A Molecular Tag for Drug-Regulated Synthesis of Specific Proteins

用于药物调控合成特定蛋白质的分子标签

• 批准号: 8733708
• 负责人: Michael Z. Lin
• 金额: $ 31.75万
• 依托单位: STANFORD UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-09-05 至 2016-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8733708
• 关键词: Address; Alleles; Animals; Antibodies; Binding; Biological; Biological Response Modifier Therapy; Biology; Biomedical Research; Cell Therapy; Cells; Chimeric Proteins; Data; Degradation Pathway; Development; Disease; Dose; Drug usage; Elements; Endoplasmic Reticulum; Epilepsy; Event; Excision; Exhibits; Free Energy; Generic Drugs; Genes; Genetic; Genetic Transcription; Golgi Apparatus; Human; Investigation; Kinetics; Lead; Life; Mediating; Memory; Methods; Molecular; Mus; Nervous system structure; Neurons; Neurotransmitters; Organism; Pathology; Pathway interactions; Peptide Hydrolases; Pharmaceutical Preparations; Physiological; Physiology; Process; Production; Promoter Regions; Protease Inhibitor; Protein Biosynthesis; Protein Engineering; Protein Secretion; Protein Synthesis Induction; Proteins; RNA; RNA Folding; Regulation; Research; Research Personnel; Ribosomes; Role; Seizures; Signal Transduction; Somatomedins; Speed; Stimulus; Structure; Synapses; System; Tertiary Protein Structure; Testing; Therapeutic; Time; Transcriptional Regulation; Transgenic Mice; Translational Research; Translations; Work; Yeasts; base; extracellular; gene therapy; genome wide association study; high throughput screening; human disease; in vivo; interest; long term memory; meetings; novel; promoter; protein degradation; protein expression; protein folding; protein function; rapid technique; research study; response; small molecule; thermostability; tool; transcription factor; treatment strategy

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.

描述（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、快速和可推广的方法，可以用药物阻止特定蛋白质的这一基本过程，这将是生物医学和转化研究中非常有用的工具。通过允许感兴趣的蛋白质的条件表达，这将有利于蛋白质功能的基础研究。它将提供一种在体内调节基因和细胞疗法产生蛋白质的方法。控制特定蛋白质合成的方法也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，这是不可能的研究路线。在拟议的工作中，我们将开发一种新的可推广的方法，用于使用小分子药物关闭遗传标记蛋白质的合成，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强大的降解信号，经历自催化去除。默认情况下，蛋白质将从降解信号中释放出来并正常发挥功能。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上的降解信号，导致其快速降解。我们将进行实验，以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生产。我们将进一步使用这种方法来测试一个长期存在的假设，即特定的突触调节蛋白的合成是需要在转基因小鼠的记忆巩固。如果成功，这些实验将开创性地建立一种全新的方法来控制蛋白质的生产，这种方法快速，稳健，简单，可推广。拟议的研究将在生物医学研究中产生广泛的好处，通过提供一种通用的方法来调节蛋白质的表达，允许更快的动力学比转录控制方法，但像转录调控，产生功能蛋白质没有一个永久的融合标签。因此，这项工作可能有助于对蛋白质功能的研究，包括全基因组筛选，并提供了一种在体内严格控制基因和细胞疗法的方法。这项工作还将产生第一个实验工具，能够解决正常生理和疾病中特定蛋白质合成事件的重要性。因此，所提出的实验有可能在我们控制蛋白质功能的能力方面取得重大进展，以阐明生物机制和控制生物疗法。

项目成果

StaPLs: versatile genetically encoded modules for engineering drug-inducible proteins.

• DOI: 10.1038/s41592-018-0041-z
• 发表时间: 2018-07
• 期刊: Nature methods
• 影响因子: 48
• 作者: Jacobs CL;Badiee RK;Lin MZ
• 通讯作者: Lin MZ