Light-activated proteolysis as a tool to analyze intracellular protein function

描述（由申请人提供）：后基因组时代的下一个重大挑战之一是蛋白质组在空间和时间上的功能分析，这对理解正常和病理细胞行为至关重要。在复杂的细胞内过程中直接分析蛋白质功能需要一种方法，可以在活细胞的选择性区域实时、快速和特异性地灭活感兴趣的蛋白质。这种方法是不存在的。目前研究细胞内蛋白功能的方法有严重的局限性，要么是非特异性的，要么缺乏足够的空间和时间分辨率。例如，由于RNA干扰（RNAi）依赖于细胞内缓慢的蛋白质周转，因此它有助于检测长期表型，但不允许直接，急性分析蛋白质功能。小分子抑制剂并不广泛适用，因为在活细胞实验中往往难以确定特异性，而且设计非酶蛋白功能抑制剂具有挑战性。此外，这两种方法都只能应用于整个细胞，而不能用于分析空间限制的细胞内过程。最后，光消融和发色团辅助激光失活（CALI）利用高功率照明进行非特异性、不可逆的蛋白质破坏。该项目的目标是通过开发一种创新的、通用的、遗传编码的方法来解决这一挑战，通过这种方法，感兴趣的蛋白质可以在全细胞或细胞内区域被特定的光激活蛋白水解破坏，作为一种分析活细胞中蛋白质功能的新工具。由于我们建议使用光来切换蛋白酶活性，因此实验可以完全在配备充足的显微镜上进行，从而通过使用图案照明对细胞内蛋白质失活进行前所未有的高时间和空间控制。这种技术将彻底改变细胞生物学，并将对短时间内发生的细胞内过程的分析产生异常高的影响，并且依赖于直接调节蛋白质活性而不是基因表达变化。实现这一目标的策略将包括两个主要步骤：1)将植物趋光蛋白的光感结构域与小核糖核酸病毒3C蛋白酶的高特异性结合起来，设计和优化光激活位点特异性蛋白酶；2)通过对感兴趣的蛋白酶敏感蛋白进行基因工程验证可行性，并分析在RNAi沉默感兴趣基因内源性功能的活细胞中光激活靶蛋白失活的功能后果。我们将通过生成蛋白酶敏感版本的两种多结构域细胞骨架蛋白，talin和EB1，并通过构建蛋白酶敏感的激酶结构域来验证我们的方法的可行性和多功能性。

DESCRIPTION (provided by applicant): One of the next great challenges of the postgenomic era is functional analysis of the proteome in space and time, which will be essential to understand normal and pathological cell behavior. Direct analysis of protein function in complex intracellular processes requires a method to acutely, rapidly and specifically inactivate proteins of interest in real time and in selective regions of live cells. Such a method does not exist. Current methods to investigate intracellular protein function have severe limitations, and are either non-specific or lack sufficient spatial and temporal resolution. For example, because RNA interference (RNAi) relies on slow intracellular protein turnover, it is useful to detect long-term phenotypes, but does not allow direct, acute analysis of protein function. Small molecule inhibitors are not broadly applicable because specificity is often hard to establish in live cell experiments, and it is challenging to design inhibitors of non-enzymatic protein functions. In addition, both of these methods can only be applied to whole cells and are not useful to analyze spatially restricted intracellular processes. Finally, photoablation and chromophore-assisted laser inactivation (CALI) employ non-specific, non-reversible protein destruction using high power illumination. The objective of this project is to address this challenge by developing an innovative, versatile, genetically-encoded method by which a protein of interest can be disrupted by specific light-activated proteolysis in either whole cells or intracellular regions as a novel tool to analyze protein function in live cells. Because we propose to use light to toggle protease activity, experiments can be carried out entirely on an adequately equipped microscope allowing unprecedented high temporal and spatial control of intracellular protein inactivation by using patterned illumination. Such a technique would revolutionize cell biology, and would have an exceptionally high impact on the analysis of intracellular processes that occur on short time scales, and rely on direct regulation of protein activity rather than gene expression changes. The strategy to achieve this objective will involve two major steps: 1) Design and optimize a light-activated site-specific protease by combining the photosensory domain of plant phototropins with the exceptionally high specificity of picornavirus 3C proteases; and 2) Validate feasibility by genetically engineering protease-sensitive proteins of interest, and analyze functional consequences of light-activated target protein inactivation in live cells in which endogenous function of the gene of interest has been silenced by RNAi. We will test our approach by generating protease-sensitive versions of two multi-domain cytoskeleton proteins, talin and EB1, and by constructing a protease-sensitive kinase domain to demonstrate feasibility and versatility. PUBLIC HEALTH RELEVANCE: This project aims to build a novel tool to inactivate specific proteins in live cells with high spatial and temporal control by developing a light-activated site-specific protease in combination with a protease-sensitive version of a target protein of interest. A method to specifically, rapidly and locally disrupt intracellular protein function does not currently exist, and development of such a tool will have a high impact on the analysis of intracellular protein function in many fields of biomedical research. Detailed analysis of protein function in live cells is required to understand normal and pathological processes in cells, and will lead to the development of novel drugs and therapeutic strategies.