A Molecular Tag for Drug-Regulated Synthesis of Specific Proteins

描述（由申请人提供）：核糖体合成蛋白质是生物体表达遗传信息的基本步骤。一种简单、可靠、快速、可推广的方法来阻止药物对特定蛋白质的这一基本过程，将是生物医学和转化研究中非常有用的工具。它允许有条件地表达感兴趣的蛋白质，这将有利于蛋白质功能的基础研究。它将为调节体内基因和细胞疗法的蛋白质生产提供一种手段。一种控制特定蛋白质合成的方法还将允许评估特定蛋白质的可诱导合成如何促进特定生物反应或疾病，这是一种不可能的调查方法。在这项工作中，我们将开发一种新的通用方法，使用小分子药物来关闭遗传标记蛋白的合成，并使用这种方法来解决新蛋白质合成在神经系统适应中的作用的突出问题。我们将表达感兴趣的蛋白质作为融合到一个有效的降解信号，经历自催化去除。默认情况下，蛋白质会从降解信号中释放出来，正常发挥功能。应用特定的无毒细胞渗透性药物将保留随后合成的蛋白质的降解信号，导致其快速降解。我们将进行实验来验证该方法在原代细胞和动物中的实用性，以确定该方法所依赖的蛋白质降解途径，并将该策略扩展到控制分泌蛋白的产生。我们将进一步使用这种方法来验证一个长期存在的假设，即转基因小鼠的记忆巩固需要合成特定的突触调节蛋白。如果成功，这些实验将在建立一种快速、稳健、简单和可推广的控制蛋白质生产的全新方法方面具有开创性。拟议的研究将在生物医学研究中有广泛的好处，因为它提供了一种通用的方法来调节蛋白质表达，这种方法比转录控制方法允许更快的动力学，但是，像转录调节一样，产生没有永久融合标签的功能性蛋白质。因此，这项工作可能促进蛋白质功能的研究，包括全基因组筛选，并提供一种严格控制体内基因和细胞治疗的手段。这项工作还将产生第一个能够解决正常生理和疾病中特定蛋白质合成事件重要性的实验工具。因此，拟议的实验有可能在我们控制蛋白质功能以阐明生物机制和控制生物疗法的能力方面取得重大进展。

DESCRIPTION (provided by applicant): The synthesis of proteins by ribosomes is a fundamental step in the expression of genetic information by living organisms. A simple, reliable, rapid, and generalizable method for stopping this fundamental process for a specific protein with a drug would be an enormously useful tool in biomedical and translational research. It would benefit basic studies of protein function by allowing conditional expression of proteins of interest. It would provide a means for regulating protein production from gene and cellular therapies in vivo. A method for controlling synthesis of specific proteins would also allow assessment of how inducible synthesis of specific proteins contributes to particular biological responses or to disease, a line of investigation which has not been possible. In the proposed work, we will develop a novel generalizable method for shutting off synthesis of genetically tagged proteins using a small-molecule drug, and use this method to address outstanding questions on the role of new protein synthesis in nervous system adaptation. We will express proteins of interest as fusions to a potent degradation signal that undergoes autocatalytic removal. By default, the proteins will be released from the degradation signal and function normally. Application of a specific non-toxic cell-permeable drug will preserve the degradation signal on subsequently synthesized proteins, leading to their rapid degradation. We will perform experiments to validate the utility of this method in primary cells and animals, to determine the protein degradation pathways on which this method relies, and to extend the strategy to controlling production of secreted proteins. We will further use this method to test a long-standing hypothesis that synthesis of specific synapse-regulating proteins is required for memory consolidation in transgenic mice. If successful, these experiments will be groundbreaking in establishing a completely new method for controlling protein production that is rapid, robust, simple, and generalizable. The proposed research will have broad benefits in biomedical research by providing a generic method for regulating protein expression that allows more rapid kinetics than transcriptional control methods but, like transcriptional regulation, produces functional proteins without a permanent fusion tag. This work may thus facilitate studies on protein functions in general, including genome-wide screens, and provide a means for tightly controlling gene and cellular therapies in vivo. This work will also produce the first experimental tools capable of addressing the importance of specific protein synthesis events in normal physiology and disease. Thus the proposed experiments have the potential to produce a major advance in our ability to control protein function for elucidating biological mechanisms and for controlling biological therapies.