Experimental evolution of ligand-receptor relationships

描述(申请人提供)：这项研究的目标是开发一种实验进化系统，在该系统中激素受体进化出对新配体的亲和力。人类雌激素受体α(ERpha)和两种主要的乳腺癌药物是ERpha拮抗剂或反应调节剂将作为测试案例。这一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--这比目前使用的定向突变和文库筛选方法是一个重大进步，后者需要艰苦地生产和评估新功能的突变受体。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关于疾病进展的应用问题，因为它将在实验室中概括肿瘤细胞进化耐药的过程，并随着进化时间的推移，正常细胞中的受体蛋白进化出新的功能。该系统将由一个工程酵母菌株组成，其生长速度取决于由雌激素反应元件控制的基因的表达，而这些基因的表达反过来又取决于添加到培养基中的配体对重组人ERα的激活。当在实验室培养时，具有更好地被配体激活的变异ER的酵母将通过突变产生；在存在限制生长剂量的新配体的情况下，这些变异将由于自然选择而增加频率。经过许多代，使用新的配体作为高亲和力激动剂的内质网将会进化。进化受体的基因序列将以频繁的间隔获得，它们的功能将通过报告基因分析来表征，它们的三维结构将被建模，它们的进化动力学将被评估。通过重复大量的实验和对照重复，这个系统将允许严格测试关于内质网结构-功能关系和受体-配体共同进化的动力学的假设。这个系统最终可以扩展到研究几乎任何受体-配体相互作用的协同进化，包括任何激素、生长因子或神经递质与其受体之间的相互作用，以及宿主和病原体蛋白之间的相互作用。

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.