme mechanism; intermolecular interaction; molecular cloning; peptide chemical synthesis; protein purification; protein sequence; protein structure function; steroid 17alpha monooxygenase; steroid biosynthesis

DESCRIPTION (provided by applicant): Many enzymes that synthesize steroid hormones are strongly bound to membranes. This membrane anchor is important for activity but complicates biochemical and biophysical studies. P450c17 (17- hydroxylase/17, 20-1yase) occupies a critical position in steroidogenesis, regulating the flux of precursors down pathways to mineralocorticoids, glucocorticoids, and sex steroids. The 17, 20-1yase activity of P450c17 is stimulated 10-fold by cytochrome b5 (b5), but the mechanism of this stimulation is not known. The action of b5 is part of the physiology of adrenarche and gonadal development, and this b5-P450c17 interaction looms as an ideal target for pharmacological inhibition of sex steroid synthesis. Abnormally high 17, 20-1yase activity underlies common androgen excess states including polycystic ovary syndrome, which affects 5-10% of reproductive-aged women, and 21-hydroxylase deficiency, which affects 1/14,000 live births. Detailed knowledge of the fine structure of these proteins and identification of the residues responsible for the b5-P450c17 interaction would advance our understanding of the complex mechanisms that regulate 17, 20-1yase activity and will ultimately facilitate rational design of highly specific, non-steroidal inhibitors of sex steroid biosynthesis, for treating androgen excess and breast and prostate cancer. We have developed a system of fusion proteins that link human P450c17 with cytochrome b5 to bury the hydrophobic surface of P450c17 in a protein-protein interaction rather than in the membrane. Several initial versions of these fusion proteins are active when expressed in yeast or HEK-293 cells. We will build a series of constructs with portions of the membrane-spanning regions deleted, with the goal of preserving at least partial activity while improving solubility and optimizing expression. We will determine if the fusion proteins are active in HEK-293 cells and yeast. Our approach allows subcloning, using purification tags, into vectors that permit high-level expression in E coil or Sf9 cells for purification. As a second goal, we will use computer modeling and site-directed mutagenesis to identify the residues on b5 that stimulate the 17, 20- lyase activity of P450c17. The work performed in this pilot and feasibility study will thus identify fusion proteins that are suitable for crystallization and structure determination studies and will ascertain which region(s) of b5 stimulate high 17, 20-lyase activity of P450c17. In addition, this approach might be generally applicable to structural studies of membrane-bound proteins.

描述（由申请人提供）：许多合成类固醇激素的酶与膜牢固结合。这种膜锚对于活动很重要，但使生化和生物物理研究变得复杂。 P450c17（17-羟化酶/17, 20-1yase）在类固醇生成中占据关键地位，调节前体流向盐皮质激素、糖皮质激素和性类固醇的路径。 P450c17 的 17, 20-1yase 活性被细胞色素 b5 (b5) 刺激 10 倍，但这种刺激的机制尚不清楚。 b5 的作用是肾上腺初现和性腺发育生理学的一部分，这种 b5-P450c17 相互作用有望成为药理学抑制性类固醇合成的理想靶标。异常高的 17、20-1y 酶活性是常见雄激素过多状态的基础，包括影响 5-10% 育龄妇女的多囊卵巢综合征和影响 1/14,000 活产的 21-羟化酶缺乏症。对这些蛋白质精细结构的详细了解以及对 b5-P450c17 相互作用的残基的鉴定将促进我们对调节 17, 20-1yase 活性的复杂机制的理解，并最终促进高度特异性的非甾体药物的合理设计。性类固醇生物合成抑制剂，用于治疗雄激素过多以及乳腺癌和前列腺癌。我们开发了一种融合蛋白系统，将人 P450c17 与细胞色素 b5 连接起来，将 P450c17 的疏水表面埋藏在蛋白质-蛋白质相互作用中，而不是埋藏在膜中。这些融合蛋白的几个初始版本在酵母或 HEK-293 细胞中表达时具有活性。我们将构建一系列删除部分跨膜区域的构建体，目的是在提高溶解度和优化表达的同时保留至少部分活性。我们将确定融合蛋白在 HEK-293 细胞和酵母中是否有活性。我们的方法允许使用纯化标签亚克隆到允许在大肠杆菌或Sf9细胞中高水平表达以进行纯化的载体中。作为第二个目标，我们将使用计算机建模和定点诱变来识别 b5 上刺激 P450c17 17, 20-裂解酶活性的残基。因此，在该试点和可行性研究中进行的工作将鉴定适合结晶和结构测定研究的融合蛋白，并将确定 b5 的哪些区域刺激 P450c17 的高 17、20-裂解酶活性。此外，这种方法可能普遍适用于膜结合蛋白的结构研究。