Optogenetics for all: A general method for optical control of protein activity

所有人的光遗传学：蛋白质活性光学控制的通用方法

• 批准号: 8564060
• 负责人: Michael Z. Lin
• 金额: $ 80.25万
• 依托单位: STANFORD UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-09-30 至 2018-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8564060
• 关键词: Apoptosis; Biological; Biology; Cell Therapy; Cell physiology; Cells; Dissociation; Dreams; Genes; Green Fluorescent Proteins; Implant; Light; Location; Mediating; Methods; Nervous system structure; Optical Methods; Optics; Physiology; Proteins; Regulation; Resolution; Signal Transduction; Translating; Variant; in vivo; interest; migration; optogenetics; protein protein interaction; screening

DESCRIPTION (provided by applicant): Optical control of genetically defined protein activities, i.e. optogenetic regulation of proteins, has long been a dream in biology. If we could develop a generalizable method to optically control activities of proteins of interest, it could profoundly transform biological experimentation and impart new capabilities to gene- and cell-based therapies. For instance, the cellular functions that proteins coordinate, such as survival, apoptosis, differentiation, migration or connectivity (in the nervous system) could be controlled in vivo to study organismal physiology with micron-level resolution. Therapeutically implanted cells could be similarly controlled by light to precisely focus treatment at desired anatomical locations. There are countless other possible applications. Given these advantages of optical protein regulation, considerable efforts have been expended to adapt known light-responsive signaling domains to regulate mammalian proteins. However, existing strategies require extensive screening to create light-responsive proteins, or rely on protein relocalization to indirectly regulate activity. As a result, these methods have been used to control only a few proteins. Thus there exists a need for a method to create light-regulated proteins of interest that is generalizable. We have recently discovered a new class of light-mediated protein-protein interaction, and translated this discovery into a generalizable method for controlling protein activities with light. We hypothesized that fluorescent proteins (FPs) could undergo light-dependent conformational changes that drive changes in oligomerization state. Indeed, we found that tetrameric and dimeric variants of the reversibly photoswitching green FP Dronpa undergo dissociation as they are switched from bright to dark states by cyan light. We then discovered that fusing Dronpa domains at both ends of an enzymatic domain of interest cages it in the dark but allows uncaging upon illumina

描述（由申请人提供）：遗传上确定的蛋白质活性的光学控制，即蛋白质的光遗传学调节，长期以来一直是生物学中的梦想。如果我们能够开发出一种可推广的方法来光学控制感兴趣的蛋白质的活性，它可能会深刻地改变生物实验，并为基于基因和细胞的疗法带来新的能力。例如，蛋白质协调的细胞功能，如生存，凋亡，分化，迁移或连接（在神经系统中）可以在体内控制，以研究具有微米级分辨率的生物生理学。治疗性植入的细胞可以类似地由光控制，以将治疗精确地聚焦在期望的解剖位置。还有无数其他可能的应用。 鉴于光学蛋白质调节的这些优点，已经花费了相当大的努力来调整已知的光响应信号传导结构域以调节哺乳动物蛋白质。然而，现有的策略需要广泛的筛选来产生光响应蛋白，或者依赖于蛋白质重新定位来间接调节活性。因此，这些方法仅用于控制少数蛋白质。因此，需要一种产生感兴趣的光调节蛋白的方法， 是可推广的。 我们最近发现了一类新的光介导的蛋白质-蛋白质相互作用，并将这一发现转化为用光控制蛋白质活性的可推广的方法。我们假设，荧光蛋白（FP）可以经历光依赖性的构象变化，驱动寡聚化状态的变化。事实上，我们发现可逆光开关绿色FP Dronpa的四聚体和二聚体变体在通过青色光从亮态切换到暗态时发生解离。然后，我们发现，在感兴趣的酶结构域的两端融合Dronpa结构域将其笼在黑暗中，但允许在光照下解开，