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: 6731049
• 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 2006-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6731049
• 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密切结合。该膜锚对活动很重要，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位置，调节前体的通量向下途径，通向矿物皮质激素，糖皮质激素和性类固醇。 P450C17的17、20-1yase活性被细胞色素B5刺激10倍（B5），但该刺激的机理尚不清楚。 B5的作用是肾上腺结构和性腺发育生理学的一部分，而这种B5-P450C17相互作用隐约可作为性类固醇合成的药理抑制的理想靶标。异常高的17、20-1yase活性是雄激素过多状态的基础，包括多囊卵巢综合征，影响5-10％的生殖妇女和21-羟化酶缺乏症，这会影响1/14,000个活产。详细了解这些蛋白质的精细结构以及对B5-P450C17相互作用负责的残基的识别将提高我们对调节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类固醇抑制剂，以治疗性类固醇生物合成，以治疗和乳腺癌和乳腺癌和乳腺癌和乳腺癌。我们已经开发了一种融合蛋白系统，该系统将人P450C17与细胞色素B5联系起来，以将P450C17的疏水表面掩埋在蛋白质 - 蛋白质相互作用中，而不是在膜上。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。我们将构建一系列构造，其中删除了膜跨度区域的一部分，目的是保持至少部分活动，同时提高溶解度和优化表达。我们将确定融合蛋白是否在HEK-293细胞和酵母中活跃。我们的方法允许使用纯化标签将亚克隆进入允许在E线圈或SF9细胞中高级表达的向量进行纯化。作为第二个目标，我们将使用计算机建模和站点定向的诱变来识别刺激P450C17的17、20-裂解酶活性的B5上的残基。因此，在此试点和可行性研究中所做的工作将确定适合结晶和结构确定研究的融合蛋白，并将确定B5的哪个区域刺激了P450C17的高17、20-溶解酶活性。另外，这种方法通常适用于膜结合蛋白的结构研究。

项目成果

Ontogeny and pathway of formation of 5alpha-androstane-3alpha,17beta-diol in the testes of the immature brushtail possum Trichosurus vulpecula.

未成熟刷尾负鼠 Trichosurus vulpecula 睾丸中 5α-雄甾烷-3α,17β-二醇的个体发育和形成途径。

• DOI: 10.1071/rd05034
• 发表时间: 2005
• 期刊: Reproduction, fertility, and development
• 作者: Wilson,JeanD;Shaw,Geoffrey;Renfree,MarilynB;Auchus,RichardJ;Leihy,MichaelW;Eckery,DouglasC
• 通讯作者: Eckery,DouglasC

Role of the alternate pathway of dihydrotestosterone formation in virilization of the Wolffian ducts of the tammar wallaby, Macropus eugenii.

双氢睾酮形成的替代途径在塔马尔小袋鼠 (Macropus eugenii) 沃尔夫管男性化中的作用。

• DOI: 10.1210/en.2005-1251
• 发表时间: 2006
• 期刊: Endocrinology.
• 作者: Shaw,Geoffrey;Fenelon,Jane;Sichlau,Michelle;Auchus,RichardJ;Wilson,JeanD;Renfree,MarilynB
• 通讯作者: Renfree,MarilynB