Enabling the study of endogenous proteins in live cells

能够研究活细胞中的内源蛋白质

• 批准号: 8131587
• 负责人: ALICE Y TING
• 金额: $ 83.16万
• 依托单位: MASSACHUSETTS INSTITUTE OF TECHNOLOGY
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-09-30 至 2012-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8131587
• 关键词: Address; Affect; Biological; Biology; Biotin; Cells; Cytolysis; Disease; Enzymes; Esters; Evolution; Health; Human; Image; In Vitro; Label; Life; Ligase; Luciferases; Mass Spectrum Analysis; Methodology; Methods; Morphologic artifacts; Pattern; Physics; Physiological; Proteins; Recombinant Proteins; Recombinants; Reporter; Resolution; Technology; Thioctic Acid; Transcriptional Regulation; Translational Regulation; Ubiquitin; base; fluorophore; instrumentation; interest; mutant; protein folding; protein function; research study; small molecule; trafficking; Address; Affect; Biological; Biology; Biotin; Cells; Cytolysis; Disease; Enzymes; Esters; Evolution; Health; Human; Image; In Vitro; Label; Life; Ligase; Luciferases; Mass Spectrum Analysis; Methodology; Methods; Morphologic artifacts; Pattern; Physics; Physiological; Proteins; Recombinant Proteins; Recombinants; Reporter; Resolution; Technology; Thioctic Acid; Transcriptional Regulation; Translational Regulation; Ubiquitin; base; fluorophore; instrumentation; interest; mutant; protein folding; protein function; research study; small molecule; trafficking

Parallel advances in physics, instrumentation, and reporter technology have transformed biological imaging, such that it is now possible to visualize the localization, trafficking, interactions, and activity of many different proteins, with unprecedented spatial and temporal resolution inside living cells. However, a major challenge which affects the physiological relevance of all these experiments, has been largely un-addressed. Nearly all protein imaging experiments in living cells are performed using recombinant constructs, such as GFP, FlAsH, split ubiquitin, or luciferase fusions. Recombinant tags seriously affect biology in three ways: (1) tags can interfere with protein folding, trafficking, activity, and interactions; (2) protein over-expression leads to biological artifacts, such as changes in protein localization and interaction patterns; and (3) recombinant proteins do not have the same transcriptional and translational regulation as their endogenous counterparts, so these aspects of their biology cannot be easily studied. We propose to develop two technologies to enable the study of endogenous protein biology in living cells. First, we will develop a method to specifically and covalently label endogenous proteins of interest with small fluorophores and other probes inside living cells. This will be accomplished through in vitro evolution of two different small-molecule ligase enzymes ? biotin ligase and lipoic acid ligase ? to enable them to recognize and modify endogenous proteins. Second, we will develop a method to detect the endogenous interaction partners of specific cellular proteins. This method will be based on a promiscuous mutant of biotin ligase, which can be fused to proteins of interest, and which will release biotin-AMP ester that will covalently label any endogenous interacting partners. Biotinylated proteins can be purified and analyzed by mass spectrometry, after cell lysis. If successful, these two methodologies could transform biological studies of protein function in living cells, leading to fundamentally new discoveries of relevance to human health and disease.

物理，仪器和报告基因技术的并行进步已经改变了生物学成像，因此现在可以看到许多不同蛋白质的本地化，运输，相互作用和活性，并具有前所未有的空间和时间分辨率。但是，影响所有这些实验的生理相关性的一个主要挑战在很大程度上没有得到解决。活细胞中几乎所有蛋白质成像实验都是使用重组构建体进行的，例如GFP，Flash，分裂的泛素或荧光素酶融合。重组标签以三种方式严重影响生物学：（1）标签可以干扰蛋白质折叠，运输，活动和相互作用； （2）蛋白质过表达会导致生物伪影，例如蛋白质定位和相互作用模式的变化； （3）重组蛋白与内源性对应物没有相同的转录和翻译调节，因此其生物学的这些方面无法轻易研究。 我们建议开发两种技术，以研究活细胞中内源性蛋白质生物学的研究。首先，我们将开发一种方法，以小型荧光团和活细胞内的其他探针专门和共价标记感兴趣的内源性蛋白。这将通过两种不同的小分子连接酶的体外进化来实现？生物素连接酶和lipoic酸连接酶？使他们能够识别和修饰内源性蛋白质。其次，我们将开发一种检测特定细胞蛋白的内源相互作用伙伴的方法。该方法将基于生物素连接酶的混杂突变体，该突变体可以与感兴趣的蛋白质融合，并释放生物素 - AMP酯，该酯将共价标记任何内源性相互作用伴侣。细胞裂解后，可以通过质谱法纯化和分析生物素化蛋白。如果成功，这两种方法可以改变活细胞中蛋白质功能的生物学研究，从而从根本上与人类健康和疾病有关。