REAL-TIME QUANTITATIVE IMAGING OF INTRACELLULAR BIOTHIOL DYNAMICS

细胞内生物硫醇动力学的实时定量成像

• 批准号: 9753260
• 负责人: Jin Wang
• 金额: $ 30.62万
• 依托单位: BAYLOR COLLEGE OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-09-22 至 2021-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9753260
• 关键词: Adopted; Antineoplastic Agents; Biological; Biology; Breast Cancer Cell; Breast Cancer cell line; Breast cancer metastasis; Cancer Cell Growth; Cell Line; Cell Proliferation; Cell Survival; Cells; Cellular biology; Cessation of life; Chemicals; Chemistry; Computing Methodologies; Development; Dissociation; Drug Delivery Systems; Electrons; Enzymes; Exhibits; Fluorescent Probes; Free Energy; Glutathione; Goals; Hydrogen Bonding; Hydrogen Sulfide; Image; Investigation; Journals; Kinetics; Label; Legal patent; Libraries; Life; Malignant Neoplasms; Measurement; Measures; Mediating; Metabolism; Metals; Methods; Molecular Probes; Monitor; Mus; Nature; Organelles; Organic Chemistry; Organic Synthesis; Oxidation-Reduction; Oxidative Stress; Pathway interactions; Play; Process; Production; Property; Proteins; Reaction; Reporting; Reproducibility; Research Personnel; Resolution; Role; Series; Signal Transduction; Signaling Molecule; Specificity; Techniques; Technology; Thermodynamics; Time; Training; Transgenic Mice; Work; base; breast tumorigenesis; cancer cell; cancer initiation; cancer prevention; commercialization; computational chemistry; computer studies; design; enolate; experience; experimental study; glutaredoxin; imaging modality; in vivo; interest; ionic bond; knock-down; live cell imaging; mammary gland development; mouse model; nano; nanoparticle drug; next generation; novel; off-patent; public health relevance; quantitative imaging; quantum; ratiometric; real-time images; small molecule; success; tool; tumorigenesis

 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.

 描述（由适用提供）：该提案的目的是开发一系列特定的生物醇问题，这些问题将在进行可逆反应后将存在不同的比率光谱特性，从而通过亚细胞分辨率进行定量监测Biothiols的动力学。尽管存在用于生物成像的无数小分子荧光问题，但很少有人能提供有意义的定量结果，尤其是在检测氧化还原信号分子（如谷胱甘肽（GSH）和H2S）的任务时。我们最近的工作表明，敏感性反应的可逆性是定量监测细胞中小分子动力学的关键。比率计量问题是活细胞成像的首选，因为它们允许对探针浓度无关的分析物浓度进行定量测量。利用可逆的迈克尔添加，我们开发了COUBRO，这是活细胞中GSH定量成像的第一个荧光探针。由于探针和GSH之间的反应的可逆性，我们能够用低至50 nm的COUBRO量化MM浓度的GSH。此外，使用COUBRO测量的几个细胞系中的GSH浓度与从裂解物获得的值很好地相关。此外，我们表明这种实时成像方法具有出色的可重复性，并且能够在外部刺激后检测细胞中的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与其问题之间反应的热力学和动力学，这将指导我们的生物硫醇探针设计。我们还通过将一般编码的蛋白质技术应用于反应的小分子荧光探针来开发有机特异性H2S探针。这种通用靶向策略使我们能够推断出围绕感兴趣蛋白质的微环境中的信号分子浓度。在AIM 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问题，以监测细胞内GSH动力学。探针设计过程将由计算化学制备。在AIM 2中，我们将开发针对H2S特定反应的新的可逆化学。由于不一致的H2S水平（从NM到µM），H2S问题将开发出一系列离解常数。我们还将通过标记负责H2S产生的密钥酶和特定于某些细胞器的蛋白质来监测H2S信号传导动力学。在AIM 3中，我们将应用这些新开发的生物醇问题来研究GRX3介导的GSH代谢及其与癌细胞中H2S信号的相互作用，尤其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，并进一步阐明其在氧化还原相关的癌症信号传导和发育中的作用。