Experimental evolution of ligand-receptor relationships

描述（由申请人提供）：本研究的目标是开发一种实验进化系统，其中激素受体进化出新配体的亲和力。将使用人雌激素受体α（ER α）和两种主要的乳腺癌药物（ER α拮抗剂或反应调节剂）作为试验病例。该系统将允许使用自然选择的指数效率来评估决定雌激素受体及其配体之间相互作用的结构-功能关系-这是目前使用的定向诱变和文库筛选方法的重大进步，这些方法需要艰苦的生产和评估突变受体的新功能。该系统还将解决有关分子进化的动力学和机制基础的基本问题以及有关疾病进展的应用问题，因为它将在实验室中重现肿瘤细胞对药物产生耐药性的过程，并且随着进化时间的推移，正常细胞中的受体蛋白会进化出新的功能。该系统将由工程酵母菌株组成，其中生长速率取决于受雌激素反应元件控制的基因的表达，而这些基因的表达又取决于添加到培养基中的配体对重组人ER α的激活。当在实验室中培养时，具有更好地被配体激活的变体ER的酵母将通过突变产生;在存在生长限制剂量的新型配体的情况下，由于自然选择，这些变体的频率将增加。经过许多代，使用新配体作为高亲和力激动剂的ER将进化。不断发展的受体的基因序列将在频繁的时间间隔，其功能的特点，使用报告基因测定，其三维结构建模，并评价其演变的动力学。重复多次实验和对照重复，该系统将允许严格的测试有关ER结构-功能关系和受体-配体协同进化的动力学的假设。该系统最终可以扩展到研究几乎任何受体-配体相互作用的共同进化，包括任何激素，生长因子或神经递质及其受体之间的相互作用，以及宿主和病原体蛋白质之间的相互作用。

DESCRIPTION (provided by applicant): The goal of this research is to develop an experimental evolution system in which hormone receptors evolve affinity for new ligands. The human estrogen receptor alpha (ERalpha and two major breast cancer drugs that are ERalpha antagonists or response modulators will be used as a test case. This system will allow the structure-function relationships that determine interactions between estrogen receptors and their ligands to be evaluated using the exponential efficiency of natural selection -- a major advance over the currently used methods of directed mutagenesis and library screening, which require the painstaking production and evaluation of mutant receptors for new functions. This system will also address fundamental questions about the dynamics and mechanistic basis of molecular evolution and applied questions about disease progression, because it will recapitulate in the laboratory the processes by which tumor cells evolve resistance to drugs and, over evolutionary time, receptor proteins in normal cells evolve new functions. He system will consist of an engineered yeast strain in which growth rate depends upon the expression of genes controlled by estrogen response elements, and expression of these genes depends in turn on the activation of a recombinant human ERalpha by ligands added to the culture medium. When cultured in the laboratory, yeast with variant ERs that are better activated by the ligand will be generated by mutation; in the presence of a growth-limiting dose of a novel ligand, these variants will increase in frequency due to natural selection. Over many generations, ERs that use the novel ligand as a high-affinity agonist will evolve. The gene sequences of evolving receptors will be obtained at frequent intervals, their functions characterized using reporter gene assays, their three-dimensional structures modeled, and the dynamics of their evolution evaluated. Repeated over numerous experimental and control replicates, this system will allow the rigorous testing of hypotheses about ER structure-function relationships and the dynamics of receptor-ligand coevolution. This system can ultimately be extended to study the coevolution of virtually any receptor-ligand interaction, including those between any hormone, growth factor, or neurotransmitter and its receptor, as well as those between host and pathogen proteins.