A Novel Class of Enzyme Sensors to Elucidate the Biochemical Responses of Human Nasal Epithelial Cells to Heavy Metals

7.项目总结 重金属是一类主要的化学暴露剂，对公众有重大影响 在环境、职业和/或环境暴露后导致发病率和死亡率的健康1 吸入是最常见的接触重金属的途径之一，并已知会导致呼吸道 炎症、癌症、金属烟雾热、哮喘和体能减退2-6重要的是，众所周知 Akt激酶和/或表皮生长因子受体激酶(EGFR)的异常活性发生在 暴露于重金属后的呼吸道上皮组织。然而，由于固有的细胞 呼吸道上皮细胞的异质性和单细胞对化学物质的动态反应 重金属暴露对单细胞跨不同细胞信号动力学的影响 呼吸道上皮内的类型尚不清楚。11因此，单细胞分析技术可以 获得原代呼吸道上皮超小样本Akt和EGFR活性的多重测量 细胞，将提高我们对重金属暴露的生化机制的理解。 重要的是，这种技术可能使临床医生能够识别重金属的早期预警迹象 从非常小的、不同种类的初级样本中诱导对个体的毒性和/或疾病诱导。 我的目标是通过使用传感器为基础的化学物质来改善呼吸道上皮的生物化学研究。 细胞学。基于传感器的化学细胞术是一种单细胞分析方法，其中生物分子传感器 用来测量单个细胞的小群体中的信号动力学。12-16在这里，我建议 用于Akt和EGFR多重检测的新型荧光酶传感器的研制 在单个细胞内使用毛细管电泳法荧光检测(CE-F)。我的目标也是 改进建议的酶传感器的设计和功能；通过安装可光激活的部分 在磷酸化位点上，我希望提高传感器的膜渗透性，并获得对 16此外，我计划控制细胞内的激酶反应停止时间 通过开发一种新型的化学选择性试剂来停止细胞内的反应，并促进报告 回收利用CE-F进行分析。 使用这些新型酶传感器进行的研究将增强我们对生物化学的理解 管理来自超少量初级种群的疾病诱导和/或复原力的机制 呼吸道上皮细胞。此外，从这项建议中获得的知识将改善我们的 了解重金属暴露的生化机制，同时识别新的 开发细胞渗透性传感器的策略，以实现单细胞内的时间可控反应。

7. Project Summary Heavy metals comprise a major class of chemical exposure agents and have a significant impact on public health causing morbidity and mortality following environmental, occupational, and/or ambient exposures.1 Inhalation is one of the most common routes for heavy metal exposures, and is known to cause respiratory inflammation, cancers, metal fume fever, asthma, and reduced physical performance.2-6 Importantly, it is known that aberrant activity of Akt kinase and/or epidermal growth factor receptor kinase (EGFR) occurs within respiratory epithelial tissue following exposures to heavy metals.6-9 However, due to the inherent cellular heterogeneity of cells within the respiratory epithelium and the dynamic responses of single cells to chemical stimuli;10 the effects of heavy metal exposures on the signaling dynamics on single cells across different cell types within the respiratory epithelium is unknown.11 Therefore, a single cell analysis technology that can obtain multiplexed measurements of Akt & EGFR activity in ultra-small samples of primary respiratory epithelial cells, would improve our understanding of the biochemical mechanisms that underlie heavy metal exposures. Importantly, such a technology can potentially enable clinicians to identify early warning signs of heavy metal induced toxicity and/or disease induction in individuals from very small, heterogeneous primary samples. I aim to improve biochemical investigations of the respiratory epithelium, by employing sensor based chemical cytometry. Sensor based chemical cytometry is a single cell analysis method in which biomolecular sensors are used to measure signaling dynamics in small populations of single cells.12-16 Here, I propose the development of a novel set of fluorescent enzyme sensors to obtain multiplexed measurements of Akt & EGFR within single cells using capillary electrophoresis employing fluorescence detection (CE-F). I also aim to improve the design and functionality of the proposed enzyme sensors; by installing photoactivatable moieties on the phosphorylation sites, I expect to improve membrane permeability of the sensors, and gain control over the kinase reaction start time within cells.16 Additionally, I plan to control the kinase reaction stop time in cells by developing a novel chemo-selective reagent which halts intracellular reactions, and facilitates reporter recovery for analysis via CE-F. Studies made possible using these novel enzyme sensors will bolster our understanding of the biochemical mechanisms that govern the induction of disease and/or resilience from ultra-small populations of primary respiratory epithelial cells. Additionally, the knowledge gained from this proposal would improve our understanding of the biochemical mechanisms that underlie heavy metal exposures, while identifying novel strategies to develop cell permeable sensors to achieve temporally controlled reactions within single cells.