Fusion Proteins As Probes of P450 Structure and Function

描述（申请人提供）：许多合成类固醇激素的酶与膜牢固结合。这种膜锚对活性很重要，但使生物化学和生物物理研究复杂化。P450 c17（17-羟化酶/17，20- 1-裂合酶）在类固醇生成中占据关键位置，调节前体物质向盐皮质激素、糖皮质激素和性类固醇途径的流动。细胞色素b5（b5）可使P450 c17的17，20- 1裂合酶活性增加10倍，但其作用机制尚不清楚。b5的作用是肾上腺初显和性腺发育的生理学的一部分，并且这种b5-P450 c17相互作用隐约可见作为性类固醇合成的药理学抑制的理想靶点。异常高的17，20-羟化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活产婴儿的21-羟化酶缺乏症。这些蛋白质的精细结构的详细知识和负责的b5-P450 c17相互作用的残基的鉴定将促进我们的复杂的机制，调节17，20- 1 yase活性的理解，并最终将促进合理设计的高度特异性，非甾体类抑制剂的性类固醇生物合成，用于治疗雄激素过多和乳腺癌和前列腺癌。我们已经开发了一个系统的融合蛋白，连接人P450 c17与细胞色素b5的蛋白质-蛋白质相互作用，而不是在膜中的P450 c17的疏水表面掩埋。当在酵母或HEK-293细胞中表达时，这些融合蛋白的几个初始版本是有活性的。我们将构建一系列缺失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E coil或Sf 9细胞中高水平表达的载体中进行纯化。作为第二个目标，我们将使用计算机建模和定点诱变来鉴定刺激P450 c17的17，20-裂解酶活性的b5上的残基。因此，在该中试和可行性研究中进行的工作将鉴定适合于结晶和结构测定研究的融合蛋白，并将确定b5的哪个区域刺激P450 c17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质的结构研究。

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.