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

光激活蛋白水解作为分析细胞内蛋白质功能的工具

• 批准号: 8539033
• 负责人: Torsten Wittmann
• 金额: $ 29.52万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-09-01 至 2015-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8539033
• 关键词: Acute; Address; Biomedical Research; C-terminal; COX7A2L Protein; Cells; Cellular biology; Chimeric Proteins; Cleaved cell; Complex; Cytoskeleton; Development; Family Picornaviridae; Fluorescence Resonance Energy Transfer; Gene Expression; Genetic Engineering; Lasers; Lead; Life; Light; Lighting; Methods; Microscope; Molecular Models; Pathologic Processes; Pattern; Peptide Hydrolases; Pharmaceutical Preparations; Phenotype; Phosphotransferases; Plants; Process; Protease Domain; Protein Kinase; Proteins; Proteolysis; Proteome; RNA Interference; Regulation; Reporter; Resolution; Site; Specificity; Surface; Talin; Techniques; Testing; Therapeutic; Time; base; cell behavior; chromophore; design; gene function; high throughput screening; inhibitor/antagonist; innovation; interest; molecular modeling; novel; phototropin; protein degradation; protein function; public health relevance; research study; small molecule; tool

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.

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对时空中蛋白质组的功能分析，这对于了解正常和病理细胞行为至关重要。在复杂细胞内过程中对蛋白质功能的直接分析需要一种方法，可以实时和在活细胞的选择性区域中急性，快速，特定地灭活蛋白质。这种方法不存在。当前研究细胞内蛋白功能的方法具有严重的局限性，并且是非特异性或缺乏足够的空间和时间分辨率的。例如，由于RNA干扰（RNAI）依赖于缓慢的细胞内蛋白质周转率，因此检测长期表型很有用，但不允许直接对蛋白质功能进行直接的急性分析。小分子抑制剂并非广泛适用，因为特异性通常很难在活细胞实验中建立，并且设计非酶蛋白功能的抑制剂是具有挑战性的。另外，这两种方法只能应用于整个细胞，对于分析空间限制的细胞内过程没有用。最后，使用高功率照明，光实现和发色团辅助激光灭活（CALI）采用非特异性的，非可逆的蛋白质破坏。该项目的目的是通过开发一种创新的，多功能的，遗传编码的方法来应对这一挑战，通过该方法可以通过在整个细胞或细胞内区域特异性光激活蛋白水解来破坏感兴趣的蛋白质，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切换蛋白酶活性，所以可以完全在配备了足够的显微镜上进行实验，从而可以使用图案化的照明对细胞内蛋白质失活的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，并对短时间尺度上发生的细胞内过程的分析产生极大的影响，并依靠蛋白质活性的直接调节而不是基因表达变化。实现此目标的策略将涉及两个主要步骤：1）通过将植物光蛋白的光感（光感（光感）结构与Picornavirus 3C蛋白酶的特异性相结合，从而设计和优化了光激活的位点特异性蛋白酶；和2）通过遗传工程蛋白酶感兴趣的蛋白质验证可行性，并分析活细胞中光激活靶蛋白失活的功能后果，在这种细胞中，感兴趣基因的内源性功能已被RNAi沉默。我们将通过生成两个多域细胞骨架蛋白（Talin和EB1）的蛋白酶敏感版本，以及构建蛋白酶敏感的激酶结构域以证明可行性和多功能性来测试我们的方法。 公共卫生相关性：该项目旨在通过开发具有高空间和时间控制的活细胞中的特定细胞中的特定蛋白质来建立新的工具，通过开发光激活的位点特异性蛋白酶以及对蛋白酶感兴趣的靶蛋白的结合使用。目前不存在一种特定，快速和局部破坏细胞内蛋白质功能的方法，这种工具的开发将对许多生物医学研究领域的细胞内蛋白质功能的分析产生很大影响。需要对活细胞中蛋白质功能的详细分析来了解细胞中的正常和病理过程，并将导致新型药物和治疗策略的发展。