REAL-TIME QUANTITATIVE IMAGING OF INTRACELLULAR BIOTHIOL DYNAMICS

 描述（由申请人提供）：本提案的目的是开发一系列特定的生物硫醇探针，这些探针在经历可逆反应后将表现出不同的比率光谱特性，从而通过亚细胞分辨率的实时成像定量监测生物硫醇的动力学。尽管存在无数的小分子荧光探针开发的生物成像，很少能提供有意义的定量结果，特别是当任务检测氧化还原信号分子，如谷胱甘肽（GSH）和H2S。我们最近的工作表明，传感反应的可逆性是定量监测细胞中小分子动力学的关键。比率探针优选用于活细胞成像，因为它们允许独立于探针浓度的分析物浓度的定量测量。利用可逆迈克尔加成，我们开发了CouBro，第一个荧光探针定量成像的GSH在活细胞中。由于探针和GSH之间反应的可逆性，我们能够用低至50 nM的CouBro定量GSH的mM浓度。此外，GSH浓度在几个细胞系中，使用CouBro测量，是很好的相关性，从裂解物中获得的那些值。此外，我们表明，这种实时成像方法具有良好的再现性，能够检测细胞在外部刺激下的GSH波动。在初步研究中，我们开发了一种计算化学方法来预测生物硫醇和它们的探针之间的反应的热力学和动力学，这将指导我们的生物硫醇探针的设计。我们还开发了细胞器特异性H2S探针，通过应用遗传编码蛋白质技术，以反应为基础的小分子荧光探针。这种通用的靶向策略使我们能够推断出目标蛋白周围微环境中的信号分子浓度。在目标1中，我们将开发一系列具有快速动力学和细胞器特异性的GSH探针来监测细胞内GSH动力学。探针设计过程将通过计算化学来促进。在目标2中，我们将为H2S特异性反应开发新的可逆化学。由于报告的H2S水平不一致，范围为nM至µM，因此将开发具有一系列解离常数的H2S探针。我们还将通过标记负责H2S生产的关键酶和特定于某些细胞器的蛋白质来监测H2S信号动力学。在目标3中，我们将应用这些新开发的生物硫醇探针来研究Grx3介导的GSH代谢及其与癌细胞中H2S信号传导的相互作用，特别是在体内肿瘤发生期间。该项目的成功完成将为GSH和H2S动力学的定量成像提供全面的工具箱，并进一步阐明它们在氧化还原相关癌症信号传导和发展中的作用。

 DESCRIPTION (provided by applicant): The objective of this proposal is to develop a series of specific biothiol probes that will exhibit different ratiometric spectroscopic properties after undergoing reversible reactions, and thus quantitatively monitor the dynamics of biothiols through real-time imaging with subcellular resolution. Despite the existence of myriad small molecule fluorescent probes developed for biological imaging, very few can provide meaningful quantitative results, especially when tasked to detect redox signaling molecules, like glutathione (GSH) and H2S. Our recent work demonstrated that reversibility of sensing reactions is key to quantitatively monitoring the dynamics of small molecules in cells. Ratiometric probes are preferred for live cell imaging because they allow quantitative measurements of analyte concentrations independent of probe concentration. Taking advantage of reversible Michael additions, we developed CouBro, the first fluorescent probe for quantitative imaging of GSH in live cells. Due to the reversible nature of the reaction between the probe and GSH, we are able to quantify mM concentrations of GSH with as little as 50 nM CouBro. Furthermore, the GSH concentrations in several cell lines, measured using CouBro, are well correlated with those values obtained from lysates. In addition, we showed that this live imaging method has excellent reproducibility and is able to detect GSH fluctuations in cells upon external stimulation. In the preliminary study, we developed a computational chemistry approach to predict the thermodynamics and kinetics of reactions between biothiols and their probes, which will guide our design of biothiol probes. We also developed organelle specific H2S probes by applying genetically encoded protein technology to reaction-based small molecule fluorescent probes. This universal targeting strategy enables us to infer the signaling molecule concentration in the micro-environment around a protein of interest. In Aim 1, we will develop a series of GSH probes with fast kinetics and organelle specificity to monitor intracellular GSH dynamics. The probe design process will be facilitated by computational chemistry. In Aim 2, we will develop new reversible chemistry for H2S specific reactions. Due to inconsistently reported H2S levels, ranging from nM to µM, H2S probes with a range of dissociation constants will be developed. We will also monitor H2S signaling dynamics by labeling key enzymes responsible for H2S production and proteins specific to certain organelles. In Aim 3, we will apply these newly developed biothiol probes to investigate Grx3 mediated GSH metabolism and its interplay with H2S signaling in cancer cells, particularly during tumorigenesis in vivo. Successful completion of this project will provide a comprehensive toolbox for quantitative imaging of GSH and H2S dynamics and further elucidate their roles in redox-related cancer signaling and development.