A Molecular Tag for Drug-Regulated Synthesis of Specific Proteins

描述(申请人提供)：核糖体合成蛋白质是生物表达遗传信息的基本步骤。一种简单、可靠、快速和可推广的方法来停止药物对特定蛋白质的这一基本过程，将是生物医学和翻译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，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产生的蛋白质提供一种手段。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有助于特定的生物反应或疾病，这是一项不可能进行的研究。在拟议的工作中，我们将开发一种新的通用方法来停止使用小分子药物合成基因标记的蛋白质，并使用这种方法来解决关于新的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，作为与经历自动催化去除的有效降解信号的融合。默认情况下，这些蛋白质将从降解信号中释放出来，并正常发挥功能。应用一种特定的无毒的细胞渗透性药物将保留随后合成的蛋白质上的降解信号，导致它们快速降解。我们将进行实验，以验证这种方法在原代细胞和动物中的实用性，确定这种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质的生产。我们将进一步使用这种方法来检验一个长期存在的假设，即在转基因小鼠中，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全新的控制蛋白质生产的快速、稳健、简单和可推广的方法方面具有开创性。这项拟议的研究将在生物医学研究中获得广泛的好处，因为它提供了一种调控蛋白质表达的通用方法，这种方法允许比转录调控方法更快的动力学，但与转录调控一样，产生的功能蛋白质没有永久的融合标签。因此，这项工作可能会促进对蛋白质功能的总体研究，包括全基因组筛选，并提供一种在体内严格控制基因和细胞治疗的手段。这项工作还将产生第一批能够解决特定蛋白质合成事件在正常生理和疾病中的重要性的实验工具。因此，拟议的实验有可能在我们控制蛋白质功能、阐明生物机制和控制生物治疗方面取得重大进展。

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.