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

描述(申请人提供)：后基因组时代的下一个重大挑战之一是对蛋白质组在空间和时间上的功能分析，这将是理解正常和病理细胞行为的关键。直接分析复杂细胞内过程中的蛋白质功能需要一种方法，能够在活细胞的选择性区域实时、迅速和特异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质功能的方法具有严重的局限性，要么是非特异性的，要么是缺乏足够的空间和时间分辨率。例如，由于RNA干扰(RNAi)依赖于缓慢的细胞内蛋白质周转，因此检测长期表型是有用的，但不允许直接、尖锐地分析蛋白质功能。小分子抑制剂没有广泛的适用性，因为在活细胞实验中通常很难建立特异性，而且设计非酶蛋白功能的抑制剂是具有挑战性的。此外，这两种方法都只能应用于整个细胞，不能用于分析空间受限的细胞内过程。最后，光消融和发色团辅助激光灭活(CALI)利用高功率照明使用非特异性、不可逆转的蛋白质破坏。这个项目的目标是通过开发一种创新的、通用的、遗传编码的方法来应对这一挑战，通过这种方法，目的蛋白可以被整个细胞或细胞内特定的光激活蛋白分解所破坏，作为分析活细胞中蛋白质功能的新工具。因为我们建议使用光来触发蛋白酶活性，所以实验可以完全在配备充分的显微镜上进行，从而通过使用图案化照明来实现前所未有的高时空控制细胞内蛋白质的失活。这种技术将给细胞生物学带来革命性的变化，并将对在短时间尺度上发生的细胞内过程的分析产生异常高的影响，并依赖于对蛋白质活动的直接调节，而不是基因表达的变化。实现这一目标的策略将包括两个主要步骤：1)通过将植物趋光蛋白的光敏感区与微小核糖核酸病毒3C蛋白酶的极高特异性相结合来设计和优化光激活的位点特异性蛋白酶；2)通过对目的蛋白敏感的蛋白进行基因工程来验证可行性，并分析光激活的靶蛋白在活细胞中失活的功能后果，其中目的基因的内源功能已被RNAi沉默。我们将通过生成两个多结构域细胞骨架蛋白Talin和EB1的蛋白酶敏感版本，以及通过构建一个蛋白酶敏感的激酶结构域来验证我们的方法，以证明其可行性和多功能性。 与公共卫生相关：该项目旨在建立一种新的工具来灭活活细胞中的特定蛋白质，并具有高度的空间和时间可控性，方法是开发一种光激活的位点特定的蛋白酶，并与感兴趣的目标蛋白质的蛋白酶敏感版本相结合。目前还没有一种特异、快速、局部干扰细胞内蛋白质功能的方法，这种工具的开发将对生物医学研究的许多领域的细胞内蛋白质功能的分析产生很大影响。对活细胞中蛋白质功能的详细分析是了解细胞中正常和病理过程所必需的，并将导致新药物和治疗策略的发展。

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.